Tolerance trials with young cocoa trees infected with cocoa swollen-shoot virus

In three short-term field trials, 24 cocoa progenies were assessed for sensitivity to infection with a severe Ghanaian strain of cocoa swollen-shoot virus. Some of the progenies were derived from tolerant parents, others had themselves shown mild symptoms as young seedlings. Stem diameters, diameter increments, canopy scores and latent periods (time between inoculation and expression of symptoms) of plants infected when 12 to 22 months old were compared with similar measurements of healthy plants. During the period between 6 and 18 months after inoculation, infection reduced the stem diameter increment of the least sensitive progeny in each trial by 52, 25 and 7%, the last value increasing to 38% during the period between 24 and 30 months after inoculation. Within trials, the effects of virus infection on stem diameter increments and canopy scores were well correlated but latent periods were not significantly correlated with any other variate. In one trial, pod yield was more closely related to canopy condition at the time of cropping than to stem diameter increment 2 yr earlier. Weak correlations between pre- and post-bearing increases in diameters in these trials indicated that tolerance was not directly related to vigour. None of the progenies is considered suitable for direct use as tolerant varieties in Ghana.     

Field experiments on the resistance of cocoa to cocoa swollen-shoot virus

Progenies from crosses between different Upper Amazon cocoa types and between Upper Amazon and Amelonado were compared for their field resistance to infection with cocoa swollen-shoot virus (CSSV) virulent strain A. Among the intra-Amazon progenies, those from crosses of Scavina, Iquitos and Nanay groups showed most resistance. Progenies from crosses within these groups may have sufficient resistance to be of immediate practical value in reducing crop losses in areas where CSSV is widespread. Some Nanay progenies were more resistant than others and this provides scope for improvement by breeding. Progeny of crosses between Upper Amazon and Amelonado parents were less resistant than those from intra-Amazon crosses. The present results confirm those previously obtained in gauze-house tests on young plants.     

Studies on the possibility of increasing resistance to cocoa swollen-shoot virus by breeding

In tests using seed, the resistance of cocoa progenies to primary infection with cocoa swollen-shoot virus by mealybugs was stable to inoculum pressure and effective against several virus strains. The results suggested that the resistance would be effective throughout the cocoa growing areas of Ghana. There were indications that resistance factors from different cocoa populations could be accumulated to give progenies of higher resistance than presently available.     

A field experiment in Ghana on the tolerance of cocoa seedlings to cocoa swollen-shoot and cocoa mottle-leaf viruses

Seedings of Amelonado Cocoa and of two progenies obtained by crossing Iquitos (Upper Amazon) parents were infected with three strains of cocoa swollen-shoot virus (CSSV) and with cocoa mottle-leaf virus (CMLV). CSSV strain I A had the most severe effects on the growth and canopy condition of all varieties. All four virus isolates had more effect on the growth of Amelonado than on the Iquitos progenies, except that CSSV strain I W affected the growth of Amelonado and one of the Iquitos progenies equally, as did CMLV which caused the greatest decrease in the first crop of all varieties. The relative virulence of different CSSV strains in any one variety can be assessed from single criteria such as length of latent period or effect on growth; CMLV had a shorter latent period than CSSV Strain I A but less effect on growth.     

Methods for assessing the tolerance and resistance of different types of cocoa to cocoa swollen-shoot virus

Different types of cocoa were compared for their resistance to infection and tolerance of infection with the virulent strain A of cocoa swollen-shoot virus, inoculated either by mealybugs or manually. The method of infection did not affect the ranking of the cocoa types for resistance, except at high percentages of infection, or for tolerance. In comparison with other methods, manual inoculation simplifies the screening of cocoa types for resistance and tolerance to virus infection, and provides a more sensitive test for resistance.     

Evaluation and use of a screening method to aid selection of cocoa (Theobroma cacao) with field resistance to cocoa swollen-shoot virus in Ghana

Seeds of cocoa progenies were inoculated with cocoa swollen-shoot virus manually or by vector transfer techniques to assess the relative resistance of the progenies to infection. Assessment of progeny included in both inoculation tests and in field trials were positively correlated, indicating that the inoculation technique is suitable for the selection of progenies for testing in the field. Pure Upper Amazon progenies were more resistant than selfed Amelonado, with hybrids between Amelonado and Upper Amazons usually intermediate. Among the main Upper Amazon populations, Iquitos and Nanay clones were better sources of resistance than Parinari and Scavina clones. A survey of these populations indicated that, within populations, resistance levels do not vary greatly. A range of progenies based on the Upper Amazon female parents in existing seed gardens was screened for resistance and some were consistently more resistant than the equivalent Amazon x Amelonado hybrid now being distributed to farmers.     

Resistance of cocoa to swollen-shoot virus in Ghana. II. Assessment of seed inoculation experiments

Differences in amounts of infection following seed inoculation, by the mealybug vector (Planococcoides njalensis (Laing)) of cocoa swollen-shoot virus were used as a short-term test of virus resistance in a range of cocoa progenies. The test results were highly correlated with virus resistance as measured in field trials. The best assessment of seed inoculation experiments allowed for progeny-dependent variation in the latent period of the virus. Experiments in which estimates of progeny means varied significantly with replicate sometimes gave poor agreement with known field performance.     

Serological relationships of some viruses from cocoa (Theobroma cacao L.) in Ghana

Strains A and Anibil of cocoa swollen-shoot virus are serologically closely related. They are more distantly related to strain M and cocoa mottle-leaf virus (Kpeve isolate), which are distantly related to one another. Strains A and Anibil protected against one another in cocoa seedlings, but no protection was found with any other combination between these four virus isolates. All mealybug-transmitted cocoa viruses in West Africa are probably best considered as strains of cocoa swollen-shoot virus.     

Genetic aspects of resistance to cocoa swollen-shoot virus in Ghana

The inheritance of resistance in cocoa to infection with cocoa swollen-shoot virus was studied in seed inoculation experiments with progenies of Upper Amazon, Trinitario and West African Amelonado parents. In an eight parent diallel cross grown in two seasons and in six male × female factorial experiments, general combining abilities were much more important than specific combining abilities. The diallel cross revealed significant maternal effects but there was little evidence of specific reciprocal differences. Estimates of general combining abilities corresponded with expectation and were positively correlated with maternal effects, the latter indicating that tester parents should be used as males. The results from scaling tests with hybrids between selections from the River Nanay (a Peruvian tributary of the River Amazon) and Amelonado fitted a model with additive resistance; there was no evidence of dominance. Attempts to obtain stronger sources of resistance by hybridisation and selection were unsuccessful. In three crosses the resistance of individual seedlings was compared with that of the parents using test crosses but no evidence of transgressive segregation was obtained. In further experiments seedings of two hybrids between contrasting parents were heavily inoculated with virus so that few healthy survivors remained. Test crosses showed that these survivors and randomly selected seedlings of the same parentage did not differ as resistance sources.     

The effects of cocoa swollen-shoot virus on the growth and yield of Amelonado and Amazon cocoa (Theobroma cacao) in Ghana

The virulent strain A of cocoa swollen-shoot virus (CSSV) severely decreased the growth and yield of Amelonado cocoa (Theobroma cacao) trees kept free of capsids (Distantiella theobroma and Sahlbergella singularis) and the dieback fungus (Calonectria rigidiuscula) in Ghana. Fifteen per cent of graft-inoculated Amelonado trees showed symptoms within 4 months, and 48, 80 and 100% within 6, 12 and 20 months, respectively. Infected trees, whether shaded or unshaded, began to decline 6 months after infection, and deteriorated rapidly during the next 27 months by which time 16 % had died and most others were moribund; fertilizer applications had no significant effect on the rate at which infected trees deteriorated. Yields of pods and dry cocoa were greatly reduced 2 yr after infection and were very low after 3 yr; yields were significantly reduced by virus infection but there were no significant further effects of applying fertilizer. These results confirm that CSSV strain A alone is very damaging and often eventually lethal to Amelonado trees in Ghana, and indicate that the conflicting results obtained previously in Ghana and Nigeria were probably due to differences in the virulence of the CSSV strains tested. In contrast, the virus had much less effect on cocoa trees of the Amazon type; only 3% of graft-inoculated Amazon trees showed symptoms within 4 months, and 43, 84 and 97% after 1, 2 and 3 yr, respectively. Slight deterioration of tree canopies was first detected c. 15 months after infection and, although it continued slowly during the next 21 months, the decline was much less severe than that of Amelonado trees. Yields of both unshaded and shaded trees were apparently reduced by virus infection, but yield losses were much smaller than those of Amelonado trees. These results support the present objectives of controlling the spread of CSSV in Ghana by roguing infected trees, and selecting cultivars with greater tolerance to infection for future use.     

Resistance of cocoa to swollen-shoot virus in Ghana. I. Field trials

In Ghana, field resistance to cocoa swollen-shoot virus (CSSV) in young, bearing trees was assessed by monitoring natural spread by mealybug vectors from adjacent graft-inoculated line sources. Apparent rates of virus spread were estimated by regressing proportions of infected trees on time from inoculation. In some hybrids between parents derived from Upper Amazonian material, spread was only 25–40% that in currently recommended varieties. This resistance is the most effective, feasible measure to reduce economic losses and the resistant hybrids could be made available quickly for large scale use. The results from trials in which virus spread most rapidly were the easiest to interpret. Spread of CSSV is erratic, necessitating large experiments, and in trials with equal replication the largest plots gave the greatest precision. Neighbouring plot adjustment proved unsatisfactory as it improved precision but appeared to reduce accuracy.     

Additive inheritance of resistance to pod rot caused by Phytophthora palmivora in cocoa

Summary Quantitative inheritance of resistance to Phytophthora pod rot (Ppr) was studied in cocoa hybrid progeny from 12 Trinitario x Amazonian crosses and their reciprocal crosses. The crossing scheme was similar to a factorial design. Disease was assessed by the number and percentage of infected pods on each tree. Highly significant differences due to general combining abilities (GCA) were obtained for all characters, except for the GCA of Trinitario on total pod production. Differences for specific combining ability (SCA) were not significant for all characters. There were no significant differences between reciprocal crosses. The Trinitario clone K82 provided the only source for the hybrid progenies of strong Ppr resistance to the hybrid progenies, while K20 provided moderate resistance. Other parental clones — KA2-101, KA5-201, KEE 2, KEE 5, and KEE 52 — produced progenies which were susceptible to Ppr. It is evident that resistance to Ppr in cocoa is inherited additively. Maternal and cytoplasmic effects were assumed to have no influence on inheritance of resistance. It is also concluded that resistance to Ppr of the kind shown by K82 is likely to be horizontal resistance. Breeding for high-yielding cultivars combined with Ppr resistance is the most effective way of controlling Ppr of cocoa on the crops of growers with small holdings in Papua New Guinea.     

Genetic mapping of resistance factors to Phytophthora palmivora in cocoa.

Phytophthora palmivora causes pod rot, a serious disease on cocoa widespread throughout the producing regions. In order to ascertain the genetic determination of cocoa resistance to P. palmivora, a study was carried out on two progenies derived from crosses between a heterozygous, moderately resistant Forastero clone, T60/887, and two closely related and highly susceptible Forastero clones, one completely homozygous, IFC2, and one partially heterozygous, IFC5. The cumulative size of both progenies was 112 individuals. Plants were subjected to natural and artificial inoculation of P. palmivora in C te d'Ivoire. The genetic maps of T60/887 and of IFC5 were constructed using amplified fragment length polymorphism (AFLP) markers and microsatellites. The map of T60/887 comprised 198 markers assembled in 11 linkage groups and representing a total length of 793 cM. The map of IFC5 comprised 55 AFLP markers that were assembled into six linkage groups for a total length of 244 cM. Ratio of rotten over total number of fruit under natural infection was measured for each tree over two harvests. Artificial inoculations were performed on leaves and pods. These tests were weakly correlated with the pod rot rate in the field. Five quantitative trait loci (QTLs) of resistance were detected for T60/887 but none were common between the three traits measured. Stability and reliability of the experimental procedures are discussed and revealed the difficult use of these artificial tests on adult trees for a good prediction of field resistance.     

Yeast diversity of Ghanaian cocoa bean heap fermentations

The fermentation of the Theobroma cacao beans, involving yeasts, lactic acid bacteria, and acetic acid bacteria, has a major influence on the quality of the resulting cocoa. An assessment of the microbial community of cocoa bean heap fermentations in Ghana resulted in 91 yeast isolates. These were grouped by PCR-fingerprinting with the primer M13. Representative isolates were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences and partial actin gene sequences leading to the detection of 15 species. Properties of importance for cocoa bean fermentation, namely sucrose, glucose, and citrate assimilation capacity, pH-, ethanol-, and heat-tolerance, were examined for selected isolates. Pichia kudriavzevii ( Issatchenkia orientalis ), Saccharomyces cerevisiae , and Hanseniaspora opuntiae formed the major components of the yeast community. Hanseniaspora opuntiae was identified conclusively for the first time from cocoa fermentations. Among the less frequently encountered species, Candida carpophila, Candida orthopsilosis, Kodamaea ohmeri, Meyerozyma ( Pichia ) caribbica, Pichia manshurica, Saccharomycodes ludwigii , and Yamadazyma ( Pichia ) mexicana were not yet documented from this substrate. Hanseniaspora opuntiae was preferably growing during the earlier phase of fermentation, reflecting its tolerance to low pH and its citrate-negative phenotype, while no specific temporal distribution was recognized for P. kudriavzevii and S. cerevisiae .     

Genetic variation in resistance to vascular-streak dieback in cocoa (Theobroma cacao)

Summary Genetic variation in disease resistance to vascular-streak dieback (Vsd) was studied in 54 cocoa hybrid progenies from Trinitario x Amazonian crosses. The disease was assessed by the number of Vsd infected branches per tree, total length of infected branches, average depth of disease penetration within branches and percentages of infected and dead plants. Highly significant differences due to general combining ability for both Trinitario female parents and Amazonian male parents were obtained for all characters. Percent of dead plants was the only character where specific combining ability was also important. Gene effects were predominantly additive for most characters, indicating that selection for progenies resistant to Vsd is effective in the present breeding population. There is strong evidence that resistance to Vsd is in the form of horizontal resistance. It is polygenic and largely inherited as additive genes. Therefore, breeding for resistance is an effective means of controlling Vsd in Papua New Guinea.     

Assessment of the value of doubled haploids as progenitors in cocoa (Theobroma cacao L.) breeding

In order to evaluate twelve doubled haploids (DHs) of Theobroma cacao L. used as parents, a trial was set up in C?te d'Ivoire. Several traits were observed, such as yield, vigour, yield/vigour ratios, resistance to the black pod disease caused by Phytophthora, percentage of flat beans and mean weight of 100 cocoa beans. Out of the three progenies derived from crosses between two DHs, two showed severe drawbacks. A reduction of the heterogeneity within these progenies was occasionally observed for some of the traits, but failed to be consistent. When tested as female parents in combination with diploid testers, some of the DHs showea significantly higher combining value than their parents for traits such as the mean weight of 100 beans and the yield/canopy surface ratio. The results showed the potential of DHs to improve selected parents in only one cycle of selection but more crosses between two DHs need to be tested in order to evaluate potential of the resulting F(1) progenies.     

No OTA in fresh cocoa beans

The presence of ochratoxin A (OTA) in cocoa and chocolates has been reported. There is no previously published data available on the source and development of OTA producing moulds and OTA itself in cocoa,i.e. where the mycotoxin enters the cocoa supply chain. A selection of fresh and undamaged cocoa pods from various growing regions was examined for mycotoxin OTA content. In addition, a small selection of damaged or mouldy cocoa pods was included in the examination. It was shown that the ripening phase of healthy cocoa pods from the tree up to being harvested was not a critical period for the occurrence of the mycotoxin OTA. The mycotoxin OTA was not detectable in any of the analysed cocoa pods. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

A method of inoculating young cocoa plants with basidiospores of Oncobasidium theobromae

A method is described for collecting basidiospores of Oncobasidium theobromae onto agar containing 35% w/v sucrose and preparing infective spore suspensions. When cocoa seedlings and clonal cuttings were inoculated, large differences in percentage infection were demonstrated between seedling progenies of resistant and susceptible trees. Resistant clonal cuttings were generally difficult to infect. The value of the technique for resistance screening is discussed.     

Characterisation and genetic mapping of resistance and defence gene analogs in cocoa (Theobroma cacao L.)

Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS –like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management.     

Biodiversity conservation in cocoa production landscapes: an overview

Cocoa agroforests that retain a floristically diverse and structurally complex shade canopy have the potential to harbour significant levels of biodiversity, yet few studies have documented the plant and animal species occurring within these systems or within landscapes dominated by cocoa production. In this special issue, we bring together nine studies from Latin America, Africa and Asia that document the contribution of cocoa agroforestry systems to biodiversity conservation, and explore how the design, management and location of these systems within the broader landscape influence their value as habitats, resources and biological corridors. Tree diversity within the cocoa production systems is variable, depending on management, cultural differences, location and farm history, among other factors. Animal diversity is typically highest in those cocoa agroforests that have high plant diversity, structurally complex canopies, and abundant surrounding forest cover. In general, both plant and animal diversity within cocoa agroforests is greater than those of other agricultural land uses, but lower than in the original forest habitat. There are several emerging threats to biodiversity conservation within cocoa production landscapes, including the loss of remaining forest cover, the simplification of cocoa shade canopies and the conversion of cocoa agroforestry systems to other agricultural land uses with lower biodiversity value. To counter these threats and conserve biodiversity over the long-term, land management should focus on conserving native forest habitat within cocoa production landscapes, maintaining or restoring floristically diverse and structurally complex shade canopies within cocoa agroforests, and retaining other types of on-farm tree cover to enhance landscape connectivity and habitat availability.