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.     

Assessing resistance to Crinipellis perniciosa using cocoa callus

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

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.     

Factors influencing the production of basidiocarps and the deposition and germination of basidiospores of Crinipellis perniciosa, the causal fungus of witches' broom on cocoa (Theobroma cacao)

The production of basidiocarps by Crinipellis perniciosa on detached, dead witches'brooms from cocoa was assessed in relation to temperature, light, cocoa clone, age of broom and type of tissue, in cabinets with a daily cycle of 8 h wet and 16 h dry. More basidiocarps formed and matured at 20–25°C than at 25–30°C. In the latter regime the pilei were smaller and white, instead of the usual crimson colour, and the stipes were longer. No basidiocarps formed at 30–35°C. At 20–25°C. more basidiocarps formed and matured with light at 100 μE m^-2 s^-1 during the wet period than at 10 μE m^-2 s ^-1. Only one basidiocarp and five primordia developed on 20 brooms kept in the dark. Brooms from 10 cocoa clones at Pichilingue. Ecuador, differed in basidiocarp productivity. most basidiocarps forming on brooms from Seavina and least on ICS clones. The numbers of basidiocarps produced on brooms aged 1.2.3 or 4 months when detached from cocoa trees were similar but time to initiation of the first primordium differed considerably. More basidiocarps formed at nodes than internodes.
The discharge of basidiospores was optimal at 20–25°C and 80% RH: germination was optimal in water agar films. Neither process was dependent on light.     

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.     

Witches' broom disease on cocoa in Rondonia, Brazil: basidiocarp production on detached brooms in the field

Fruiting of Crinipellis perniciosa was assessed in relation to various climatic factors using dead witches' brooms detached from cocoa, either suspended in the canopy or laid on leaf litter under cocoa. Induction of basidiocarp production occurred over approximately 60 days with rain. Basidiocarps were finally observed on about 85% of brooms in all samples examined and induction of fruiting required at least 17 rainy days, although brooms with a minimum age of 2 years produced basidiocarps about 25 days before brooms with a minimum age of 1 year. Groups of brooms showed distinct cycles of basidiocarp production, and up to 50% of fruiting occurred on dead, attached leaves, Fruiting was reduced on brooms on leaf litter and basidiocarps survived for less time than when brooms were suspended in canopy. Brooms were most productive with moderate amounts of wetness per day, and less than 4 h or more than 20h was inhibitory. No fruiting occurred at mean air temperatures higher than 30°C or lower than 20°C. The microclimate of the litter layer is discussed in relation to reduction of fruiting caused by pruning of brooms.     

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.     

Comparison of Crinipellis perniciosa isolates from Brazil by ERIC repetitive element sequence-based PCR genomic fingerprinting

Fifty isolates of Crinipellis perniciosa originating from Theobroma cacao , Heteropterys acutifolia and Solanum lycocarpum , from six states within Brazil, were characterized through ERIC-PCR, representing the first application of this method for molecular characterization within C. perniciosa . Phenetic analysis of banding patterns revealed a separation of isolates on the basis of host of origin, with T. cacao -derived isolates showing only a 0·2 similarity level to a cluster comprising the isolates from H. acutifolia and S. lycocarpum . Considerable intraspecific variability was observed within C. perniciosa isolates from T. cacao , with distinct groups observed correlating with geographical origin. Given that a number of isolates from T. cacao from the Amazon region grouped with isolates from Bahia state, this work discusses the possibility that current C. perniciosa populations pathogenic on T. cacao in Bahia originated from the Amazon region, rather than from alternative host plants.     

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.     

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.     

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.     

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

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

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.     

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.     

Biochemical and pathogenic differences between Kenyan and Brazilian isolates of pseudomonas syringae pv. garcae

Isolates of Pseudomonas syringae pv. garcae from Kenya and Brazil differed in pathogenic and biochemical characters. In inoculations on Coffea arabica var. SL28 from Kenya, only the Kenyan isolates were virulent. The Kenyan isolates were not bacteriocin producers while the Brazilian isolates were active producers comparable to P. s. syringae from lilac ( Syringa vulgaris ). Pigment production separated the two types of P. s. garcae isolates distinctly. The Kenyan isolates produced the UV fluorescent yellow-green siderophore while the Brazilian isolates produced a nonfluorescent brown diffusible pigment on King's B medium. API-20NE diagnostic kits were largely ineffective in distinguishing between biochemical reactions of P. s. garcae isolates from Kenya and Brazil or between these and P. s. syringae . Syringomycin activity on lemon and Geotrichum candidum distinguished P. s. syringae from P. s. garcae isolates. It is concluded that P. s. garcae (as represented by the seven cultures from the National Collection of Plant Pathogenic Bacteria, Harpenden, UK) exists in at least two strains, the Kenyan isolates comprising one strain while the Brazilian isolates comprise one or more distinct strains.     

Genetic Diversity in Colletotrichum gloeosporioides from Stylosanthes spp. at Centers of Origin and Utilization

ABSTRACT Using molecular markers, this work compares the genetic diversity in Colletotrichum gloeosporioides infecting species of the tropical forage legume Stylosanthes at the center of origin in Brazil and Colombia with that of Australia, China, and India, where Stylosanthes spp. have been introduced for commercial use. There was extensive diversity in the pathogen population from Brazil, Colombia, China, and India. The Australian pathogen population was least diverse probably due to its geographical isolation and effective quarantine. The extensive diversity in China and India means that threats from exotic pathogen races to Stylosanthes pastures can potentially come from countries outside the South American center of origin. In Brazil and India, both with native Stylosanthes populations, a high level of genetic differentiation in the pathogen population was associated with sites where native or naturalized host population was widely distributed. There was limited genetic diversity at germplasm evaluation sites, with a large proportion of isolates having identical haplotypes. This contrasts recent pathogenicity results for 78 of the Brazilian isolates that show hot spots of complex races are more common around research stations where host germplasm are tested, but few are found at sites containing wild host populations. For a pathogen in which the same races arise convergently from different genetic backgrounds, this study highlights the importance of using both virulence and selectively neutral markers to understand pathogen population structure.     

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.     

Witches' broom disease of cocoa in Rondonia, Brazil: infection of vegetative flushes and flower cushions in relation to host phenology

Formation of vegetative brooms and cushion infections were assessed in relation to host phenology and fructification of Crinipellis perniciosa over 2 years. Vegetative growth occurred in distinct periods or'flushes'based on an endogenous cycle of 8 weeks. Correlation analysis showed that growth patterns of the sample trees were very similar. There were four main flushes in 1984, three in 1985, and the first two in each year coincided with periods of fruiting of the fungus. Total yearly infection increased from 13% in 1984 to 32% in 1985 when there were more periods considered suitable for infection and production of basidiocarps. Many infections on young vegetative tissue produced more than one hypertrophied shoot (broom) and 11 new infections were produced from every fruiting broom in 1984 compared with 3.2 in 1985. The total number of vegetative brooms increased from about 160 per tree in September 1983 to about 650 in September 1985, despite natural broom loss of 68% every 12 months. Infection of flower buds (cushions) was related to flowering intensity and periodicity, and a small proportion of infected cushions produced brooms. The implications of these results are discussed in relation to methods of control.     

Identification of an A2 population of Phythophthora andina attacking tree tomato in Peru indicates a risk of sexual reproduction in this pathosystem

Tree tomato, Solanum betaceum, is an Andean fruit crop previously shown to be attacked by Phytophthora andina in Ecuador and Colombia. Blight‐like symptoms were discovered on tree tomato plants in the central highlands of Peru in 2003 and shown to be caused by P. andina. Isolates of P. andina, collected from three different plantations in Peru over a 6‐year time span (2003–2008), were compared genetically with P. andina isolates from Colombia and Ecuador to test whether the pathogen population is geographically structured in the Andes. Restriction fragment length polymorphism (RFLP), mitochondrial DNA and simple sequence repeat (SSR) genetic markers, and mating type behaviour indicated that the Peruvian P. andina population from tree tomato is genetically distinct from populations infecting tree tomato in Colombia (CO‐1) and Ecuador (EC‐3, Ia, A1), but is more similar to the population infecting solanaceous hosts of the Anarrhichomenum complex (EC‐2, Ic, A2). Such geographic substructuring within this pathogen species could result from spatial isolation. Most strikingly, in contrast to the Ecuadorian and Colombian P. andina isolates from tree tomato, the Peruvian isolates have the A2 mating type. The presence of both mating types in the Andean population of P. andina attacking tree tomato indicates a risk of sexual reproduction and the presence of long‐lasting oospores in this pathosystem.     

Variations in isolates of Mycogone perniciosa and in disease symptoms in Agaricus bisporus

Eight isolates of Mycogone perniciosa , five from Agaricus bisporus and three from Agaricus arvensis , were studied. One isolate of Mycogone rosae was also included. Aleuriospore and phialospore morphology varied among the isolates as did other characteristics, but M. rosae was the only isolate to produce a red colouration of the medium. Growth was also variable, with three isolates of M. pemiciosa growing at about half the rate of the fastest. The slow-growing isolates contained virus-like particles, 36 nm diameter, and produced sclerodermoid mushrooms. The fast-growing isolates did not contain virus-like particles and caused cap spotting, a symptom not previously described for M. perniciosa. M. rosae produced characteristic cap spots and no scierodermoid mushrooms. A comparison of two isolates of St. perniciosa. one from A. bisporus and one from A arvensis , showed a much greater yield reduction as a result of symptoms caused by the isolate from A. bisporus. The isolate of M. rosae had no significant effect on yield.
Restriction fragment banding patterns of ribosomal DNA showed no differences among the seven isolates of M. perniciosa from England, but the isolate from China was slightly different. The single isolate of M. rosae was distinct from M. perniciosa.