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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Morphological variation in Planococcoides njalensis occurring on cocoa in Ghana

A study was made to determine the extent of morphological variation in Planococcoides jalensis (Laing), the most important mealybug vector of cocoa swollen shoot virus (CSSV) in West Africa, and to resolve if more than one Planococcoides species is present. Fourteen populations of P. njalensis collected on cocoa from different localities in Ghana and one population each on Gliricidia sepium (Jacq.) Steud and Coffea canephora Pierre were compared by measured and meristic female characters using two types of morphometric analysis, Discriminant Functional Analysis and Principal Component Analysis. The analyses showed that P. njalensis exhibited a wide range of intraspecific morphological variation, particularly in meristic characters, but failed to identify new Planococcoides species. The results are discussed in relation to the control of the mealybug vectors of CSSV and to the suitability of Gliricidia as a shade tree for cocoa.     

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.     

Improved methods for the purification and detection of cocoa swollen shoot virus

The extraction of cocoa swollen shoot virus (CSSV) from cocoa leaves with pectinase, purification from the concentrated extracts by filtration through Celite and Sepharose 2B, and concentration of the virus by isopycnic CsCl centrifugation are described. The pectinase and Celite treatments effectively removed mucilage and particulate host plant materials, and enhanced the release of virus particles, Isopycnic CsCl centrifugation resulted in 10–100-fold concentration of particles, but with apparent loss of infectivity. After extraction with pectinase, CSSV particles were regularly seen by electron microscopy in sap. Immunosorbent electron microscopy (ISEM) considerably enhanced the detection of CSSV in sap. The particles were bacilliform or bullet-shaped and of various lengths. The common dimensions were 142 × 27 nm.     

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.     

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.     

Evidence from the virobacterial agglutination test for the existence of eight serogroups of cocoa swollen shoot virus

Many isolates of cocoa swollen shoot virus (CSSV) have been found in Ghana. Relationships between these isolates have been based on symptom expression and limited serological information. This paper reports on the serological relationships between 44 accessions of CSSV using the virobacterial agglutination test. The CSSV group is differentiated into eight groups using seven antibody 'types'. The largest group comprising those isolates closely related to CSSV 1A is sub-divided into four further groups. These groupings are compared with previous results. Differences are seen between accessions of the same isolate which may be due to contamination of the source plants. These serological results can be used for studying mixed isolate infections as well as for determining the most closely related CSSV mild isolate for appropriate cross-protection against severe isolates.     

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 case of mistaken identity? Vaccinia virus in a live camelpox vaccine

Live-attenuated (LA), and inactivated adjuvant (IA) camelpox virus (CMLV) vaccines are produced in several countries worldwide. A tissue culture attenuated CMLV isolated (Jouf-78) is used to produce an LA vaccine in Saudi Arabia (Hafez et al., 1992). DNA extracts from the Saudi LA vaccine were used as positive controls for a routine ATIP PCR produced fragments longer than 881 bp. PCR-amplified ATIP sequences were similar to vaccinia virus (VACV) Lister strain. PCR and sequence analysis of two extracellular enveloped virus (EEV)-specific (A33R and B5R), and two intracellular mature virus (IMV) (L1R and A27L) othrologue genes from the vaccine DNA extracts confirmed the finding. CMLV sequences were not detected in vaccine DNA extracts. A VACV Lister strain imported from Switzerland was used in control experiments during initial testing of the Saudi LA vaccine. High antigenic similarity between VACV and CMLV, and a possible contamination event during production may have caused this issue. Environmental and health impact studies were recommended because early VACV vaccines produced in some European countries contained nonhighly attenuated strains that were not adequately screened for adventitious agents.     

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.     

Pure yeast culture fermentation of cocoa (Theobroma cacao L): effect on yield of sweatings and cocoa bean quality

Cocoa sweatings, the pale yellowish liquid that drains off during cocoa fermentation, is the breakdown product of the mucilage surrounding the fresh cocoa bean, and constitutes about 10% of the weight of the cocoa fruit. On average, about 1.9 million l of sweatings are produced annually in Ghana during the cocoa harvesting season. It has been shown to be a suitable medium for the production of wines, alcohol, marmalade, jam and syrup. Its rapid collection in high yields and quality is the first step to its utilization on a commercial scale. Thus pure yeast culture fermentation of cocoa under controlled temperature conditions and its effect on yield of sweatings and final cocoa bean quality was investigated. Cocoa fermentations employing Saccharomyces chevalieri or Kluyveromyces fragilis alone gave significantly higher yields of sweatings (p 0.05) than controls. The initial rates of sweating by the two strains were also very high but dropped to a constant minimum value after 12h of fermentation. In contrast, fermentations employing Torulopsis candida or Candida norvengensis alone as well as different combinations of all the yeast strains did not give any significant difference in yield compared to controls (p 0.05). Fermentations using S. chevalieri alone or other combinations in which S. chevalieri was present gave beans with acceptable quality based on different quality indices used for grading cocoa beans commercially.     

Studies on Particle Components of Cacao Swollen Shoot Virus

Virions of the cacao swollen shoot virus (CSSV) strain 1A were purified and used for studies of its particle components. CSSV virions had a buoyant density of 1.365 g/cm³ in buffered CsCl. Following SDS polyacrylamide gel electrophoresis (PAGE), CSSV-specific proteins were identified in electroblot immunoassays (EBIA) with cross-absorbed polyclonal antibodies and especially well with monoclonal antibodies (MABs) to CSSV-1A. Based upon EBIA experiments with a selected MAB, CSSV virions appeared to have one capsid protein species with a relative molecular mass (M), of about 43 kd that was shown to be not glycosylated. However, this protein is sensitive to proteolytic degradation as degradation products ranging from 37 to 33 kd were found in addition to the 43 kd protein. Studies on the viral genome of CSSV revealed that CSSV virions contain a DNA of about 7. 5 kbp. Nucleic acid probes obtained by cloning parts of the viral genome yielded specific hybridization reactions with extracts and preparations from plants infected with strain 1A of CSSV but not with those from non-inoculated plants. One clone of 738 bp was sequenced and shown to contain a motif similar to the putative RNA binding domain of pararetroviruses. Based upon particle morphology and properties of the virion components, CSSV can be grouped with other nonenveloped bacilliform viruses for which the name badnaviruses has been proposed.     

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.