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.     

Infection of non‐host model plant species with the narrow‐host‐range Cacao swollen shoot virus

Cacao swollen shoot virus (CSSV) is a major pathogen of cacao (Theobroma cacao) in Africa, and long‐standing efforts to limit its spread by the culling of infected trees have had very limited success. CSSV is a particularly difficult virus to study, as it has a very narrow host range, limited to several tropical tree species. Furthermore, the virus is not mechanically transmissible, and its insect vector can only be used with difficulty. Thus, the only efficient means to infect cacao plants that have been experimentally described so far are by particle bombardment or the agroinoculation of cacao plants with an infectious clone. We have genetically transformed three non‐host species with an infectious form of the CSSV genome: two experimental hosts widely used in plant virology (Nicotiana tabacum and N. benthamiana) and the model species Arabidopsis thaliana. In transformed plants of all three species, the CSSV genome was able to replicate, and, in tobacco, CSSV particles could be observed by immunosorbent electron microscopy, demonstrating that the complete virus cycle could be completed in a non‐host plant. These results will greatly facilitate the preliminary testing of CSSV control strategies using plants that are easy to raise and to transform genetically.     

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.     

Detection and Comparison of some Ghanaian Isolates of Cacao Swollen Shoot Virus (CSSV) by Enzyme-Linked Immunosorbent Assay (ELISA) and Immunoelectron Microscopy (IEM) Using an Antiserum to CSSV Strain 1A

Various isolates of Cacao Swollen Shoot Virus (CSSV) were detected without difficulty in leaves of Theobroma cacao L. by ELISA and immunosorbent electron microscopy (ISEM) using an antiserum to severe strain 1A. Many isolates were detected with relatively high values at dilutions of 1:30, whereas some other isolates were hardly or not at all detected at this dilution. Strain 1A was detected at dilutions of up to 1: 2560 of crude leaf extracts. All isolates yielding high reactions seem to be serologically closely related to strain 1A. Strains of the mottle-leaf type (AD 191, AD 196, AD 7, AD 36, AD 135, Kpeve) and others were poorly detected; their relationship to strain 1A is discussed. A close correlation was found between results obtained by ELISA and ISEM.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Inventory and abundance of mealybug species in immature and mature cocoa farms in Côte d'Ivoire

From 2013 to 2018, surveys were conducted in counties not previously surveyed in order to determine species of mealybugs present in the cocoa orchard in Côte d'Ivoire as well as their abundance according to the age of cocoa trees. Immature and mature cocoa trees were inspected to hand‐height in 5 and 29 counties infected with Cacao swollen shoot virus (CSSV). In each cocoa farm, mealybugs were searched for on fruits, leaves, flowers, twigs and trunks. Mealybug species were identified, and colonies were counted. Five mealybug species were identified on immature cocoa trees: Ferrisia virgata, Formicococcus njalensis, Planococcus citri, Planococcus kenyae and Pseudococcus longispinus. In addition to these species, four species, Dysmicoccus brevipes, Maconellicoccus hirsutus, Phenacoccus hargreavesi and Pseudococcus jackbeardsleyi were identified on mature cocoa trees. On immature cocoa trees, Fo. Njalensis, Pl. citri and Ps. longispinus comprised were, respectively, 35%, 33% and 19% of colonies, respectively. On mature cocoa trees, Fo. Njalensis and Pl. citri comprised 63.2% and 21.0%, and others species 15.8%. Nevertheless, the abundance of mealybug species varied according to the age of cocoa trees. The preferred organs of mealybugs were pods (74.1%) followed by twigs (13.4%) and flowers (7.4%). Previously, the mealybug Paracoccus burnerae (Brain) was found on Theobroma cacao, which is the first record for this species in Côte d'Ivoire and on this host‐plant.     

Analysis of multiparasitism by Eupelmus vuilleti (Craw) (Eupelmidae) and Dinarmus basalis (Rond) (Pteromalidae) in the presence of one of their common hosts,Bruchidius atrolineatus (Pic) (Coleoptera Bruchidae)

Bruchidius atrolineatus (Pic) is a tropical beetle (Coleoptera Bruchidae) that develops during the larval and pupal stages in the seeds of a legume Vigna unguiculata (Walp). Two species of Hymenoptera, Dinarmus basalis (Rond) and Eupelmus vuilleti (Craw), solitary ectoparasitoids of the larvae and pupae of B. atrolineatus, were introduced successively in the presence of their hosts, varying the interval between the two introductions. When D. basalis females were introduced 24 h, 3 days or 7 days after E. vuilleti, multiparasitism was low. The females had low fecundity, and their eggs were not distributed randomly over the different available hosts. When E. vuilleti females were introduced second, they oviposited on the different hosts availabe and did not avoid multiparasitism. The presence of hosts already parasitised by D. basalis increased the reproduction of E. vuilleti, and the fecundity of the females was higher than in control batches with E. vuilleti alone. E. vuilleti seems capable of detecting the ovipositor shafts drilled by the D. basalis females, and by introducing its own ovipositors killing the D. basalis eggs or larvae. When interspecific competition was occurring the number of E. vuilleti adults emerging from the seeds was no different from that observed in control batches with E. vuilleti alone, and there were always fewer D. basalis adults than in control batches (D. basalis alone). This interspecific competition reduces the influence of the two parasitoids in the biological control of bruchid populations.     

Biology of Nomioides variegatus (Olivier) (Hymenoptera: Halictidae)

The nesting biology, life history and pollen foraging of the minute Nomioides variegatus (Olivier) is described and illustrated. All Nomioides biological studies are reviewed. Nesting and foraging were observed over two years in a sandy natural area in the Rhône River delta region of southern France. Nest and cell structure was obtained from soil blocks removed from the nesting area and examined in the laboratory. Females, which were on average only 4.2 mm long, nested solitarily in loose aggregations in areas free of vegetation. Cells were placed from 6 to 12 cm deep, each terminating a lateral burrow off the main burrow. Cell walls were lined with a water proof wax-like lining. The pollen-nectar provision was a flattened sphere. The posterior end of the egg was embedded on the dorsal surface of the provision. Larval and pupal development was rapid, proceeding to adults by August, the over-wintering stage. Pollen foraging patterns were analyzed from both pollen provisions and fecal material for different nests (3) and cells (8). Nomioides variegatus is polylectic, but individuals bees exhibited a high degree of foraging consistency for the provisioning of single cells. Eleven different pollen types representing 10 plant families were found. In 1996, Ecballium elaterium (Cucurbitaceae) was the dominant pollen present in all samples but it was absent in 1997. Tamarix gallica (Tamaricaeae) was the dominant pollen type in 1997 and absent in 1996. A third pollen, Centaurium (Gentianaceae) was present in both years and represented 70% of some samples.     

Polymorphism in mitochondrial DNA (mtDNA) of yak (Bos grunniens)

Mitochondrial DNAs (mtDNA) from 21 yaks (Bos grunniens) were assayed for restriction fragment length polymorphisms by using 20 restriction endonucleases, six of which (AvaI, AvaII, BglII, EcoRI, HindIII, and HpaI) detected polymorphism. Four different mtDNA haplotypes were identified. Combining this with previous reports about the mtDNA RFLPs of B. indicus and B. taurus, there are obvious differences in mtDNA polymorphism between the yak and other Bos species. We estimated that the divergence times between the ancestor of B. grunniens and the ancestor of B. taurus or B. indicus were about 1.2–2.2 and 1.01–2.02 million years ago, respectively.     

Interspecific competition among larvae ofHemipyrellia ligurriens (Calliphoridae) andBoettcherisca formosensis (Sarcophagidae) (Diptera)

1. The effects of larval rearing density and species relative proportions on life-history parameters of two necrophagous Diptera, Hemipyrellia ligurriens (Wiedemann) (Calliphoridae) and Boettcherisca formosensis Kirner and Lopes (Sarcophagidae), were investigated in mixed cultures. Larval rearing density had a significant effect on larval to adult survivorship, duration of immature development, adult size and relative performance (measured by the composite index of performance, r′) of both species. However, species relative proportions affected adult size of both flies and the duration of immature development of B. formosensis only.
2. B. formosensis had a higher survivorship than H. ligurriens in all mixed cultures and showed a similar survivorship pattern to that in pure cultures. By contrast, survivorship of H. ligurriens was lower in mixed than in pure cultures.
3. H. ligurriens adults reared from mixed cultures were smaller than those from pure cultures of comparable density, but B. formosensis adults from pure and mixed cultures were of similar size.
4. The results suggest that competition between B. formosensis and H. ligurriens larvae was asymmetric and the former was the superior competior.
5. At low larval densities in mixed cultures, the presence of H. ligurriens enhanced the performance (as measured by r′) of B. formosensis, a consequence of suspected interspecific facilitation of larval growth.

     

Effect of Paranosema (Nosema) locustae (Microsporidia) on morphological phase transformation of Locusta migratoria manilensis (Orthoptera: Acrididae)

Field and laboratory studies demonstrated that Paranosema (Nosema) locustae had significant effects on the morphological phase transformation of Locusta migratoria manilensis (Meyen 1835). In the field, spraying P. locustae on gregarious locusts caused a substantial population reduction by 16 days after treatment, with most of the surviving locusts being phase solitaria. However, the effects of P. locustae on locust phase transformation began before direct mortality had caused a substantial reduction in locust density: locust numbers were still high at day 10, but locusts had already transformed to phase transiens. Laboratory assays showed that while a low dose of P. locustae had no effect on phase transformation, at a higher dose of 1×10⁵ spores/mL, locusts had F/C ratios that were significantly (P<0.05) more solitaria than untreated locusts, with locusts having ratios that were either phase solitaria or on the solitaria side of phase transiens. In a second laboratory experiment that analysed the effects of locust density on phase transformation by P. locustae, there was no obvious effect of density on female locusts 10 days later as all were solitaria at all locust densities. At day 16, female locusts were transiens at higher densities, but were solitaria at 4/cage. With males there were lesser effects. These results provide new evidence for P. locustae having sub-lethal effects on locust phase transformation at a wide range of locust densities.     

Distribution of native legumes (Leguminoseae) in frequently burned longleaf pine (Pinaceae)-wiregrass (Poaceae) ecosystems

Legume species distribution and abundance and selected environmental variables were quantified across a complex gradient (varying in both water-holding capacity and fertility) for frequently burned longleaf pine (Pinus palustris)–wiregrass (Aristida stricta) ecosystems. Legumes were present in all months; however, abundance peaked in June and was minimal after killing frosts in October. Legume species were prominent in the flora (43 species encountered) ubiquitous (94% of 2-m² subplots had at least one legume species), and abundant (nearly 120 000 stems/ha). Although most species were widely distributed throughout the gradient, Lespedeza angustifolia was distinctly associated with the more hydric end of the gradient, while both Petalostemon pinnatum and Galactia microphylla were located in the more xeric extreme. The percentage variation in species that could be accounted for by environmental variation was low (27%). Of the variation that could be accounted for, a number of environmental variables were important, including soil moisture, pine basal area (i.e., light), and bivalent base cations (e.g., Ca²⁺). Although gradients in resource availability among sites did not affect the distribution of species or abundance of legumes strongly, variation in resources are likely to regulate N₂-fixation rates of the various native legume species, and thereby affect ecological functions such as maintenance of N capital and productivity.     

Trophodynamics of Protomyctophum (Myctophidae) in the Scotia Sea (Southern Ocean)

This study investigated spatial and temporal patterns in distribution, population structure and diet of Bolin's lanternfish Protomyctophum bolini, Tenison's lanternfish Protomyctophum tenisoni and gaptooth lanternfish Protomyctophum choriodon in the Scotia Sea using data collected by midwater trawl during spring, summer and autumn. Protomyctophum bolini was the most abundant species of the genus encountered throughout the Scotia Sea with the greatest concentrations occurring around the Antarctic Polar Front (APF). This species had a life cycle of 2+ years, but spatial differences in population structure were apparent as the I‐group was absent from all regions south of the APF, suggesting that the species does not recruit in the Scotia Sea. Protomyctophum tenisoni occurred mostly in waters characteristic of the APF and was absent from the southern Scotia Sea. It had a limited size range, but there was clear size‐related sexual dimorphism with males significantly larger than females. The species had a life cycle of c. 2 years, but the I‐group (c. 1 year old, 1 November to 31 October the next year) occurred only in regions close to the APF suggesting that recruitment is restricted to these waters. A seasonal southward migration for P. choriodon is likely as the species occurred mostly to the south‐west of South Georgia in summer, but extended to the sea‐ice sectors in autumn. Protomyctophum choriodon had a life cycle of 4+ years in the Scotia Sea and the population was dominated by age classes >3 years old. Larval stages were absent during the surveys for all species. Diurnal variations in vertical distribution were apparent for all three species. Interspecific variations in diet were evident, but all species were primarily copepod feeders, with Metridia spp., Rhincalanus gigas and Calanus simillimus generally dominating their diet. Small euphausiids, principally Thysanoessa spp., were also an important component of their diets, particularly for P. choriodon which had the largest body size. The spatial and temporal variations in diet for both P. bolini and P. tenisoni were broadly consistent with underlying abundance patterns within the mesozooplankton community.     

Phagostimulation of Lytechinus variegatus (Lamarck) (Echinodermata: Echinoidea)

Artificial agarose foods containing either starch, glycogen, carragheenan, alginate, casein, gelatin, albumin, or peptones at a concentration of 1% dry weight were not phagostimulatory. Artificial foods containing sugars, especially galactose, at a concentration of 0.005 M were phagostimulatory. Of the common amino acids, only L‐phenylalanine was phagostimulatory at this concentration. The initiation of feeding postures (movement of the spines, tube feet, peristome, and Aristotle's lantern) of Lytechinus variegatus was little affected by the concentration of plant extracts. Many aspects of this behavior were attributable to physical rather than chemical stimulation. Feeding rates of L. variegatus upon artificial agarose foods containing either Thalassia testudinum or Gracilaria verrucosa were directly dependent upon plant tissue concentration. All artificial agarose food‐items were initially moved to the mouth and fed upon, however the time spent feeding was directly dependent upon plant type and tissue concentration. Retention of the artificial food‐items upon the test after feeding was not influenced by plant type or tissue concentration. Differences in consumption of foods by L. variegatus are determined by the degree of persistence in feeding upon encountered items and not be selection before initiating feeding or by retention of food‐items for subsequent feedings.