Biodiversity and biogeography of the cacao (Theobroma cacao) pathogen Moniliophthora roreri in tropical America

Moniliophthora roreri , the cause of moniliasis or frosty pod rot, occurs on the neotropical rainforest genera Theobroma and Herrania . While this basidiomycete has had devastating effects on the cacao tree ( T. cacao ) in tropical America, where it is confined, little is known of its biogeography and intraspecific genetic variability. Here, AFLP and ISSR profiles of 94 isolates of M. roreri from across its geographic range in Central/South America were analyzed. The study provided limited evidence to support the hypothesis that M. roreri is capable of sexual reproduction. The highest levels of genetic diversity occurred in Colombia and not in Ecuador as originally believed. The fungus was broadly divided into five genetic groups. Two of these have a wide geographic range: Bolívar group (north of Santander in Colombia, eastern Venezuela, peripheral Ecuador, Peru), and Co-West group (western Colombia, central Ecuador, Central America). The other groups are all apparently endemic to Colombia (Co-East and Co-Central groups) or north-western Ecuador ( Gileri group). We speculate that central/north-eastern Colombia may represent the centre of origin for M. roreri . Sequence data from the internal transcribed spacer region of the nuclear rDNA repeat were congruent with the AFLP/ISSR results, dividing M. roreri into two broad groups: the Orientalis group, comprising most isolates from the Co-East, Co-Central and Bolívar groups, and the Occidentalis group, comprising isolates from the Co-West and Gileri groups. The spread of M. roreri into new areas and countries mediated by human activity is discussed.     

Foliar resistance of cacao (Theobroma cacao) to Phytophthora palmivora as an indicator of pod resistance in the field: interaction of cacao genotype, leaf age and duration of incubation

The effects were studied of four leaf development stages (LDS) and three durations of incubation (DI) on the accuracy of leaf-disc tests on eight cacao (cocoa) clones (C) for predicting field resistance to phytophthora pod rot caused by Phytophthora palmivora . The clones were known to possess different general combining abilities (GCA) for pod resistance in the field, evaluated monthly at harvest over a 9-year period. Disease severity (DS) was affected strongly by C, DI and LDS, with increasing levels of significance. Two- and three-way interaction effects were smaller than the clone effect, but still significant. Clone effects were most significant for LDS3 (i.e. leaves 50–60 days old) and for DI5 and DI7 (observations made 5 and 7 days after inoculation, respectively). Coefficients of rank correlation between DS and field results were significant for seven of the 12 treatments, with highest values obtained again for treatments LDS3/DI5 ( r = 0·87) and LSD3/DI7 ( r = 0·93). Pooling of data for different LDS and DI treatments did not further improve the correlation with field results. However, these correlations were improved (from an average of 0·74 to 0·88) when the GCA values for field resistance were based on weekly observations, carried out in one year, including losses of pods and cherelles. It was concluded that, when carried out in a standardized manner and under optimal conditions, the leaf-disc test may explain 75–90% of the genetic variation for field resistance of cacao genotypes to P. palmivora .     

Frosty pod of cacao: a disease with a limited geographic range but unlimited potential for damage

ABSTRACT Moniliophthora roreri, the cause of frosty pod rot (FP), is a specialized fungal pathogen (family Marasmiaceae) that invades only actively growing pods of cacao, Theobroma cacao, and related species of Theobroma and Herrania. FP damages pods and the commercially important seeds that some of these species produce. M. roreri was confined to northwestern South America until the 1950s. Its appearance in Panama in 1956 signaled a change in its geographic distribution. Now, it is found in 11 countries in tropical America. The fungus is currently in an active dispersal phase, possibly due to an increase in human-mediated spread. FP is more destructive than black pod (Phytophthora spp.) and more dangerous and difficult to control than witches' broom, caused by Moniliophthora (Crinipellis) perniciosa. The aggressiveness of M. roreri, its capacity to survive different environmental conditions, its rapid natural dispersal, its propensity for man-mediated dispersal, and the susceptibility of most commercial cacao genotypes, all indicate that FP presents a substantial threat to cacao cultivation worldwide.     

Molecular characterization of fungal endophytic morphospecies isolated from stems and pods of Theobroma cacao

Endophytic fungi were isolated from healthy stems and pods of cacao ( Theobroma cacao ) trees in natural forest ecosystems and agroecosystems in Latin America and West Africa. These fungi were collected for screening as a potential source of biocontrol agents for the basidiomycetous pathogens of cacao in South and Central America, Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches' broom). Many of these isolates were morphologically unidentifiable as they failed to form fruiting structures in culture, or only produced arthrosporic stages. Affinities with basidiomycetes were suspected for many of these based on colony morphology. Fifty-nine of these morphologically unidentifiable isolates were selected for molecular identification by DNA extraction and sequence analysis of nuclear ribosomal DNA (rDNA). The large subunit (LSU) was chosen for initial sequencing because this region has been used most often for molecular systematics of basidiomycete fungi, and comprehensive LSU datasets were already available for sequence analyses. Results confirmed that the majority of the isolates tested belonged to the Basidiomycota, particularly to corticoid and polyporoid taxa. With LSU data alone, identification of the isolates was resolved at varying taxonomic levels (all to order, most to family, and many to genus). Some of the isolates came from rarely isolated genera, such as Byssomerulius , whilst the most commonly isolated basidiomycetous endophyte was a member of the cosmopolitan genus Coprinellus (Agaricales). The role of these fungi within the host and their potential as biological control agents are discussed.     

Foliar resistance of cacao (Theobroma cacao) to Phytophthora palmivora as an indicator of pod resistance in the field: the effect of light intensity and time of day of leaf collection

Resistance of cacao leaves to Phytophthora palmivora was studied with regard to the time of leaf collection (morning, afternoon) and the degree of exposure of the leaves to light in the field (low, medium and high). The efficiency of leaf disc inoculations in predicting field resistance of nine clones was compared with that of detached and attached pod inoculations. Significant effects were observed, with leaves exposed to high light intensity and collected early in the afternoon showing highest susceptibility. The effect of time of leaf collection was reduced when leaves were stored overnight and leaf discs prepared and inoculated the following day, as compared to inoculations on the day of collection. Interactions between the main factors were significant, though less substantial than the clone effects. The most significant correlations with pod resistance ( r  = 0·70 to 0·97) were obtained for leaves collected early in the morning and exposed to intermediate shade conditions in the canopy. For other treatments, the correlations with pod resistance were still positive ( r  = 0·23 to 0·83) but often not significant. Pod inoculations in the laboratory were better correlated with field resistance ( r  = 0·92) than pod inoculations in the field ( r  = 0·72). Detached pod inoculations were also better correlated with leaf disc inoculations than those of attached pods. The results confirm the validity of laboratory inoculations of leaves and pods to assess field resistance to Phytophthora . Standardization of the leaf disc test is essential to obtain reliable results.     

Phytophthora resistance in cacao (Theobroma cacao): Influence of pod morphological characteristics

Twelve diverse cacao ( Theobroma cacao ) genotypes were assessed for pod resistance to Phytophthora palmivora at the penetration and post-penetration stages of infection using two inoculation methods. Correlation analysis between a number of pod characteristics (stomatal frequency, stomatal pore length, surface wax, thickness of exocarp/endocarp, hardness of exocarp/mesocarp, moisture content) and resistance indicated a strong relationship between resistance to lesion establishment (lesion frequency) and the joint effect of stomatal frequency and pore length. The epidermal impressions of the pod surfaces bearing germinating zoospores of P. palmivora provided evidence that penetration occurs through stomata, epidermal hair base, scar and by direct penetration. A poor correlation was obtained between the pod characteristics studied and post-penetration resistance, suggesting that this resistance, assessed by lesion size, is not governed by morphological or physical characteristics of the pod, but probably by biochemical factors. The importance of these findings in breeding of cacao for resistance to P. palmivora is discussed.     

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.     

Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan

A total of 651 isolates of cucumber corynespora leaf spot fungus ( Corynespora cassiicola ) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC₅₀) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL⁻¹ and from 0·04 to 0·59 μg mL⁻¹, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL⁻¹. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC₅₀ values ranging from 1·1 to 6·3 μg mL⁻¹, and a very highly resistant (VHR) group consisting of 25 isolates with EC₅₀ values higher than 24·8 μg mL⁻¹. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.     

Canopy-microclimate effects on the antagonism between Trichoderma stromaticum and Moniliophthora perniciosa in shaded cacao

The collective impact of several environmental factors on the biocontrol activity of Trichoderma stromaticum ( Ts ) against Moniliophthora perniciosa ( Mp ), the cause of cacao witches' broom disease, was assessed under field conditions of shaded cacao ( Theobroma cacao ) in south-eastern Bahia, Brazil. Biocontrol experiments were performed adjacent to an automated weather station, with sensors and Ts -treated brooms placed at different canopy heights. Sporulation occurred at the same dates for all Ts isolates, but in different quantities. Broom moisture >30%, air temperature of approximately 23 ± 3°C, relative humidity >90%, solar radiation intensities <0·12 KW m⁻² and wind speed near zero were the key environmental parameters that preceded Ts sporulation events. A multiple logistic regression indicated that these weather variables combined were capable of distinguishing sporulation from non-sporulation events, with a significant effect of wind speed. Analyses of environmental factors at ground level indicated similar pre-sporulation conditions, with a soil moisture content above a threshold of 0·34 m³ m⁻³ preceding all sporulation events. The sporulation of five selected Ts isolates was compared at four different canopy heights. Isolates responded differently to weather variation in terms of sporulation and antagonism to Mp at different canopy levels, indicating that different microclimates are established along the vertical profile of a shaded cacao plantation. The potential of these findings for development of predictive mathematical models and disease-management approaches is discussed.     

A disease and production index (DPI) for selection of cacao (Theobroma cacao) clones highly productive and tolerant to pod rot diseases

Frosty pod rot (FPR) (Moniliophthora roreri), along with black pod rot (Phytophthora species) and witches’ broom disease (Moniliophthora perniciosa) constitute the main phytosanitary problems limiting cacao (Theobroma cacao) production causing severe yield losses. One of the main sought after methods of pod rot management is the selection of tolerant cacao genotypes. Typically, the selection is carried out through the quantification of the percentage of diseased pods (PDP). However, PDP does not consider the relative productivity, or production potential (PT) of the genotype. Production potential can vary among cacao genotypes. Consequently, genotypes with similar PT can have similar or vastly different disease tolerance levels as measured by PDP. The disease and production index (DPI) was developed to integrate a genotype's tolerance to M. roreri and other diseases as measured by PDP, with its PT. Here, we evaluated the number of healthy pods, number of diseased pods, and weight of fresh seed for 29 clones grown in replicated five-tree plots over 4 years. The data obtained was used to calculate PDP and DPI for each clone for three different disease combinations: frosty pod rot alone, pod rots other than frosty pod rot, and the combination of all pod rots. Multivariate analysis verified that DPI discriminated between clones based on productivity and disease tolerance. Surprisingly, there was a close ranking of clones between resistance to FPR and resistance to all other pod rots. The DPI can be used in breeding programmes focused on the selection of high yielding disease-tolerant cacao genotypes.     

PCR‐based identification of cacao black pod causal agents and identification of biological factors possibly contributing to Phytophthora megakarya's field dominance in West Africa

Among the Phytophthora species that cause black pod of cacao, P. megakarya is the most virulent, posing a serious threat to cacao production in Africa. Correct identification of the species causing the black pod and understanding the virulence factors involved are important for developing sustainable disease management strategies. A simple PCR‐based species identification method was developed using the species‐specific sequences in the ITS regions of the rRNA gene. A phylogenetic tree generated for 119 Phytophthora isolates, based on the 60S ribosomal protein L10 gene and rDNA sequence, verified the PCR‐based identification assay and showed high interspecific variation among the species causing black pod. Phytophthora megakarya isolates were uniformly virulent in an assay using susceptible cacao pod husks inoculated with zoospores, while the P. palmivora isolates showed greater divergence in virulence. The virulence of P. megakarya was associated with earlier production of sporangia and an accelerated induction of necrosis. While zoospore germ tubes of both species penetrated pods through stomata, only P. megakarya produced significant numbers of appressoria. A hypersensitive‐like response was observed when attached SCA‐6 pods were inoculated with P. palmivora. SCA‐6 pods became vulnerable to P. palmivora when wounded prior to zoospore inoculation. Phytophthora megakarya was more aggressive than P. palmivora on attached SCA‐6 pods, causing expanding necrotic lesions with or without wounding. Phytophthora megakarya is predominant in the Volta region of Ghana and it remains to be seen whether it can displace P. palmivora from cacao plantations of Ghana as it has in Nigeria and Cameroon.     

Genetic characteristics of Fusarium verticillioides isolates from maize in Costa Rica

Thirty-nine isolates of Fusarium verticillioides from maize seeds from three regions of Costa Rica were classified on fertility, fumonisin production, vegetative compatibility and pathogenicity. The identity of the isolates was verified by sexual crosses with standard tester strains and by isozyme analysis. Twenty-three isolates (59%) were mating type A⁻ and 16 (41%) were A⁺; 29 (74%) were female fertile. The isolates produced high amounts of fumonisin B₁ when grown on sterilized maize grits, 32 isolates producing more than 1000 μg g⁻¹, as determined by TLC, and 7 less than 1000 μg g⁻¹. Vegetative compatibility tests by pairing nit mutants identified 34 vegetative compatibility groups (VCGs), of which 29 had one member and 5 had two members. Isolates belonging to the same VCG were obtained from the same seed sample. Two pathogenicity tests with different inoculation methods were performed: on toothpick inoculation of 7-week-old maize stalks, 71% of the isolates were pathogenic according to the length of the necrosis formed in the stalk, and on sand inoculation of maize seedlings all the isolates were pathogenic, according to shoot length and dry weight production. Differences in aggressiveness between some of the isolates were recorded. It is concluded that natural populations of F. verticillioides in Costa Rica consist of genetically diverse, highly fertile and pathogenic isolates that represent a potential risk for disease development and fumonisin accumulation in maize crops.     

Development of a marker assisted selection program for cacao

ABSTRACT Production of cacao in tropical America has been severely affected by fungal pathogens causing diseases known as witches' broom (WB, caused by Moniliophthora perniciosa), frosty pod (FP, caused by M. roreri) and black pod (BP, caused by Phytophthora spp.). BP is pan-tropical and causes losses in all producing areas. WB is found in South America and parts of the Caribbean, while FP is found in Central America and parts of South America. Together, these diseases were responsible for over 700 million US dollars in losses in 2001 (4). Commercial cacao production in West Africa and South Asia are not yet affected by WB and FP, but cacao grown in these regions is susceptible to both. With the goal of providing new disease resistant cultivars the USDA-ARS and Mars, Inc. have developed a marker assisted selection (MAS) program. Quantitative trait loci have been identified for resistance to WB, FP, and BP. The potential usefulness of these markers in identifying resistant individuals has been confirmed in an experimental F(1) family in Ecuador.     

An optimized screening method for identifying levels of resistance to Crinipellis perniciosa in cocoa (Theobroma cacao)

The effects of host age, leaf number, host type (clone or seedling), pathogen spore concentration and incubation time on inoculation with Crinipellis perniciosa (witches' broom disease of cocoa) were studied in greenhouse experiments using susceptible cocoa genotypes. Three methods of inoculation (agar-drop, water-drop and spray) were also tested. An optimized inoculation method was selected and tested for its repeatability as well as its ability to discriminate between various levels of resistance to C. perniciosa in cocoa. The optimized method (350 000 viable basidiospores per mL, 60 h incubation, agar-drop technique) produced 100% infection repeatedly, on both clonal and seedling plants of a susceptible genotype. Seedling age (2–12 months) and leaf number did not significantly affect the percentage of plants with symptoms or broom characteristics. This method discriminated effectively between the various levels of resistance in 14 cocoa genotypes and is recommended as an inoculation method to identify levels of resistance in germplasm collections. Symptom severity was shown to be a better measure of resistance than infection success.     

A qualitative host-pathogen interaction in the Theobroma cacao-Moniliophthora perniciosa pathosystem

The aim of this study was to test whether resistance of clones of Theobroma cacao (cocoa) varied between isolates of Moniliophthora (formerly Crinipellis ) perniciosa , the cause of witches' broom disease. Developing buds of vegetatively propagated T. cacao grown in greenhouses in the UK were inoculated with 16 000 spores of M. perniciosa per meristem in water, under conditions where water condensed on the inoculated shoot for at least 12 h after inoculation. The proportion of successful inoculations varied between clones and was inversely correlated with time to symptom production or broom formation. A specific interaction was demonstrated among three single-spore isolates of M. perniciosa and the clone Scavina 6 (SCA 6) and a variety of susceptible clones. Isolates Castenhal-I and APC3 were equally likely to infect SCA 6 and the other clones, but isolate Gran Couva A9 never infected SCA 6, although it was as virulent on the other clones. The interaction was maintained when the wetness period was extended to 70 h. Offspring of SCA 6 × Amelonado matings were all susceptible to both Castenhal-I and GC-A5, with no evidence of greater variability in susceptibility to GC-A5 than Castanhal-I. This suggests recessive inheritance of a single homozygous factor conferring resistance to GC-A5, from SCA 6. The progenies were slightly more susceptible to Castanhal-I than GC-A5. The implications for managing the disease are discussed.     

Management of late leaf spot of groundnut (Arachis hypogaea) with chlorothalonil-tolerant isolates of Pseudomonas aeruginosa

Fifteen groundnut-associated bacterial isolates that inhibited by > 90% the in vitro conidial germination of Phaeoisariopsis personata , causal agent of late leaf spot disease of groundnut, were applied as a prophylactic spray (10⁸ cfu mL⁻¹) and tested for control of the disease in the glasshouse. Two groundnut seed-associated bacterial isolates, GSE 18 and GSE 19, identified as Pseudomonas aeruginosa , reduced the lesion frequency (LF) by up to 70%. A 90-day-old peat-based formulation of P. aeruginosa GSE 18 reduced LF measured 15 days postinoculation by up to 60%. Both P. aeruginosa GSE 18 and GSE 19 were tolerant to chlorothalonil (Kavach®) up to 2000  µ g mL⁻¹ in LB broth. In glasshouse trials, GSE 18 and GSE 19 tested in combination with reduced concentrations of chlorothalonil were highly efficient in management of the disease. The disease was completely controlled by chlorothalonil (> 250  µ g mL⁻¹), and in the presence of GSE 18 or GSE 19, 100  µ g mL⁻¹ chlorothalonil was equally effective. Application of rifamycin-resistant mutants of GSE 18 or GSE 19 together with chlorothalonil significantly increased the survival of these isolates in the groundnut phylloplane. In the field, a combination of GSE 18 and 500  µ g mL⁻¹ chlorothalonil reduced disease severity comparable to 2000  µ g mL⁻¹ chlorothalonil alone. Use of chlorothalonil-tolerant pseudomonads together with a quarter concentration of the recommended field dose of chlorothalonil doubled pod yield compared with the untreated unsprayed control.     

Molecular and metabolic changes of cherelle wilt of cacao and its effect on Moniliophthora roreri

Young Theobroma cacao pods, known as cherelles, are commonly lost to physiological thinning known as cherelle wilt. Cherelles are susceptible to frosty pod rot caused by Moniliophthora roreri. We studied the cherelle wilt process and its impact on M. roreri infection using microscopic, metabolite, and gene expression analyses. Wilt was associated with increased levels of tricarboxylic acid cycle intermediaries and decreased levels of major metabolites. Expression changes of cacao ESTs in response to wilt suggest induction of the polyamine, ethylene, and jasmonic acid biosynthetic pathways and regulation of abscisic acid and cytokinin levels. M. roreriinfection caused little alteration of cherelle physiology. M. roreri responded to the late stage of wilt by altering the expression of M. roreri ESTs associated with metabolite detoxification and host tissue degradation. The environment of the wilting cherelles may truncate the disease cycle of frosty pod rot, by limiting M. roreri sporulation and stopping the lifecycle.     

Genetic variation in populations of the cacao wilt pathogen, Ceratocystis cacaofunesta

Ceratocystis cacaofunesta (=  Ceratocystis fimbriata ) causes a lethal wilt disease of cacao ( Theobroma cacao ) in Latin America. Polymorphic microsatellite markers, (CAT)₅ nuclear DNA fingerprints and Hae III mitochondrial DNA fingerprints were used to compare genetic diversity among isolates of C. cacaofunesta collected from populations in western Ecuador, Costa Rica, Colombia, and Rondônia and Bahia in Brazil. Microsatellite markers and nuclear DNA fingerprints separated Ecuadorian isolates from isolates of the other four populations, and these two major groups correspond to genetic lineages already identified from ITS-rDNA sequences and intersterility groupings. Mitochondrial DNA fingerprints also demonstrated substantial diversity and split the Ecuadorian isolates into two groups. All marker types showed limited variation in the Colombian, Costa Rican and Bahian populations, as might be expected for introduced populations that have gone through recent genetic bottlenecks. In contrast, the Rondonian and western Ecuadorian populations showed gene diversity values similar to natural populations of other Ceratocystis species. The Rondonian population was the only sampled population in the native range of T. cacao (the Upper Amazon), and the putatively introduced populations were more closely related to the Rondonian population than to the western Ecuadorian population. The Ecuadorian population is in an area with other native Theobroma species, which may serve as natural hosts.     

Diversity and pathogenicity of the Ceratocystidaceae associated with cacao agroforests in Cameroon

Knowledge of the diversity and ecology of plant pathogenic fungi in cacao agroforests and surrounding natural ecosystems can inform the development of sustainable management strategies for new cacao disease outbreaks. This study investigated the occurrence of fungi related to the Ceratocystidaceae and their nitidulid beetle vectors in cacao agroforests in Cameroon, under diverse agroecological conditions. The fungi and their vectors were collected from artificially induced stem wounds on cacao and associated shade trees. Collections were also made from abandoned cacao pod husks and other tree wounds within and around plantations. Fungal isolates were identified using DNA sequence‐based phylogenies and morphological comparisons, and two representatives of each species were evaluated for pathogenicity on cacao. Five species of Ceratocystidaceae were recovered, including Huntiella chlamydoformis sp. nov., H. pycnanthi sp. nov. and H. moniliformis, as well as Thielaviopsis cerberus and T. ethacetica. The incidence of these fungi appeared to be influenced by the prevailing agroecological conditions. Nitidulid beetles in the genus Brachypeplus were found to be their most common insect associates on cacao. Both T. ethacetica and H. pycnanthi produced extensive lesions after inoculation on branches of mature cacao trees, while T. ethacetica also caused pod rot. Although their impact remains unknown, fungi in the Ceratocystidaceae and their nitidulid beetle vectors are common and probably contribute to the parasitic pressure in Cameroonian cacao agrosystems.     

Isolation of actinomycetes and endospore-forming bacteria from the cacao pod surface and their antagonistic activity against the witches’ broom and black pod pathogens

In this study, actinomycetes and endospore-forming bacteria were isolated from the surface of cacao pods. The activity of these microorganisms againstCrinipellis perniciosa andPhytophthora palmivora, causal agents of witches’ broom and black pod diseases of cacao, respectively, was investigated. A total of 336 isolates of actinomycetes and endosporeforming bacteria were tested on a detached pod assay againstC. perniciosa. The screening procedure used proved to be fast and inexpensive, allowing the selection of five actinomycetes as the most promising isolates for the biocontrol ofC. perniciosa. Under laboratory conditions the actinomycetes were able to inhibit 100% ofC. perniciosa basidiospore germination. However, under field conditions the selected actinomycetes were unable to protect cacao pods against both pathogens. In these experiments, inhibition ofC. perniciosa ranged from 6% to 21% in relation to the control, whereas there was no inhibition of black pod caused byP. palmivora. Formulations need to be improved in order to enhance the activity of the actinomycetes against cacao pathogens in the field. Molecular identification of the selected isolates showed that they are species of the genusStreptomyces.