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 .     

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.     

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.     

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.     

The infection process of Moniliophthora perniciosa in cacao

The development of the basidiomycete Moniliophthora perniciosa in resistant and susceptible Theobroma cacao genotypes was analysed. The infection process leading to broom formation in shoot apexes was characterized by studying the kinetics of basidiospore germination, mode of penetration and colonization of the pathogen. Both resistant and susceptible cacao genotypes were inoculated with M. perniciosa and kept in the greenhouse for 90 days, explants were collected, treated for histological studies and meristematic tissues were observed by electron and light microscopy. Variation in the kinetics of germination between the cacao genotypes was detected 4 h after inoculation. The fungal penetration occurred through the star‐shaped trichome base, natural openings on the cuticular surface and stomata. Host responses between genotypes were found to be different. Compared with non‐infected plants, the swelling of all the stem tissues was evident at 60 days after inoculation. In the susceptible genotype, typical symptoms developed and fungal colonization was more intense than in resistant genotypes, which showed little or no fungal colonization. The investigations reported herein provide an important step in understanding the pattern of pre‐ and post‐penetration events of M. perniciosa in cacao genotypes with different levels of resistance to this disease.     

The occurrence of Xylosandrus compactus and its associated fungi on cacao from South Sulawesi,Indonesia: A preliminary study of an emerging threat to the cacao industry

Journal of Plant Diseases and Protection - Xylosandrus compactus is one of the significant beetle pests on perennial crops, which has become an increasing problem on cacao productivity and...     

Evaluation of cacao (Theobroma cacao) clones against seven Colombian isolates of Moniliophthora roreri from four pathogen genetic groups

Artificial pod inoculation was used to compare the relative aggressiveness of seven Colombian isolates of Moniliophthora roreri (the causal agent of moniliasis or frosty pod disease), representing four major genetic groupings of the pathogen in cacao (cocoa), when applied to five diverse cacao genotypes (ICS-1, ICS-95, TSH-565, SCC-61 and CAP-34) at La Suiza Experimental Farm, Santander Department, Colombia. The following variables were evaluated 9 weeks after inoculation of 2- to 3-month-old pods with spore suspensions (1·2 × 10⁵ spores mL⁻¹): (i) disease incidence (DI); (ii) external severity (ES); and (iii) internal severity (IS). IS was found to be of greatest value in classifying the reaction of the host genotype against M. roreri . Genetic variation reported between isolates and cacao genotypes was not matched by similar diversity in their aggressiveness. All isolates were generally highly aggressive against most cacao genotypes, with only two isolates showing reduced IS and ES reactions. There was considerable variation between clones in the IS and ES scores, but one cultivated clone (ICS-95) displayed a significant level of resistance against all seven isolates. This clone may be useful in cacao breeding initiatives for resistance to moniliasis of cacao.     

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.     

The effects of mild cacao swollen shoot virus strains N1 and SS365B on growth and yield of cacao

A field experiment started at the Cocoa Research Institute of Ghana in 2007 to investigate the effects of the cacao swollen shoot virus (CSSV) mild stains N1 and SS365B on growth and yield of mixed hybrid cacao (Theobroma cacao L.). Growth was significantly influenced by the duration of infection with the two CSSV mild strains. Slicing analyses showed no significant differences in stem circumference among treatments up to the third year of field data collection. However, from the fourth to the seventh year, growth of N1-inoculated cacao plants was significantly higher than that of SS365B-inoculated plants. Overall, growth did not differ significantly between cacao plants inoculated with mild strain and the non-inoculated plants. Contrary to some previous reports, yield of mild strain-inoculated cacao plants was significantly lower than that of the non-inoculated plants. Polymerase chain reaction was used to detect the presence of CSSV in randomly selected field samples. The implications of these results (based on field data collected from 2008 to 2014) in the management of cocoa swollen shoot virus disease in Ghana are discussed.     

Oomycete species associated with Theobroma cacao crops in Colombia

The study of oomycetes associated with crops is highly important due to the economic losses they might cause. In cacao, the genus Phytophthora has been extensively studied, but little is known about other genera and species of oomycetes associated with this plant. This study aimed to determine the oomycetes’ diversity present in Colombian cacao crops. A total of 146 isolates were obtained from diseased plants and soil in 11 departments. Analysis of internal transcribed spacer (ITS) and cytochrome oxidase subunit I (coxI) sequences was performed along with the assessment of morphological characteristics. Nine species were identified, distributed in four genera: Phytophthora (P. palmivora, 54%; P. nicotianae, 1%), Phytopythium (Phy. chamaehyphon, 15%; Phy. cucurbitacearum, 9%; Phy. vexans, 7%; Phy. helicoides, 1%), Globisporangium (G. splendens, 3%), and Pythium (Py. delicense, 1%; Py. inflatum,1%). Additionally, an unidentified and possibly new species of Phytophthora (5%) and three unidentified species of Phytopythium (3%) were found. This is the first report of Globisporangium, Phytopythium, and Pythium in cacao crops of Colombia and the first report of the species Phy. chamaehyphon in the country. Interestingly, some isolates of Phytopythium spp. were isolated from necrotic leaves and vascular section of stems, which may suggest a role in cacao diseases traditionally associated with Phytophthora. Also, it is proposed that the new species of Phytophthora may be contributing significantly to black pod disease in Colombian cacao crops, and we highlight that the study of P. palmivora is urgent because of its distribution all over the country.     

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.     

Relationships Between Black Pod and Witches'-Broom Diseases in Theobroma cacao

ABSTRACT Field observations were conducted from 1998 to 2001 at the International Cocoa Genebank, Trinidad, to evaluate 57 cacao clones for resistance to black pod (BP) and witches'-broom (WB) diseases (caused by Phytophthora sp. and Crinipellis perniciosa, respectively). Each month ripe pods were harvested and the number of healthy and diseased was recorded. The number of brooms on vegetative shoots was recorded three times a year on selected branches. Twenty-three clones showed less than 10% of infection for both BP and WB on pods. Among those, eight clones showed an absence of brooms on the observed branches: IMC 6, MAN 15/60 [BRA], PA 67 [PER], PA 195 [PER], PA 218 [PER], PA 296 [PER], PA 303 [PER], and POUND 32/A [POU]. Broad-sense heritability was estimated at 0.38 and 0.57 for WB disease on pods and shoots, respectively, and at 0.51 for BP disease. Genetic correlation between WB disease on pods and on shoots was low and estimated at 0.39, whereas the correlation between WB and BP diseases on pods was 0.48. To choose putative parents for breeding schemes, it is suggested that clones are first assessed for their level of resistance to WB on shoots, and the most promising individuals are screened for BP with a detached pods test. Further studies are needed to confirm whether the level of resistance to WB on pods can be predicted using an early test on seedlings.     

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.     

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.     

Trichoderma harzianum produces nonanoic acid, an inhibitor of spore germination and mycelial growth of two cacao pathogens

An isolate of Trichoderma harzianum Rifai from an infected cacao pod produces and secretes nonanoic (pelargonic) acid into a liquid culture medium. Nonanoic acid (NA) was very inhibitory to spore germination and mycelial growth of two cacao pathogens, Crinipellis perniciosa Stahel and Moniliophthora roreri Cif. H.C. Evans. It was highly active causing 75% inhibition of spore germination in an in vitro assay at a rate as low as 0.09 μM for M. roreri and 0.92 μM for C. perniciosa. Mycelial growth was comparatively less sensitive to inhibition, but still there was a 75% reduction in growth with 0.62 μM in M. roreri and 151 μM NA in C. perniciosa. In contrast, NA did not affect Trichoderma mycelial growth or spore germination at concentrations that were inhibitory to the pathogens. 6-pentyl-α-pyrone was also produced and secreted into the medium by T. harzianum, however; it was not antagonistic to the cacao pathogens. Although a number of metabolites produced by Trichoderma spp. have been identified in the past, this is the first report of NA production and secretion by any Trichoderma. The results suggest that NA may play a role in the successful use of some Trichoderma spp. isolates in the biocontrol of fungal diseases of plants.     

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.     

Molecular Fingerprinting Suggests Two Primary Outbreaks of Witches' Broom Disease (Crinipellis perniciosa) of Theobroma cacao in Bahia, Brazil

Crinipellis perniciosa (Stahel) Singer is the causal agent of witches' broom disease in the Sterculiaceae, Solanaceae, and Bixaceae families. The disease is endemic to the Brazilian Amazon, and was first reported infecting Theobroma cacao (cocoa) in the State of Bahia, Brazil, in 1989. Random amplified polymorphic DNA (RAPD) analyses were performed on 46 isolates of C. perniciosa from cocoa that were collected from 15 counties in Bahia and the Brazilian Amazon. A total of 258 RAPD loci from 20 primers and three mixed primers were analyzed. Of these loci, 108 (42%) were polymorphic, with an average of 4.7 polymorphic loci per primer produced. Genetic similarities were estimated using Nei and Li's index and UPGMA clustering. Bootstrap analysis divided the phenogram into four significantly different clusters: two groups contained isolates from Ariquemes and from Ouro Preto, Rondônia, and the other two separated the isolates from Bahia into two major groups of C. perniciosa, classified as Group 1 (G1) and Group 2 (G2). The two groups of isolates from Bahia differed for their genetic similarity with the isolates from the Brazilian Amazon. The geographic distribution of the groups in Bahia suggests two independent focal points of introduction. Ongoing programs to screen for resistant cocoa genotypes should consider both groups of isolates.     

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.     

Incidence of Phytophthora pod rot disease of cacao at different heights in the tree

The rate of infection of pods byP. palmivora at the lower trunk of a cacao tree is always higher than of those in the canopy. This is often held as a proof that the soil is the primary source of infection. An experiment has indicated that climatic conditions at the beginning of the rainy season in Western Nigeria do not favour infection of pods in the canopy, which may also explain the said phenomenon.Samenvatting In Nigeria evenals in andere landen is waargenomen dat doorP. palmivora aangetaste cacaokolven in het begin van de regentijd vooral voorkomen aan de stam en dat het aantastingspercentage van de stamvruchten over het gehele seizoen genomen aanzienlijk hoger light dan dat van de vruchten in de kroon. Een proef waarbij ongeveer 10% van de stamvruchten en ook van de vruchten in de kroon werd geïnoculeerd bij het begin van de regentijd, wees uit dat in deze periode van het jaar de klimaatomstandigheden in de kroon waarschijnlijk ongunstig zijn voor infectie.Een hoog percentage aangetaste stamvruchten in genoemde periode mag derhalve niet zonder meer gezien worden als bewijs dat de grond in het begin van de regentijd de enig belangrijke besmettingsbron van de ziekte zou zijn.