Evaluation of attract-and-kill strategy for management of cocoa pod borer,Conopomorpha cramerella,in Malaysia cocoa plantation

Abstract

In South-East Asia, cocoa production is dramatically affected by cocoa pod borer (CPB) infestations. As an alternative tool to chemical control, the efficacy of attract-and-kill strategy (CPB sex-pheromone as attractant and Delta trap without sticky liner sprayed with cypermethrin solution as killing station) was evaluated and compared with current standard CPB management approach as control treatment during two main cocoa harvest seasons in Malaysia (with 100 µg and 33.3 µg CPB-pheromone loading per station, respectively). In both seasons, attract-and-kill strategy was highly effective at reducing male flight activity (p?<?0.05) in attract-and-kill plots comparing with standard CPB management plots. For the percentage of CPB-infested pods, the attract-and-kill strategy (100 µg) was as good as the conventional pesticide spray applications of cypermethrin (p?=?0.083) in first season. However, it was significantly (p?=?0.021) reduced in the second season with lower pheromone loading (33.3 µg), indicating that this semiochemical based strategy is far superior to and more feasible than the currently applied conventional synthetic pesticide treatment and is therefore a good alternative in CPB integrated pest management.     

Assessing resistance to Crinipellis perniciosa using cocoa callus

Callus cultures from cocoa clones reported to have different susceptibilities to Brazilian isolates of Crinipellis perniciosa , causal fungus of witches' broom disease, including Catongo (susceptible). Scavina-6, CAB 64 and CAB 67 (resistant) were inoculated with basidiospores of C. perniciosa and the subsequent development of mycelia was assessed. Generally, on Catongo callus, mycelium similar to that found in green brooms (parasitic type), developed abundantly but on callus from Scavina-6 and the two CAB clones, mycelial growth was relatively poor and tended to be similar to that in dry brooms and on agar media (saprotrophic type). There was often considerable variation in fungal development on individual callus cultures from the same clone, but there was an overall consistency between successive experiments in ranking the callus material in terms of fungal development. However, growth of isolates from Colombia and Trinidad was similar on callus from Scavina-6 which in the field is known to reac differentially to these two isolates. Experiments with isolates from Colombia indicated that inoculum from basidiocarps produced on brooms which were kept for long periods in cabinets to induce fruiting had a decreased ability to colonize callus.     

Molecular analysis of the major Phytophthora species on cocoa

The internally transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene cluster of 161 isolates of Phytophthora species involved in pod rot, stem canker and leaf blight of cocoa were analysed to determine inter- and intraspecific variation in this disease complex. The species P. palmivora , P. megakarya , P. capsici , P. citrophthora and P. nicotianae could all be clearly distinguished by PCR amplification of the ITS region followed by restriction analysis with Hae III, Hinf I, Pvu II and Alu I. This method provided a relatively rapid identification procedure for these species, and was able to distinguish isolates that had previously been misidentified by morphological methods. Sequence analysis showed that the four main cocoa-associated species formed two distinct groups, one comprising P. capsici and P. citrophthora , and the other P. palmivora and P. megakarya . Detailed sequence analysis and comparison with published literature suggested that P. capsici isolates from cocoa may be closely related to P. tropicalis , a species recently described from Cyclamen and Dianthus .     

Phylogeny of the frosty pod rot pathogen of cocoa

Morphological, cytological and molecular evidence is presented which confirms that the frosty pod rot pathogen of cocoa, formerly classified as the mitosporic fungus Moniliophthora roreri (Deuteromycota), belongs to the hymenomycetous genus Crinipellis (Basidiomycota) and that two varieties should now be recognized: Crinipellis roreri var. roreri and the new variety C. roreri var. gileri . The latter was collected on Theobroma gileri , an endemic tree of submontane forests in north-west Ecuador, and can be distinguished from Ecuadorian and Peruvian isolates from cocoa ( T. cacao ) on the basis of spore morphology, incompatibility and nucleotide sequence data. As with var. roreri , meiosis is shown to occur within the dispersive and infective spore stage of var. gileri and these meiospores are interpreted to represent a much modified probasidium. In addition, in a field inoculation experiment, an isolate from T. gileri proved to be noninfective to cocoa pods when compared with positive control strains isolated from T. cacao in western Ecuador and T. bicolor in eastern Ecuador. It is concluded that var. gileri is the vestigial progenitor of the frosty pod rot pathogen of cocoa, with a host range and distribution restricted to T. gileri in the mesic forests of north-west South America.     

Long-term control of Phytophthora diseases of cocoa using trunk-injected phosphonate

Pod rot and stem canker, caused by Phytophthora palmivora , are serious diseases of cocoa causing pod losses of up to 63% and the death of up to 10% of trees annually on Kar Kar Island. Papua New Guinea. Trials were conducted on commercial cocoa plantations to compare the effectiveness of potassium phosphonate when applied as trunk injections, trunk paints and foliar sprays, and trunk injections of Aliette CA and of Ridomil 250EC, with conventional Ridomil-based spray programmes. The results show that, in trials conducted over 5 years, injections of potassium phosphonate give the highest healthy pod yield and lowest incidence of Phytophthora pod rot and stem canker when compared with Ridomil-based spray programmes or trunk injections of Ridomil 250EC or Aliette CA. The increase in ripe healthy pod yields was due to both pod rot and stem canker control. The level of control is independent of the seasonal timing of injection. Optimum control was achieved with annual injections of 15 g a. i. per tree, and varied with tree size and disease severity. Injections of phosphonate will, under the range of conditions found in our trials. provide the most cost-effective control of Phytophthora diseases.     

Activity of Bacillus thuringiensis toxins against cocoa pod borer larvae

Twelve Cry proteins from Bacillus thuringiensis Berliner were tested in bioassays on cacao plantations in Indonesia for activity against the larvae of cocoa pod borer (Conopomorpha cramerella (Snellen)), an insect pest of the cacao tree. Through the damage caused by their feeding, the larvae of cocoa pod borer cause the pods of the cocoa tree to ripen prematurely. They are difficult to control with conventional measures. Preliminary assays identified five toxins that were more active than others. In two subsequent bioassays the activity of selected toxins was determined more accurately. Three Cryl proteins with relatively little homology were all found to be toxic, opening perspectives for controlling cocoa pod borer by expression of Cry proteins in transgenic plants.     

Black pod: diverse pathogens with a global impact on cocoa yield

ABSTRACT Pathogens of the Straminipile genus Phytophthora cause significant disease losses to global cocoa production. P. megakarya causes significant pod rot and losses due to canker in West Africa, whereas P. capsici and P. citrophthora cause pod rots in Central and South America. The global and highly damaging P. palmivora attacks all parts of the cocoa tree at all stages of the growing cycle. This pathogen causes 20 to 30% pod losses through black pod rot, and kills up to 10% of trees annually through stem cankers. P. palmivora has a complex disease cycle involving several sources of primary inoculum and several modes of dissemination of secondary inoculum. This results in explosive epidemics during favorable environmental conditions. The spread of regional pathogens must be prevented by effective quarantine barriers. Resistance to all these Phytophthora species is typically low in commercial cocoa genotypes. Disease losses can be reduced through integrated management practices that include pruning and shade management, leaf mulching, regular and complete harvesting, sanitation and pod case disposal, appropriate fertilizer application and targeted fungicide use. Packaging these options to improve uptake by smallholders presents a major challenge for the industry.     

Inter- and intraspecific morphometric variation and characterization of Phytophthora isolates from cocoa

Difficulties in the accurate identification of the Phytophthora species responsible for black pod disease of cocoa continue to hamper effective disease control. A re-evaluation of morphological characters ( Brasier & Griffin, 1979 ) and a detailed morphometric analysis of 161 Phytophthora isolates largely associated with black pod disease of cocoa from 17 countries worldwide have shown considerable inter- and intraspecific variation. Stable and more reliable parameters for the identification of the species responsible for the disease have been determined. Colony characteristics such as pattern and growth rate on V8 agar are reasonably characteristic for the cocoa Phytophthora species, and can be used to make preliminary identification to species level. Significant sporangial character variation was found within isolates of species from the same and different sources, highlighting the difficulties in making accurate identification on the basis of raw morphological data. Pedicel length was found to be the most consistent species-linked sporangial characteristic. Cluster plots of length/breadth ratios of sporangia versus reciprocals of sporangial pedicel length clearly separated all isolates into distinct species groups ( P. capsici , P. citrophthora , P. palmivora and P. megakarya ) and can be used reliably to identify accurately those pathogens involved in black pod disease outbreaks.     

Chemical control of vascular-streak dieback disease of cocoa in Papua New Guinea

High-volume foliage sprays of 18 compounds including 13 systemic fungicides in field trials at 4-week intervals did not control vascular-streak dieback (VSD) of cocoa caused by Oncobasidium theobromae in Papua New Guinea. Soil drenching with carbendazim and other systemics was also ineffective, possibly because insufficient carbendazim may have been taken up into the xylem to inhibit the fungus. Stem paints with four systemic fungicides in aqueous formulations were also ineffective, but 4-weekly stem painting of the hardened bark of young seedlings with 10 g/l propiconazole in 25% white oil caused a highly significant reduction in VSD incidence under field conditions. The treatment had no curative effect. Some phytotoxicity was observed but this was not apparent when the concentration of propiconazole was doubled and the treatment interval lengthened to 8 weeks. The ineffectiveness of systemic fungicides as soil drenches and foliage applications was probably due to failure of these compounds to accumulate at the shoot apex where infection occurs. A stable and non-toxic formulation containing surfactants was developed which may improve control by aiding bark penetration by propiconazole or related compounds.     

Pathogenic variability amongst isolates of Crinipellis perniciosa from cocoa (Theobroma cacao)

Dry witches' brooms from cocoa were imported from various areas within Bolivia, Brazil, Colombia, Ecuador, Trinidad and Venezuela. Basidiocarps of Crinipellis perniciosa were induced to form on these brooms and seedlings of different types of cocoa were inoculated with basidiospores either on the hypocotyl or cotyledon bud. Host reactions were assessed mainly by recording stem base swelling and broom development at the cotyledon node (hypocotyl inoculations) or the extent of swelling and branching of shoots (cotyledon bud inoculations).
Results from 30 experiments indicated considerable diversity amongst isolates in inducing disease symptoms, but suggested that two groups or populations of C. perniciosa exist on cultivated cocoa. One group (A), comprising isolates from Bolivia and Pichilingue (Ecuador) and most isolates tested from Colombia, induced severe symptoms on cocoa with Scavina 6 as one parent; the other group (B), comprising isolates from Brazil, Trinidad and Venezuela, did not. Within these groupings variants could be further distinguished by particular host reactions. Isolates from Ecuador, especially from the Oriente, a centre of diversity for Theobroma cacao, showed a range of pathogenicity comparable to that found amongst isolates from cultivated cocoa over a much wider area.     

Development stage-dependent susceptibility of cocoa fruit to pod rot caused by Phytophthora megakarya

Pod rot causes up to 30 % losses in world cocoa production. In order to predict the risk evolution of disease, it is important to take into consideration the developmental stage of fruits. In fact, it has been shown that the risk of attack by pod rot depends amongst others on the developmental stage of fruits. We proposed here to estimate the susceptibility at different stages. Susceptibility of fruit to disease was investigated at three fruit developmental stages (cherelle, young pod and adult pod); disease severity was assessed in laboratory conditions, on detached, artificially inoculated fruits, while disease incidence was assessed in the field, under natural inoculum pressure. In both assessment fruits at the cherelle stage were the most susceptible whereas the young and adult fruits were equally susceptible. The vertical position of the fruits on the tree did not influence their susceptibility. Estimates of the fruit susceptibility and of the infectious potential were derived from the severity and incidence measurements, using a model assuming that the number of spores on a fruit follows a Poisson distribution with the mean, the density of spores per fruit as the parameter. The estimated parameter values allowed the evaluation of the probability of attack of a fruit by the disease, which could be implemented in a disease warning system.     

Control of vascular-streak dieback of cocoa with triazole fungicides and the problem of phytotoxicity

The effectiveness of a range of systemic fungicides against vascular-streak dieback of cocoa, caused by Oncobasidium theobromae , was evaluated over 2 years in Papua New Guinea. Monthly foliar sprays of the triazole fungicides hexaconazole, tebuconazole and triadimenol all gave good protection against the disease. The relative efficacy of these chemicals was, in order, tebuconazole (EC₅₀ 0–90 mg a.i. per plant), hexaconazole (EC₅₀ 2.35 mg a.i. per plant), triadimenol (EC₅₀ 4.17 mg a.i. per plant). However, the relative severity of plant growth inhibition induced by these compounds had the same ranking. Growth inhibition was reduced by extending the interval between treatments, but this also reduced the efficacy of disease control. Propiconazole stem paints (EC₅₀ 2502 mg a.i. per plant) were markedly less effective than these sprays. Neutralized phosphorous acid sprays and paints showed no activity against the disease.     

Growth rates of, and mycelial interactions between, isolates of Crinipellis perniciosa from cocoa

Growth of 87 single-basidiospore isolates of Crinipellis perniciosa , derived from witches' brooms on cocoa in 10 localities throughout South America and the Caribbean, was examined at 25°C on five agar media (Czapek-Dox, prune extract, potato-dextrose, V8 and carboxymethylcellulose). Interactions (designated intermingling or mutually antagonistic) between paired mycelia of 64 isolates were determined on V8 plates.
Six somatic-compatibility groups were identified comprising isolates from; (1) Pichilingue and Rio Palenque in Ecuador, Chigorodo and Manizales in Colombia; (2) Sucua (Ecuador); (3) Manaus (Brazil); (4) Ouro Preto (Brazil); (5) Castanhal (Brazil); (6) Trinidad and Tobago. This geographical separation of isolates was supported by results of the growth tests; growth of isolates within each compatibility group differed from other groups on at least one of the five media.
Separation of isolates by these methods did not conflict with previous results from tests of pathogenicity and could be useful in selecting isolates for screening cocoa lines for resistance to C. perniciosa.     

A novel method for producing basidiocarps of the cocoa pathogen Crinipellis perniciosa using a bran-vermiculite medium

A novel method for the production of basidiocarps from the mycelia ofCrinipellis perniciosa is described. This involved the colonization of a bran-vermiculite medium with pure culture of the fungus, prior to application of a peat-based casing. Basidiocarp production was induced by hanging the cultures in a cabinet where they were subjected to a daily cycle of wetting and drying. The method was successfully and reproducibly used to fruit isolates of all four known biotypes of the fungus within 10–16 weeks of inoculation.     

Changes in morphology and measurement of cytokinin levels during the development of witches' brooms on cocoa

The anatomy of diseased stems from brooms induced on cocoa ( Theobroma cacao ) by the fungus Crinipellis perniciosa was examined using the scanning electron microscope, and the amounts of selected cytokinins were measured by immunoassay.
Although the diseased stems were wider in diameter with a lower dry weight:fresh weight ratio than stems of healthy plants, the overall internal organization of tissues remained unchanged, and all the cell types were present. However, the vascular tissue was less differentiated in diseased stems since xylem vessels were absent and the ratio of phloem fibres and sieve tubes to phloem parenchyma was reduced. Cell sizes in diseased stems were larger for all tissues but cell numbers were unchanged except for xylem where fewer cells were seen.
Cytokinins (zeatin, zeatin riboside, isopentenyl adenine and isopentenyl adenosine) were measured in healthy plants and diseased stems at specific stages of disease development. Of these cytokinins only zeatin riboside was present in significantly greater amounts in diseased tissue. The likely role of plant growth regulator balance in stem enlargement and proliferation in witches' broom disease of cocoa is discussed.     

Host specialization of Verticillium dahliae, with emphasis on isolates from cocoa (Theobroma cacao)

Isolates of Verticillium dahliae from cocoa (four Brazilian and one Ugandan) were screened against cocoa, aubergine, tomato, cotton, and pepper. Isolates originating from hosts other than cocoa were also inoculated. In general, all inoculated crops were systemically invaded by isolates from cocoa. Isolates from each host tended to be more aggressive on their original host. All isolates from cocoa were pathogenic to cocoa, but exhibited various degrees of aggressiveness to other crops. They induced severe symptoms on aubergine, but few symptoms on tomato, although colonization occurred up to the stem apex in both cases. Likewise, symptomless invasion of pepper was found with some Brazilian isolates. The Ugandan isolate was significantly more aggressive on cotton and pepper than the Brazilian isolates.
A Brazilian isolate from cocoa from the State of Bahia was also used to inoculate 12 common weed species from the same geographical area. Four species showed wilt symptoms, while V. dahliae was readily recovered from the stems of a further four species. The role of alternative hosts on disease spread in cocoa growing areas is discussed.