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.     

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.     

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.     

The resistance and tolerance of different cocoa varieties to cocoa swollen-shoot virus in Ghana

The resistance and tolerance to infection with cocoa swollen-shoot virus (virulent strain A) of forty-five and thirty-eight cocoa progenies respectively was estimated using a manual inoculation method. Resistance was estimated from the proportion of inoculated seedlings which developed symptoms, and tolerance from the severity of these symptoms during a 4-month period. Resistance was found among Nanay, Iquitos and Scavina, all Upper Amazon types; and an inter-Nanay cross was outstandingly resistant and offers scope for improvement by breeding. Some Amazon-Amazon and Trinitario-Amazon progenies snowed tolerance, especially those with Trinitario T9/21 as a parent. Some progenies seem sufficiently promising for field testing to select varieties suitable for planting in those areas in Ghana where swollen-shoot disease is epidemic.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Functional yeast starter cultures for cocoa fermentation

The quest to develop a performant starter culture mixture to be applied in cocoa fermentation processes started in the 20th century, aiming at achieving high-quality, reproducible chocolates with improved organoleptic properties. Since then, different yeasts have been proposed as candidate starter cultures, as this microbial group plays a key role during fermentation of the cocoa pulp-bean mass. Yeast starter culture-initiated fermentation trials have been performed worldwide through the equatorial zone and the effects of yeast inoculation have been analysed as a function of the cocoa variety (Forastero, Trinitario and hybrids) and fermentation method (farm-, small- and micro-scale) through the application of physicochemical, microbiological and chemical techniques. A thorough screening of candidate yeast starter culture strains is sometimes done to obtain the best performing strains to steer the cocoa fermentation process and/or to enhance specific features, such as pectinolysis, ethanol production, citrate assimilation and flavour production. Besides their effects during cocoa fermentation, a significant influence of the starter culture mixture applied is often found on the cocoa liquors and/or chocolates produced thereof. Thus, starter culture-initiated cocoa fermentation processes constitute a suitable strategy to elaborate improved flavourful chocolate products.     

Phenology and Stem Diameter Increment Seasonality in a Costa Rican Wet Tropical Forest

The relationship between phenology and tree stem diameter increment is largely unexplored in tropical species, especially in wet tropical forests. To explore links between these phenomena, we measured stem diameter increment and phenology of ten canopy tree species from a range of functional types in the Atlantic lowlands of Costa Rica to test for seasonal and interannual patterns. We measured stem diameter increment using band dendrometers and visually assessed leaf and reproductive phenology monthly from 1997 to 2000. We categorized the species into groups based on patterns of leaf exchange and reproduction. Species were either deciduous with synchronous or asynchronous leaf drop, or evergreen with continuous or seasonal leaf flushing. Flowering occurred supra-annually, annually, or continuously. Of the ten species studied, four species, Cecropia insignis, Dipteryx panamensis, Lecythis ampla, and Simarouba amara , had consistent seasonal stem diameter increment patterns in both years. Dipteryx panamensis and L. ampla were deciduous with synchronized leaf drop . Cecropia insignis was evergreen and produced new leaves continuously. Simarouba amara , also evergreen, exchanged leaves over a brief period once a year. We tested whether stem diameter increment was correlated to phenology using logistic regression. Leaflessness significantly explained patterns in stem diameter increment but reproductive phenology did not. Deciduous trees were 2.6–9.3 times more likely to grow less than average the month following leaffall than in months when trees had full crowns.     

The virobacterial agglutination test as a rapid means of detecting cocoa swollen shoot virus

The virobacterial agglutination (VBA) test was developed as a means of detection of cocoa swollen shoot virus (CSSV). Identification of CSSV-infected Theobroma cacao in the field has only been possible by visual examination of symptoms, by virus transmission using mealybugs and by grafting to induce symptom expression in Amelonado cocoa seedlings. Detection of latent infection has not been possible even using enzyme-linked immunosorbent assays (ELISA). The VBA test successfully detected CSSV in infected sap diluted to 1/2560. Antisera to a range of mild and severe CSSV isolates were tested, and the results suggest a close relationship between seven isolates (1A, Bosomtwi, Bosomuoso, Nkrankwanta, Nsaba, Seidi-Nkawie and SS365B) while the mild isolate N1 appears to be less closely related. The VBA test was compared with both direct and indirect ELISA in the field. Only VBA detected all the cocoa trees which were known to be infected and additionally identified infection in many symp-tomless trees.     

A small-scale screening technique for evaluating fungicides against Phytophthora palmivora pod rot of cocoa

Plots of clonal Trinitario cocoa were sprayed with fungicides in the field. Ripe pods were harvested from the trial plots and artificially inoculated with Phytophthora palmivora using various techniques, the most suitable of which proved to be the zoospore spot method. Percentage infection results from the spot inoculation tests agreed well with the field percentage infection results and it was concluded that field spraying followed by zoospore spot inoculation of detached pods is a suitable preliminary screening technique for fungicides. Three systemic fungicides metalaxyl, aluminium tris (ethyl phosphonate) and DPX-3217 in a mixture with captafol and copper oxychloride were compared with the protectant cuprous oxide. Metalaxyl was found to be the most effective of the systemic fungicides and when applied at the rate of 0·44 g ai/ha every 8 wk it gave a similar reduction in percentage infection to 4 weekly applications of cuprous oxide at the rate of 4·4 kg Cu⁺⁺/ha during the wetter part of the year when pod rot is most severe.     

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.     

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.     

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.     

Kinetic analysis of strains of lactic acid bacteria and acetic acid bacteria in cocoa pulp simulation media toward development of a starter culture for cocoa bean fermentation

The composition of cocoa pulp simulation media (PSM) was optimized with species-specific strains of lactic acid bacteria (PSM-LAB) and acetic acid bacteria (PSM-AAB). Also, laboratory fermentations were carried out in PSM to investigate growth and metabolite production of strains of Lactobacillus plantarum and Lactobacillus fermentum and of Acetobacter pasteurianus isolated from Ghanaian cocoa bean heap fermentations, in view of the development of a defined starter culture. In a first step, a selection of strains was made out of a pool of strains of these LAB and AAB species, obtained from previous studies, based on their fermentation kinetics in PSM. Also, various concentrations of citric acid in the presence of glucose and/or fructose (PSM-LAB) and of lactic acid in the presence of ethanol (PSM-AAB) were tested. These data could explain the competitiveness of particular cocoa-specific strains, namely, L. plantarum 80 (homolactic and acid tolerant), L. fermentum 222 (heterolactic, citric acid fermenting, mannitol producing, and less acid tolerant), and A. pasteurianus 386B (ethanol and lactic acid oxidizing, acetic acid overoxidizing, acid tolerant, and moderately heat tolerant), during the natural cocoa bean fermentation process. For instance, it turned out that the capacity to use citric acid, which was exhibited by L. fermentum 222, is of the utmost importance. Also, the formation of mannitol was dependent not only on the LAB strain but also on environmental conditions. A mixture of L. plantarum 80, L. fermentum 222, and A. pasteurianus 386B can now be considered a mixed-strain starter culture for better controlled and more reliable cocoa bean fermentation processes.     

Epidemiology of vascular-streak dieback of cocoa

Inoculation studies provided further evidence that Oncobasidium theobromae is the cause of vascular-streak dieback of cocoa and that the fungus can penetrate undamaged, soft leaves. Spores are forcibly ejected from basidia at night and dispersed by wind. They are rapidly killed by exposure to normal day-time conditions and require free water for germination. Spore dispersal is probably very limited under plantation conditions and disease spread occurs along a steep gradient into new plantings. Disease increase in young plantations was found to occur at a rate of 0.31 per unit per month; in one block 100% of seedlings were infected and 55% were killed within 33 months of planting. The disease was most damaging in seedlings younger than 18 months old, although there was evidence that it also caused substantial yield loss in mature susceptible cocoa. Sporophore production and infection rate were correlated with rainfall incidence. The unusual grographical distribution of the disease and aspects of its control are discussed.