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.     

Spatial genetic structure and dispersal of the cacao pathogen Moniliophthora perniciosa in the Brazilian Amazon

Moniliophthora perniciosa is the causal agent of witches’ broom in Theobroma cacao (cacao). Three biotypes of M. perniciosa are recognized, differing in host specificity, with two causing symptoms on cacao or Solanaceae species (C‐ and S‐biotypes), and the third found growing endophytically on lianas (L‐biotype). The objectives of this study were to clarify the genetic relationship between the three biotypes, and to identify those regions in the Brazilian Amazon with the greatest genetic diversity for the C‐biotype. Phylogenetic reconstruction based on the rRNA ITS regions showed that the C‐ and S‐biotypes formed a well‐supported clade separated from the L‐biotype. Analysis of 131 isolates genotyped at 11 microsatellite loci found that S‐ and especially L‐biotypes showed a higher genetic diversity. A significant spatial genetic structure was detected for the C‐biotype populations in Amazonia for up to 137 km, suggesting ‘isolation by distance’ mode of dispersal. However, in regions containing extensive cacao plantings, C‐biotype populations were essentially ‘clonal’, as evidenced by high frequency of repeated multilocus genotypes. Among the Amazonian C‐biotype populations, Acre and West Amazon displayed the largest genotypic diversity and might be part of the centre of diversity of the fungus. The pathogen dispersal may have followed the direction of river flow downstream from Acre, Rondônia and West Amazon eastward to the rest of the Amazon valley, where cacao is not endemic. The Bahia population exhibited the lowest genotypic diversity, but high allele richness, suggesting multiple invasions, with origin assigned to Rondônia and West Amazon, possibly through isolates from the Lower Amazon population.     

Genetic and Biological Diversity of Trichoderma stromaticum, a Mycoparasite of the Cacao Witches'-Broom Pathogen

ABSTRACT The witches'-broom disease, caused by the basidiomycete Crinipellis perniciosa, is the most limiting factor for cacao cultivation in Brazil. Trichoderma stromaticum is a mycoparasite of the witches'-broom pathogen of cacao that is currently being applied in the field to manage the disease in Bahia State, Brazil. In this work, molecular and traditional methods were used to study the genetic and biological diversity of this mycoparasite. Ninety-one isolates, mostly collected from farms not sprayed with the fungus, were analyzed by amplified fragment length polymorphisms (AFLP), which showed that two genetic groups (I and II) of T. stromaticum occur in Bahia State. This classification of T. stromaticum into two distinct AFLP groups was also in agreement with several other characteristics, including growth on agar media at different temperatures and sporulation on infected stem segments (broom pieces) and rice grains. Group II favors higher temperatures compared with group I. The genetic and biological differences of the isolates, however, were not evident in field experiments, where sporulation was evaluated on the surface of brooms under natural conditions. Our results show that there is considerable genetic and biological diversity within T. stromaticum in Bahia and other cacao-growing regions of South America that are affected by the witches'-broom disease. This diversity could be explored in the development of efficient biological control agents against the disease. Factors that may affect the application and performance of this biocontrol agent in the field, such as sporulation on rice substrate and on the brooms and growth at various temperatures, are discussed.     

Production and Germination of Conidia of Trichoderma stromaticum, a Mycoparasite of Crinipellis perniciosa on Cacao

ABSTRACT Growth characteristics of the fungus Trichoderma stromaticum, a mycoparasite on the mycelium and fruiting bodies of Crinipellis perniciosa, the causal agent of witches'-broom disease of cacao, were evaluated under controlled environmental conditions. The ability of T. stromaticum to produce conidia and germinate on dry brooms was evaluated at three constant temperatures (20, 25, and 30 degrees C) and two constant relative humidities (75 and 100%). T. stromaticum produced abundant conidia on brooms at 100% relative humidity and incubation temperatures of 20 and 25 degrees C, but none at 30 degrees C. Sporulation of T. stromaticum was not observed at 75% relative humidity at any temperature. At 100% relative humidity and either at 20 or 25 degrees C, treatment of brooms with T. stromaticum suppressed C. perniciosa within 7 days. In contrast, at 30 degrees C, treatment with T. stromaticum had no effect on the pathogen in brooms maintained at either 75 or 100% relative humidity. Mycelium of C. perniciosa grew from brooms at all temperatures at 100% relative humidity. Conidial germination on broom tissue approximated 80% at temperatures from 20 to 30 degrees C. Results suggest that applying T. stromaticum under high-moisture conditions when the air temperature is below 30 degrees C may enhance the establishment of this mycoparasite in cacao plantations.     

Random amplified polymorphic DNA (RAPD) analysis of Crinipellis perniciosa isolates from different hosts

DNA sequence polymorphisms among isolates of Crinipellis perniciosa , causing witches' broom disease in several genera and species in the Sterculiaceae, Solanaceae and Bixaceae, were revealed by random amplified polymorphic DNA (RAPD) banding patterns. Comparisons were made among isolates from Theobroma cacao (cultivars SCA6 and SVB), T. grandiflorum, T. obovatum, T. subincanum, Herrania sp. (all collected near Belém, Brazil, at three plots within two kilometres of each other), and Bixa orellana and Solanum rugosum (150 and 1440 km from Belém, respectively). Genetic similarities, as determined by Sorensen's coefficient ( Sc_ij ; fraction of shared bands), ranged from 1·00 ( T. cacao SCA6 and T. grandiflorum ) to 0·56 ( S. rugosum and T. subincanum). Results indicate that proximity is more important than host species for determining the genetic relationships among isolates, that there were two independent sources of inoculum at the site near Belém, and that the isolate from B. orellana is most closely related to the isolate from T. cacao SVB. Comparisons were also made among multispore cultures from different basidiocarps on the same T. cacao SVB broom, as well as monospore cultures from the same S. rugosum basidiocarp. Banding patterns were similar among basidiocarps on the same broom, but differences were detected among monospore cultures from the same basidiocarp.     

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.     

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 and Pathogenicity of the Fungus Crinipellis perniciosa on Interaction with Cacao Leaves

ABSTRACT We investigated developmental changes in the primary mycelium of Crinipellis perniciosa upon its interaction with immature and mature leaves of Theobroma cacao. On nutritive medium, the primary mycelium grew significantly slower in the presence of host tissue than without host tissue. In the absence of the cacao leaves, incomplete phase transition occurred after 5 days, wherein older hyphae progressed to the dikaryotic state and growing tips remained monokaryotic. Phase transition occurred between 3 and 5 days on mature leaves, 10 and 12 days on meristematic leaves, and required 2 weeks on T. cacao callus tissue. The biotrophic mycelia were able to invade immature and mature cacao leaves without open wounds or stomata. Club-shaped hyphal tips and the formation of adhesive structures were induced by cuticle extracts and suggest host recognition. The initial cuticular disintegration at the site of penetration was followed by blister formation and complete digestion of leaves by the primary mycelium. The data suggest specific interactions between host and pathogen that control the onset of the necrotrophic phase of the fungus. The data further indicate that primary mycelium rather than spores can be used to study C. perniciosa pathogenicity.     

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.     

Ceratocystis wilt of cacao-a disease of increasing importance

ABSTRACT Ceratocystis cacaofunesta (formerly C. fimbriata) causes a lethal wilt disease of cacao (Theobroma cacao) in the Caribbean and Central and South America. Recent studies employing phylogenetics, intersterility, and host range separate the cacao pathogen from other strains of the C. fimbriata complex. Ceratocystis wilt has been managed through genetic resistance, but the disease is an emerging problem in Bahia, Brazil, where it was recently introduced. Genetic studies indicate that populations of the fungus in Costa Rica, Colombia, and Bahia may have been introduced on cacao cuttings; whereas populations in Rond?nia, Brazil, and western Ecuador appear to be native. The fungal genotype present in Bahia is similar to those found in Rond?nia and may have been introduced on propagative material with witches' broom resistance.     

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.     

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.     

Interaction of Moniliophthora perniciosa biotypes with Micro‐Tom tomato: a model system to investigate the witches' broom disease of Theobroma cacao

The miniature tomato (Solanum lycopersicum) cultivar Micro‐Tom (MT) has become an important platform to investigate plant–pathogen interactions. In the case of the witches' broom disease of Theobroma cacao (cacao), the existence of Moniliophthora perniciosa isolates pathogenic to Solanaceae (S‐biotype) may enable the use of MT to circumvent limitations of the cacao host, whereas the availability of a non‐infective cacao C‐biotype allows the evaluation of contrasting responses of MT. Infection of MT by the S‐biotype led to stem swelling and axillary shoot growth to form broom‐like symptoms similar to the biotrophic phase in cacao, but the infected tissues did not progress to necrosis. Conversely, inoculation with the C‐biotype did not cause typical symptoms, but reduced plant height, appearing as a non‐host interaction. Histopathological characterization of the S‐biotype infection of MT by light and electron microscopy indicated limited germ tube penetration, preferentially through wounds at the base of trichomes or actively through the epidermis. No intracellular mycelium was observed, corroborating the lack of the necrotrophic stage of the pathogen. The analysis of gene expression during the interaction between the S‐ or C‐biotype with MT indicated that expression of plant defence‐associated genes differs for kinetics and intensity between a compatible or incompatible M. perniciosa–MT interaction. The pattern of spore germination and low rate of mycelia penetration suggests that the S‐biotype is not a fully adapted tomato pathogen, but possibly a case of broken non‐host resistance, and evidence suggests the occurrence of a non‐host MT response against the C‐biotype.     

Cacao diseases-the trilogy revisited

ABSTRACT This paper reviews the significant advances by the diseases themselves, as well as by the scientists, in the intervening period since the disease trilogy was first delimited in 1989. The impact of these diseases, black pod, witches' broom, and frosty pod rot, has increased dramatically. In addition, there have been radical changes in the taxonomic profiles of these pathogens, which have been based on both traditional (morphological, cytological) and modern (molecular) approaches. Black pod is caused by a complex of Phytophthora species, in which P. palmivora still is the most important worldwide. However, recent invasion of the principal cacao-growing countries of West Africa by the more virulent P. megakarya has been cause for concern. The latter evolved in the ancient forests straddling the Cameroon-Nigerian border as a primary coloniser of fallen fruit. Conversely, frosty pod rot, caused by Moniliophthora roreri, and witches' broom, caused by M. (Crinipellis) perniciosa, both neotropical diseases, are hemibiotrophic, coevolved pathogens. Respectively, M. roreri arose on Theobroma gileri in submontane forests on the north-western slopes of the Andes, whereas M. perniciosa developed as a complex of pathotypes with a considerably wider geographic and host range within South America; the cacao pathotype evolved on that host in the Amazon basin. The inter-relationships of these vicariant species and their recent spread are discussed, together with control strategies.     

Genetic Variability and Host Specialization in the Latin American Clade of Ceratocystis fimbriata

ABSTRACT The Ceratocystis fimbriata complex includes many undescribed species that cause wilt and canker diseases of many economically important plants. Phylogenetic analyses of DNA sequences have delineated three geographic clades within Ceratocystis fimbriata. This study examined host specialization in the Latin American clade, in which a number of lineages were identified using sequences of the internal transcribed spacer (ITS) region of the rDNA. Three host-associated lineages were identified from cacao (Theobroma cacao), sweet potato (Ipomoea batatas), and sycamore (Platanus spp.), respectively. Isolates from these three lineages showed strong host specialization in reciprocal inoculation experiments on these three hosts. Six cacao isolates from Ecuador, Trinidad, and Columbia differed genetically from other cacao isolates and were not pathogenic to cacao in inoculation tests. Further evidence of host specialization within the Latin American clade of Ceratocystis fimbriata was demonstrated in inoculation experiments in growth chambers using sweet potato, sycamore, Colocasia esculenta, coffee (Coffea arabica), and mango (Mangifera indica) plants; inoculation experiments in Brazil using Brazilian isolates from cacao, Eucalyptus spp., mango, and Gmelina arborea; and inoculation experiments in Costa Rica using Costa Rican isolates from cacao, coffee, and Xantho-soma sp. Hosts native to the Americas appeared to be colonized by only select pathogen genotypes, whereas nonnative hosts were colonized by several genotypes. We hypothesize that local populations of Ceratocystis fimbriata have specialized to different hosts; some of these populations are nascent species, and some host-specialized genotypes have been moved to new areas by humans.     

Population structure and migration of the witches’ broom pathogen Moniliophthora perniciosa from cacao and cultivated and wild solanaceous hosts in southeastern Brazil

Moniliophthora perniciosa, causal agent of witches’ broom disease in cacao plantations in South America and the Caribbean Islands, has co‐evolved with its host cacao, but the pathogen has also emerged in many solanaceous hosts in Brazil, including economically important food crops and wild species. This study was carried out to: (i) determine the existence of host subpopulations of M. perniciosa in Brazil; (ii) estimate gene and genotypic diversity of M. perniciosa host subpopulations infecting solanaceous hosts in southeastern Bahia and Minas Gerais states, Brazil; and (iii) estimate the amount and directionality of historical migration of M. perniciosa subpopulations. Up to 203 M. perniciosa isolates collected from solanaceous hosts with symptoms from Bahia and Minas Gerais states in Brazil and from Theobroma spp. (cacao) and Herrania spp. were characterized with 11 microsatellite markers. Factorial correspondence analyses, minimum‐spanning network and Bayesian clustering revealed genetic clusters associated with their host of origin. Significant subpopulation differentiation was evident (Φ_ST = 0.30, P ≤ 0.05) among M. perniciosa host subpopulations. Most of the multilocus microsatellite genotypes (MLMGs) were host‐specific, with few MLMGs shared among subpopulations. Pairwise comparisons among M. perniciosa host subpopulations were significant, except between jurubeba (Solanum paniculatum) and cultivated solanaceous subpopulations. The combined analyses rejected the null hypothesis that M. perniciosa in Brazil is a single genetic population not structured by host. These findings support a scenario of introduction and subsequent adaptation to solanaceous hosts that should be taken into consideration to improve mitigation and management of M. perniciosa.     

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.     

Analysis of molecular diversity in <Emphasis Type="Italic">Crinipellis perniciosa</Emphasis> with AFLP markers

Crinipellis perniciosa causes a serious disease of cacao known as witches broom (WB). Heritable resistance to witches broom has been used in cacao improvement programs. SCA6 and SCA12 are highly resistant and are the most commonly used parents in the breeding schemes. However, SCA hybrids are not resistant to witches broom in all production areas. Presumably, different populations of C. perniciosa cause these variable responses. Amplified fragment length polymorphism (AFLP) markers were used to assess variation and population structure in this pathogen. We examined 40 isolates of C. perniciosa and one isolate of Melanotus subcuneiformis. Nine of 64 primer pairs produced consistent and informative DNA amplification, and were used to screen all isolates. Fifteen haplotypes (AFLP fingerprints) were detected with 186 polymorphic markers. Cluster analysis grouped isolates of the C biotype (pathogenic on cacao) from Bolivia, Brazil, Ecuador and Trinidad together in a major cluster that was distinct from isolates of the S biotype (pathogenic on solanaceous hosts) and M. subcuneiformis. Isolates of the C biotype were divided further into well supported, country-specific groups. Segregation of AFLP alleles was not observed among basidiospore isolates from the same basidiome, broom, tree or field, supporting previous reports that the fungus did not outcross. The results corroborated prior conclusions that C. perniciosa was probably introduced into the Bahia state of Brazil from the Amazon basin. Representative isolates from the genetically distinct groups that were revealed will be used to examine pathogenic specialization in C. perniciosa and differential responses that have been reported in SCA6-derived germplasm.     

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.     

Activity of polygalacturonases from Moniliophthora perniciosa depends on fungus culture conditions and is enhanced by Theobroma cacao extracts

We report the first analysis of polygalacturonase regulation in the basidiomycete Moniliophthora perniciosa. Non-secreted and secreted polygalacturonase activity was obtained from M. perniciosa cultivated on bran-based solid medium or liquid media containing additional carbon sources or cacao extracts (infected or not by the fungus). Polygalacturonase activity assays were carried out under different temperatures and incubation periods. The best secreted polygalacturonase activity was obtained when the enzymatic assay was made at 50 °C for 10 min. Moreover, the polygalacturonase activity was enhanced when the fungus was cultivated on potato dextrose medium, in the presence of additional fermentable carbon sources, in the presence of cacao pulp or non infected cacao extracts.