Cocoa-based media for culturing Phytophthora palmivora (Butl.) Butl., causal agent of black pod disease of cocoa

Green cocoa pod husk agar (GCPA), ripe cocoa pod husk agar (RCPA), green cocoabean agar (GCBA), ripe cocoa bean agar (RCBA), green cocoa mucilage agar (GCMA)and ripe cocoa mucilage agar (RCMA) were prepared and assessd for their clarity andfor potential to support mycelial growth and sporulation of P. palmivora. Oatmeal agar (OMA), potato-dextrose agar (PDA), vegetable 8 juice agar (V8JA) and pineapple crown agar (PCA) were included for comparison. The highest radial growth rates of 8.3 and 7.2 mm/day were recorded, respectively, on OMA and GCPA but these were not significantly different (P ≤ 0 05) from each other. The two media also supported good aerial mycelial growth but were not clear. Radial mycelial growth rates of 6.5, 7.0 and 6.6 mm/day were obtained on GCMA, RCPA and V8JA, respectively, and these rates were also not significantly different from each other. Of the three media, only the GCMA was clear and supported the best aerial mycelial growth. In comparison, the RCMA supported a significantly lower radial growth (4.6 mm/day) of P. palmivora than the three media. Growth rates were least on RCBA, PCA and PDA but sporulation was poorest on PDA, PCA and V8JA. GCMA was found to be the best medium based on all the growth parameters and media characteristics. GCMA has been used effectively to isolate/detect P. palmivora from infected cocoa pod tissues. Apart from differences in radial growth rate, both the GCMA and RCMA were similar in all other respects and are recommended for culturing P. palmivora. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

Potential for biological control of tent building species of ants associated with Phytophthora palmivora pod rot of cocoa in Papua New Guinea

The tents of different ant species collected from young cocoa trees in their first or second year of bearing and free from visible pod rot, were tested for the presence of Phytopthtora palmivora by inoculating wounded cocoa pods with tent material. Tents of all species harboured viable P. palmivora but those consisting mainly of soil were more frequently positive than plant debris-type tents. Although Anoplolepis longipes, a dominant, ground nesting, non-tent building species sometimes transported inocula in the laboratory, it did not significantly increase black pod infection in the field. Trees infested with the dominant debris tent building species, Technomyrmex albipes, however, had significantly more black pod than those infested with A. longipes or trees without ants. A. longipes forms dense populations and can exclude other dominant ants and some cocoa pests; its introduction may be a potentially economical method of reducing the transmission of P. palmivora in redeveloped cocoa in Papua New Guinea.     

Association of Phytophthora palmivora bark canker and insect damage in cocoa in Papua New Guinea

Bark canker caused by the fungus Phytophthora palmivora has caused the death of many cocoa trees over 15 years old in Papua New Guinea in recent years. Cankers are significantly associated with channels made by the larvae of bark-damaging insects, which provide the major entry point for the pathogen. The combination of canker and insect damage may limit the economic life of cocoa to less than 15 years in affected areas.     

Inhibitory activity of plant growth regulators on Phytophthora palmivora causing cassava tuber rot

Auxins and cytokinins are implicated in a wide variety of developmental and physiological processes in plants. Phytophthora palmivora causes tuber rot in cassava growing regions of Tamil Nadu and Kerala, South India. The in vitro effect of cytokinin, benzyl amino purine (BAP) and auxins, naphthalene acetic acid (NAA) and indole acetic acid (IAA) on P. palmivora mycelium growth was investigated. The inhibitory activity varied among the growth regulators and complete inhibition of the pathogen was observed at 50, 2000 and 2500 ppm by the BAP, IAA and NAA, respectively. The effective growth regulator, BAP was also analysed on tubers before and after the invasion of the pathogen to observe its effect in tuber. Further, it was also checked against the bio-control agent Trichoderma harzianum. The study indicates that the use of BAP could be an important approach in controlling tuber rot pathogen, P. palmivora.     

Sudden death of cocoa in Papua New Guinea associated with Phytophthora palmivora cankers invaded by bark beetles

An extensive survey in 1976-82 indicated that sudden death of cocoa was widespread in Papua New Guinea with losses up to 1% per month in trees over 10-yr-old, especially in neglected plantations. Over 95% trees affected had large Phytophthora palmivora canker lesions and over 90% of the cankers were invaded by bark beetles. In an intensive single year survey of one plot 18-8% of 69 healthy trees died. Ambrosiella spp., P. palmivora and Fusarium solani were isolated from wood surrounding beetle tunnels but only the last two caused extensive lesions when inoculated into the xylem of healthy trees. The evidence obtained indicated that the sudden death syndrome is initiated by P. palmivora cankers on trunks and main branches but bark beetles are a major contributory factor and fungi associated with their tunnels may contribute to the problem. There is a risk of further outbreaks of the disease as cocoa cultivars susceptible to P. palmivora continue to be planted.     

Calcium control of differentiation in Phytophthora palmivora

A method has been developed for the preparation of zoospores from Phytophthora palmivora which allows the ionic composition of the suspension medium to be closely controlled. Sub-micromolar concentrations of calcium ions have been shown to play a key role in maintaining the zoospore state and in the transition to the cyst stage. Restriction of free Ca²⁺ to between 0.2 and 1 M resulted in zoospores which could be maintained for several hours before they finally encysted and germinated. When exposed to citrus-pectin, or 3 mM SrCl₂, or to vigorous shaking, these zoospores underwent rapid synchronous encystment. At free Ca²⁺ concentrations below 0.1 M, zoospores lysed slowly. If exposed to inducers of encystment before lysis had occurred, the zoospores failed to respond to pectin or to vigorous shaking. However, they did differentiate in response to SrCl₂ addition. Provided the free Ca²⁺ was maintained between 0.02 and 0.2 M, zoospores survived gentle centrifugation, a procedure which previously had resulted in encystment.Abbreviations IM (ion-mix) release medium containing 100 M KCl, 10 M CaCl₂, and 10 M MgCl₂     

Electrophoretic protein patterns of three species ofPhytophthora associated with black pod disease of cocoa (Theobroma cacao L.)

When electrophoretic profiles of native proteins from vegetative mycelia ofPhytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates ofPhytophthora capsici were highly homogenous and formed a single cluster. The isolates ofPhytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups.Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species ofPhytophthora encountered on cocoa. CPCRl contribution No. 914.     

Effect of cocoa pod age on egg distribution and egg parasitism of the cocoa pod borer in Malaysia

The effects of pod age on oviposition, and on egg and larval survival of the cocoa pod borer (CPB), Conopomorpha cramerella (Snellen), were investigated. CPB were allowed to oviposit on a batch of 50 pods for a period of 7 days over several exposure dates. Numbers of CPB eggs/pod and survival to larval emergence from the pods were recorded. Pods were harvested when ripe and analyzed for larval survival within the pod. The oviposition pattern varied with pod age. Older pods less than 7 weeks before ripening (WBR) were preferred, suggesting greater nutritional value or chemical attraction of the older pods. The proportion of eggs parasitized by Trichogrammtoidea bactrae fumata Nagaraja (TBF), the number of eggs lost and the proportion surviving to larval emergence from the pod were independent of pod age. Egg parasitism was low, implying that TBF was not an effective natural enemy. Mass-releases of TBF should be intensified during cropping periods with higher proportions of susceptible pods (<10 WBR). Survival of larvae in the presclerotic layer (preSCL) was high and independent of pod age. Larval mortality at the SCL (sclerotic layer) was dependent on pod age and was high in older pods. Larval density influenced the survival of larvae inside the pod and was dependent on pod age. Larval mortality associated with SCL hardness and thickness is one of the criteria for the selection of CPB resistant cocoa clones.     

Phytophthora Crown and Root Rot of Apricot Caused by Phytophthora palmivora in Turkey

Forty‐nine Phytophthora isolates were obtained from roots and crown of apricot trees with symptoms of decline grown in commercial orchards in Malatya, Elaz?? and Diyarbak?r provinces, Turkey, in 2011 and 2013. All of the recovered isolates were identified as Phytophthora palmivora on the basis of morphological characteristics. Blast analysis of ITS region sequences of rDNA of 5 isolates revealed 100% identity with a reference isolates of P. palmivora from GenBank. Isolates of P. palmivora were pathogenic on 12‐month‐old wild apricot rootstock ‘Zerdali’ plants that were wound inoculated on the roots and on the crown. This study demonstrated that P. palmivora is the cause of the crown and root rot found on apricot in Turkey. To our knowledge, this is the first report of P. palmivora on this host plant.     

A new oil-based formulation of Trichoderma asperellum for the biological control of cacao black pod disease caused by Phytophthora megakarya

In African cacao-producing countries, control of cacao black pod disease caused by Phytophthora megakarya is a priority. Introducing biological control agents as part of a P. megakarya control strategy is highly desirable, especially in a perspective of pesticide reduction. Trichoderma species are among the most used biological control agents. In Cameroon, Trichoderma asperellum formulated in wettable powder has produced positive effects against this disease. However, with this type of formulation, shelf-life and persistence of conidia on pods are limited. Our study therefore sought to develop a new T. asperellum formulation that would be more effective and better suited to the conditions of field application by small-scale producers in Cameroon. We selected a soybean oil-based oil dispersion, in which the half-life of the conidia reached 22.5 weeks, versus 5 weeks in aqueous suspension. Tested on detached pods, the formulation completely inhibited the development of the disease. When sprayed in the field on cacao clones highly sensitive to P. megakarya, the formulation resulted in 90% protection of treated pods after 1 week, and 50% after 3.2 weeks. The formulations exercised a measurable effect for up to 7 weeks, versus 2 weeks in the case of an aqueous conidial suspension and 5 weeks for that of a conventional fungicide (Kocide). Trichoderma asperellum formulated in oil dispersion has therefore great potential for the control of cacao black pod disease with less recourse to synthetic fungicides. Moreover, this formulation is well adapted to the types of sprayers used by small-scale cacao producers in Cameroon.     

The elicitin secreted by Phytophthora palmivora, a rubber tree pathogen

Palmivorein, a new member of the elicitin family, was purified from the culture filtrate of Phytophthora palmivora isolated from the rubber tree, Hevea brasiliensis. The elicitin was obtained by ammonium sulfate precipitation and further purified using ion-exchange and gel filtration. The molecular weight, isoelectric point, amino acid composition and N-terminal sequences of this molecule are reported and compared to other known elicitins. Palmivorein, as determined by SDS-PAGE, is a small protein of M(r) ca. 10,000. It is classified as an alpha-elicitin according to its acidic pI and the valine residue at position 13. Like other elicitins, the P. palmivora elicitin causes tissue necrosis on tested tobacco leaves. It also causes severe wilting and necrosis of Hevea tissue, and leaves of the susceptible rubber clone (with respect to P. palmivora) are much more sensitive to this elicitin than those that are resistant.     

Accelerated ion fluxes during differentiation in zoospores of Phytophthora palmivora

Zoospores of Phytophthora palmivora show increased fluxes of Na⁺ and Ca²⁺ 3–5 min after they have been stimulated to differentiate with pectin. Both spontaneous and pectin-induced encystment are reduced below pH 6 and accelerated above pH 7. The ionophores monensin and A23187 induce slow differentiation when added together, but not when added separately. Ethanol (0.5%) also induces slow differentiation. Amiloride and verapamil inhibit pectin-induced differentiation and also reduce the onset of the Na⁺ and Ca²⁺ flux. A requirement for Ca²⁺ for differentiation is confirmed, but a requirement for Na⁺ could not be demonstrated.     

Host preferences of the cocoa pod borer,Conopomorpha cramerella,the main threat to cocoa production in Southeast Asia

Insects ensure the survival of their offspring by depositing their eggs in suitable environments. Even generalist egg-laying insects often show preferences for specific host plants. The cocoa pod borer (CPB), Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae), successfully infests and reproduces on relatively few host plants, but has a major economic impact only on cocoa, Theobroma cacao L. (Malvaceae). Choice tests were performed in the laboratory to compare the frequency of insect visits, the duration of the visits, and the number of eggs laid on the fruits for each combination of host plants tested – that is, cocoa clones or fruits of rambutan (Nephelium lappaceum L.), Fijian longan (Pometia pinnata JR Forst & G Forst, both Sapindaceae), and langsat (Lansium domesticum Corrêa, Malvaceae). Our laboratory study showed that, when given the choice, CPB significantly favored cocoa pods over other host fruits (rambutan, langsat, or Fijian longan). Females also deposited more eggs on unripe fruits than on ripe fruits of similar size. The preference to lay eggs on specific cocoa clones in the bioassays did not reflect the level of the clone resistance to CPB damage reported from the field. Consequently, oviposition preference of female CPB does not seem to be the main factor explaining field resistance of some cocoa clones to CPB infestation.