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.     

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.     

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.     

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.     

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.     

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.     

Sensitivity of Crinipellis perniciosa to two triazole fungicides in vitro and their effect on development of the fungus in cocoa

Two triazole fungicides, hexaconazole and triadimenol, were evaluated for their effects on the growth of Crinipellis perniciosa in vitro, and for their ability to prevent broom formation on cocoa seedlings. Amounts of both fungicides required to reduce fungal growth, germ tube extension and basidiospore germination by 50% were found respectively to be less than 1, 10-150 and more than 200 mg/l. Reductions in germ tube length were associated with slower rates of cell growth, whereas growth of dikaryotic mycelium was reduced due to shorter cell lengths. Isolates of C. perniciosa showed different sensitivities to the fungicides, variation between collections from different localities being greater than between collections from the same locality. Hexaconazole gave good control of the disease applied as a spray to cocoa seedlings both before and after inoculation. Triadimenol showed good activity when used as a preinoculation soil drench and in some treatments where plants became infected no necrosis of host tissue was observed. Activity was greatly reduced by drench treatments applied 10 days after inoculation, and most infected plants formed necrotic cankers.     

Inter- and intraspecific morphometric variation and characterization of Phytophthora isolates from cocoa

Difficulties in the accurate identification of the Phytophthora species responsible for black pod disease of cocoa continue to hamper effective disease control. A re-evaluation of morphological characters ( Brasier & Griffin, 1979 ) and a detailed morphometric analysis of 161 Phytophthora isolates largely associated with black pod disease of cocoa from 17 countries worldwide have shown considerable inter- and intraspecific variation. Stable and more reliable parameters for the identification of the species responsible for the disease have been determined. Colony characteristics such as pattern and growth rate on V8 agar are reasonably characteristic for the cocoa Phytophthora species, and can be used to make preliminary identification to species level. Significant sporangial character variation was found within isolates of species from the same and different sources, highlighting the difficulties in making accurate identification on the basis of raw morphological data. Pedicel length was found to be the most consistent species-linked sporangial characteristic. Cluster plots of length/breadth ratios of sporangia versus reciprocals of sporangial pedicel length clearly separated all isolates into distinct species groups ( P. capsici , P. citrophthora , P. palmivora and P. megakarya ) and can be used reliably to identify accurately those pathogens involved in black pod disease outbreaks.     

Effects of the fungus Crinipellis perniciosa, causal agent of witches' broom disease, on cell and tissue cultures of cocoa (Theobroma cacao L.)

The symptoms of witches' broom disease in cocoa, caused by the Basidiomycete fungus Crinipellis perniciosa , are pronounced swelling of the terminal and axillary buds followed in the long term by necrosis of this tissue. The direct effect of C. perniciosa on cocoa cells was examined under controlled conditions by growing primary and secondary phase cultures of the fungus separately and also with callus cultures and with cell suspensions. Both primary and secondary phase mycelium reduced growth of callus cultures by about 47% after one week compared with the controls. However, cell suspensions containing primary phase mycelium showed initial growth double that of the uninfected controls after 5 days, but then growth was reduced below that of the control and particularly when the primary phase became secondary phase mycelium. This change in fungal development coincided with the time that the cell culture reached the stationary growth stage. Cell cultures inoculated with stationary phase mycelium showed the same growth as the control after 5 days but then growth was reduced to 50% of the control after 19 days incubation and remained at this low level subsequently. The inhibitory effect of secondary phase mycelium was examined by incubating callus and cell suspensions with culture filtrate from liquid cultures of the secondary phase. Inclusion of 50% by volume of culture filtrate from the secondary phase in the growth medium for callus and cell suspensions, respectively, resulted in a reduction in growth of the plant tissue cultures. Addition of fungal culture filtrates also led to loss in potassium and loss of viability of cell suspensions and of isolated cells as represented by protoplasts. The necrotrophic mode of the secondary phase may be achieved through the production of phytotoxins acting on the host cell membrane.     

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.     

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.     

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.     

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.     

Cocoa production in Ecuador in relation to dry-season escape from pod rot caused by Crinipellis perniciosa and Moniliophthora roreri

Cropping and disease patterns were observed over a period of 4 years in individual cocoa trees at three contrasting sites near Quevedo, Ecuador, with the aim of evaluating the importance of dry season production and the consequent avoidance of pod disease caused by Crinipellis perniciosa and Moniliophthora roreri. Distinction of the diseases on necrotic pods was aided by splitting open pods after removal from the tree. The year was divided into wet season (April to September) and dry season (October to March) harvest periods. The 3-month lag between the actual wet and dry seasons and these harvest periods allowed time for symptom development. Combined disease losses ranged from 19·0% to 62·1 % at the various sites. At all sites and in each year, more ripe healthy pods were harvested in the dry season harvest period than in the wet season harvest period. In general, pod losses caused by C, perniciosa were curtailed more sharply by the dry season than those caused by M. roreri , which at one site caused as much damage in the dry season as in the wet season. The proportion of the annual production falling in the dry season harvest period varied among sites and between years at a given site. A comparison of trees from two progenies growing side by side showed consistent differences in cropping patterns, and identified certain trees that were productive, yielded consistently in the dry season harvest period, and were little affected by M, roreri. Analysis of long-term rainfall data for the Quevedo region indicated that years which lack a normal dry season do occur, but only once every 10 years on average. Further exploitation of disease escape through selection and breeding appears to be both feasible and appropriate.     

Limited morphological,physiological and genetic diversity of Phytophthora palmivora from cocoa in Papua New Guinea

In Papua New Guinea (PNG) cocoa (Theobroma cacao) is one of the most important cash crops grown in the tropical lowland and island regions. As in most cocoa‐growing areas, phytophthora black pod and canker cause significant yield losses. Cocoa breeding activities in PNG are focused in East New Britain province where disease control recommendations are also developed. This study tested the hypothesis that there was no diversity in the Phytophthora palmivora population causing black pod on cocoa by characterizing the variation in pathogen populations within and between the five major cocoa‐growing areas. Diseased pods were sampled hierarchically from the five locations and additional isolates were collected from soil, stem and leaf lesions, or retrieved from culture collections. Morphological characters showed continuous variation within the range described for P. palmivora. Genetic analysis revealed that the isolates belonged to one dominant clonal lineage, with restricted distributions of several other subpopulations. Lowest diversities were found in the geographically isolated Karkar Island and East Sepik province. Soil isolates showed greater genetic diversity than isolates from cocoa lesions. Intra‐farm variation was as much as inter‐farm or inter‐province variation. Both mating types were detected, although no strong evidence of sexual recombination was observed. The analysis revealed limited geographic, temporal or host specialization, suggesting continuous selection for pathogenicity from a genetic pool of P. palmivora. These findings have significant implications on the deployment of cocoa genotypes, enforcement of inter‐province quarantine and sustainable disease management strategies.     

Characteristics of Pestalotiopsis Associated with Hardy Ornamental Plants in the UK

Pestalotiopsis isolates obtained from the foliage, stem-base and roots of hardy ornamentals grown on commercial nurseries in the UK were identified and characterised according to pathogenicity and colony morphology. All 18 isolates were identified as Pestalotiopsis sydowiana on the basis of conidia morphology, and confirmation of identification was made by experts at CABI Bioscience. Isolates were pathogenic on the host from which originally isolated. Typical symptoms included foliar browning of foliage and stems, and the presence of black or greenish-black acervuli on diseased tissue. Isolates were not host specific and infected other species of hardy ornamentals. Three colony types on potato dextrose agar were distinguished according to colour and production of acervuli by individual isolates.Three selected isolates of P. sydowiana were characterised by examining the effects of growth media, temperature, pH, and water potential on hyphal extension. Isolates grew well on commonly used growth media, including PDA, Sabouraud dextrose agar (SDA), V8 juice agar (V8), malt extract agar (MEA) and Czapek Dox agar (CDA). The optimum temperature for growth on PDA was in the range 20–25°C, with little or no growth occurring below 5°C or above 30°C. Hyphal extension occurred over a pH range between 2.6–8.6, with optimum values occurring at pH 5.5. In general, decreases in osmotic and matric potential caused a reduction in growth. Hyphal extension on media adjusted osmotically as NaCl ceased between –9.9 and –10.5MPa. Isolates were more tolerant of osmotic than matric potential, with no growth occurring at –6.5MPa on media adjusted with polyethylene glycol.     

Modified agar-based media for culturing Phytophthora infestans

Studies were undertaken to evaluate locally available subtrates for use in a culture medium for Phytophthora infestans (Mont.) de Bary employing a protocol similar to that used for the preparation of rye A agar. Test media preparations were assessed for growth, sporulation, oospore formation, and long-term storage of P. infestans. Media prepared from grains and fresh produce available in Thailand and Asian countries such as black bean (BB), red kidney bean (RKB), black sesame (BSS), sunflower (SFW) and sweet corn supported growth and sporulation of representative isolates compared with rye A, V8 and oat meal media. Oospores were successfully formed on BB and RKB media supplemented with ??-sitosterol. The BB, RKB, BSS and SFW media maintained viable fungal cultures with sporulation ability for 8?months, similar to the rye A medium. Three percent and 33% of 135 isolates failed to grow on V8 and SFW media, respectively.     

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.     

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.