Vegetative compatibility and genetic analysis of Colletotrichum lindemuthianum isolates from Brazil

The causal agent of common bean anthracnose, Colletotrichum lindemuthianum, has considerable genetic and pathogenic variability, which makes the development of resistant cultivars difficult. We examined variability within and between Brazilian pathotypes of C. lindemuthianum through the identification of vegetative compatibility groups (VCGs) and by RAPD analysis. Two hundred and ninety-five nit mutants were obtained from 47 isolates of various pathotypes of the fungus collected from different regions, host cultivars and years. In complementation tests, 45 VCGs were identified. Eighteen RAPD primers were employed in the molecular analyses, producing 111 polymorphic bands. Estimates of genetic similarities, determined from the Sorence-Dice coefficient, ranged from 0.42 to 0.97; the dendrogram obtained by cluster analysis revealed 18 groups of isolates. RAPD and VCG markers presented high genotypic diversity. The number of significant associations (P=0.05) between RAPD, VCG and pathogenicity markers ranged from 0 (VCG) to 80% (pathogenicity). The test of multilocus association (rd) for RAPD markers was significantly different from zero (P<0.001), suggesting linkage disequilibrium. However, the results for VCG markers show the presence of recombination mechanisms. In conclusion, RAPD markers and VCGs were useful for detecting genetic variability among isolates of C. lindemuthianum. We found considerable diversity among isolates from the same geographic origin within a short interval; this suggests rapid evolution. There is a need for further studies to elucidate the population structure of this pathogen in agro-ecosystems.     

Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.     

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.     

Genetic heterogeneity and functional properties of intestinal bifidobacteria

AIMS: The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. METHODS AND RESULTS: A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR-ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. CONCLUSIONS: Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of ribotyping with the automated RiboPrinter System for identification and genotyping of bifidobacteria was shown in the present study.     

Diversity of pseudomonads isolated from three different plant rhizospheres

AIMS: To study the diversity of the Pseudomonas populations isolated from three different plant rhizospheres, namely pearl millet, cotton and paddy, grown in saline soils along the coastline of Southern India. METHODS AND RESULTS: The Pseudomonas populations were analysed for their biochemical characters and genetic diversity using molecular tools including RAPD and PCR-RFLP. The biochemical characterization, antibiotic resistance assay and RAPD profiles revealed a largely homogeneous population. Even in PCR-RFLP restriction studies, only two groups of isolates were seen. One group was predominant in all three rhizospheres, while the other minor group consisted of salt-sensitive isolates restricted to the paddy rhizosphere alone. CONCLUSIONS: It was observed that increasing salinity caused a predominant selection of salt-tolerant species, in particular Ps. pseudoalcaligenes and Ps. alcaligenes, irrespective of the host rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has reinstated the importance of the soil over the host plant with regard to rhizosphere populations. It has also resulted in the isolation of several salt-tolerant Pseudomonas strains, which are being screened for their biological control activity against common plant pathogens of the coastal agri-ecosystem.     

中国柱花草炭疽病原菌遗传多态性的RAPD分析

在对中国柱花草炭疽病进行广泛调查和病原采样收集的基础上,利用RAPD分子标记技术对43个代表性菌株进行了基因组DNA分析,并与276份国外菌株进行了综合聚类分析。 结果表明所用8个引物的扩增片段位于0.3～2.8kb之间, 菌株间呈现显著的DNA多态性。以柱花草起源中心——南美的柱花草炭疽菌分类为基础,中国柱花草炭疽菌可划分成3大类型即Ⅱ、Ⅲ、Ⅵ类。中国菌株与来自柱花草起源中心——南美的菌株相比之下,其生物多样性和遗传变异性则相对简单。就中国菌株而言海南菌株与广西、广东菌株相比多样性较丰富, 中国柱花草胶孢炭疽菌正在出现种内遗传分化。 从聚类结果看,通常来自于同一个地理区域或同一个寄主基因型的菌株聚成一类, 即同一RAPD聚类组内的菌株通常来自于同一寄主基因型或同一地理区域。说明来自不同寄主基因型或物种的炭疽菌在遗传基因上具有专化性,而地理上隔离的国家或地区的柱花草炭疽病原菌各自具有相对独立的进化途径。     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis

We evaluated the genetic and physiological variability of Moniliophthora perniciosa obtained from healthy and diseased branches of cacao (Theobroma cacao) plants. The diversity of the isolates was evaluated by RAPD technique and by studies of virulence and exoenzyme production. The genetic variability of endophytic and pathogenic M. perniciosa was evaluated in association with pathogenicity assays. RAPD analysis showed eight genetic groups, which were not related to plant disease status (healthy versus diseased branches). Isolates from cacao were included in three groups, excluding isolates from other host plants. Pathogenicity and enzyme analysis showed that the virulence of the isolates is not related to exoenzyme production. This is the first evidence that M. perniciosa colonizes healthy parenchymatic tissues, showing that endophytic behavior may occur in this species.     

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cry1 genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.     

Polyphasic study of the genetic diversity of lactobacilli associated with 'Almagro' eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns

AIMS: The goal of this study was to assess the genetic diversity of lactic acid bacteria (LAB) from the complex natural ecosystem present in the spontaneous fermentation of 'Almagro' eggplants by a polyphasic approach based on molecular techniques. METHODS AND RESULTS: Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to 149 Lactobacillus isolates obtained from that fermentation process. Two random primers, OPL-05 and ArgDei-For, and two rare-cutting enzymes, SfiI and SmaI, chosen after preliminary testing on the basis of band intensity and distribution, were used. RAPD and PFGE generated electrophoretic patterns suitable for strain discrimination, but further discrimination was achieved when combined numerical analysis of the results from both methods and the results previously obtained by SDS-PAGE whole cell protein analysis, was carried out. The findings indicated a considerable degree of genomic diversity in the LAB microbiota studied and especially in the Lactobacillus plantarum isolates. In terms of species assignment, the polyphasic study allowed a definite and well-founded identification of 98.7% of the isolates. CONCLUSIONS: The combined numerical analysis of RAPD and PFGE patterns represented a useful tool to discriminate the diversity of the Lactobacillus strains responsible for the spontaneous fermentation of this pickle. The species identification and strain typing results from the polyphasic study were regarded as the most exact compromise yielding the fewest contradictions based on the available data. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined numerical analysis of RAPD-PCR and PFGE patterns has not yet been employed to study the genetic diversity of LAB from an ecosystem like that found in fermenting vegetables.     

Genetic variability of green citrus aphid populations from Tunisia,assessed by RAPD markers and mitochondrial DNA sequences

The green citrus aphid Aphis spiraecola (Patch) is one of the major pests of several plant species including economically important crops such as citrus. In this study, we used random amplified polymorphic DNA (RAPD) markers and mitochondrial cytochrome oxidase subunit I sequences to assess the level and distribution of genetic diversity of A. spiraecola populations reared from Rutaceae and Rosaceae in different regions in Tunisia. RAPD analysis conducted on 141 individuals with 5 primers revealed only 50 polymorphic RAPD markers, indicating a low genetic diversity that might result from the lack of sexual phase for this species in Tunisia. Analysis of molecular variance (amova ) showed that the genetic structure was not associated with geographic location or year of collection (P = 0.70 and 0.34, respectively); however, the host‐plant had a significant effect on the partitioning of the total genetic diversity (P < 0.01). Multidimensional scaling analysis indicated that the distribution of genetic variability was significantly influenced by the host‐plant with no evidence of spatial differentiation. Based on 20 barcode sequences of the mitochondrial cytochrome‐c oxidase subunit I (COI) gene, we revealed the occurrence of two haplotypes in association with the host‐plant. Results reported here suggest the occurrence of a limited gene flow between A. spiraecola populations from Rosaceae and Rutaceae and, therefore, a possible host‐race status that could be considered in the development of an integrated controlling strategy.     

Diversity of Burkholderia isolates from woodland rhizosphere environments

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.     

Isozyme and PCR-based genotyping of epidemic Phytophthora colocasiae associated with taro leaf blight

The Oomycetous fungus Phytophthora colocasiae causing leaf blight of taro is widely distributed in India. Wide geographic range or sexual recombination provides genetic differentiation within this species. To determine how genetic variation is partitioned in P. colocasiae, 14 isolates were isolated from different regions of India, where the incidence of leaf blight is great. Molecular and biochemical techniques were employed for assessing and exploiting the genetic variability among isolates of P. colocasiae. Seven polymorphic enzyme systems revealed 23 isozyme patterns, each uniquely characterised by the presence or absence of electromorphs. Further, 10 oligodeoxynucleotide primers were selected for random amplified polymorphic DNA (RAPD) assays, which resulted in 123 polymorphic bands for 10 isolates of P. colocasiae. The data were entered into a binary matrix and a similarity matrix was constructed using a DICE similarity (SD) index. A UPGMA cluster based on SD values was generated using a NTSYS computer program. Shannon's index was used to partition genetic diversity. Similarly, isozymes and RAPDs yielded high estimates of genetic variability. Genetic diversity estimates via isozyme and RAPD pattern indicated 78.26% and 100%, respectively, total diversity among populations. This type of genetic variation in P. colocasiae indicates that variation due to asexual and/or possibly infrequent sexual mechanisms is possible and that genetic differentiation has taken place as a result of geographic isolation. The presence of larger than expected RAPD variation in isolates of P. colocasiae and the presence of distinct different zymotypes among these isolates suggests that genetic recombination (or less likely hybridisation) is at least possible in this fungus and that geographic differentiation has taken place. Even isolates obtained from the same habitat have different RAPD patterns, indicating that many populations of this fungus are made up of more than one genet and that few are derived clonally.     

Characterization and taxonomic placement of Rhizoctonia-like endophytes from orchid roots

Twenty-one Rhizoctonia-like fungal strains were isolated from the roots of four terrestrial orchid species from various locations in Hong Kong. The cultural morphology, nuclear number of the hyphal cell, pore ultrastructure, and RAPD and CAPS analyses of rDNA fragments revealed that most of these isolates were associated with the genera Ceratorhiza and Epulorhiza. RAPD analysis showed the presence of genetic diversity between the isolates from different hosts and locations. The compatibility between a selection of these Ceratorhiza and Epulorhiza isolates and 14 orchid species was determined using a symbiotic germination method. The germination and development of three orchid species, Arundina chinensis, Spathoglottis pubescens, and Spiranthes hongkongensis, were strongly stimulated by the Epulorhiza isolates. Habenaria dentata was found to form symbionts successfully with a Ceratorhiza isolate.     

Genetic differentiation, recombination and clonal expansion in environmental populations of Cryptococcus gattii in India

Cryptococcus gattii is a ubiquitous eukaryotic pathogen capable of causing life-threatening infections in a wide variety of hosts, including both immunocompromised and immunocompetent humans. Since infections by C. gattii are predominantly obtained from environmental exposures, understanding environmental populations of this pathogen is critical, especially in countries like India with a large population and with environmental conditions conducive for the growth of C. gattii. In this study, we analysed 109 isolates of C. gattii obtained from hollows of nine tree species from eight geographic locations in India. Multilocus sequence typing was conducted for all isolates using nine gene fragments. All 109 isolates belonged to the VGI group and were mating type α. Population genetic analyses revealed limited evidence of recombination but unambiguous evidence for clonal reproduction and expansion. However, the observed clonal expansion has not obscured the significant genetic differentiation among populations from either different geographic areas or different host tree species. A positive correlation was observed between genetic distance and geographic distance. The results obtained here for environmental populations of C. gattii showed both similarities and differences with those of the closely related Cryptococcus neoformans var. grubii from similar locations and host tree species in India.     

Host Specificity and Genetic Diversity of Didymella bryoniae from Cucurbitaceae in Brazil

Thirty‐seven isolates of Didymella bryoniae from three Cucurbitaceae species were collected in Brazil and tested for pathogenicity to watermelon. All isolates were pathogenic but differed in aggressiveness levels. Seven representative isolates were used in cross‐pathogenicity tests against 10 cucurbitaceous hosts. Most isolates were pathogenic to most host species tested, except to Sechium edule. Among the susceptible species, Citrullus and Cucumis species were the most susceptible hosts, while pumpkin and Luffa purgans were the most resistant. Host of origin affected the pattern of aggressiveness on each host. Isolates from watermelon were very aggressive to their original host, but much less aggressive or not pathogenic at all to some Cucurbita. Two previously described random‐amplified polymorphic DNA (RAPD)‐specific primers indicated that 81% of the isolates could be classified into the so‐called RG I group, while the remaining isolates could not be classified into any of the described RG groups. All 37 isolates were further characterized by RAPD fingerprinting and compared with three US isolates representative of RG I and RG II groups. The Brazilian D. bryoniae isolates could be separated into genetically similar clusters. The majority of the isolates were grouped in cluster DB Ia, which contained only isolates of Citrullus lanatus and Cucumis melo. Two of the American isolates used as controls clustered with this group at 68% similarity level. The DB Ib cluster included three Brazilian isolates obtained from melon and watermelon and the American representative for RG II, at a lower similarity level (43%). Two isolates from watermelon clustered with one isolate from melon in a separate group (DB II), while one single isolate from pumpkin (DB III) showed the lowest genetic similarity to all other isolates. Didymella bryoniae isolates from Brazil showed, therefore, a level of genetic diversity higher than previously reported for the species. RAPD fingerprinting allowed for geographical distinction of D. bryoniae isolates but no correlation between genetic distance, aggressiveness or origin of the isolate was found.     

Characterization of Colletotrichum lindemuthianum Isolates from the State of Minas Gerais, Brazil

Anthracnose disease of common bean (Phaseolus vulgaris), caused by Colletotrichum lindemuthianum, is responsible for extensive yield losses worldwide. This pathogen is known to vary greatly in its pathogenicity. Control strategies include chemical control and, mainly, the development of resistant cultivars, taking into account the population structure of C. lindemuthianum. The objective of this study was to investigate the pathogenic and genetic diversity and population structure among C. lindemuthianum isolates collected in Minas Gerais state, Brazil. When these isolates were inoculated on 12 differential cultivars, a total of 10 races were identified within a series of 48 isolates collected in Minas Gerais, Brazil. Races 65, 81 and 73 were the most frequent races and occurred in most of the regions. This study also detected race 337, which had not been reported previously in the literature. Random amplified polymorphic DNA (RAPD) analysis performed on the same 48 isolates revealed great genetic diversity, clustering the series into five groups at a maximum similarity value of 89.6%. There was no clear relationship between the loci sampled by RAPD markers and the pathogenic characterization. Analysis of molecular variance showed that 96.06% of the variability was contained within regions and 3.94% among regions, indicating a high exchange of genetic material among the regions of the State. Most of the variability was detected within races (75.24%). The pathogenicity and RAPD assays corroborated the broad genetic diversity of the pathogen and the results have been useful in breeding for resistance to anthracnose.     

Intraspecific genetic diversity of Oenococcus oeni as derived from DNA fingerprinting and sequence analyses.

The intraspecific genetic diversity of Oenococcus oeni, the key organism in the malolactic fermentation of wine, has been evaluated by random amplified polymorphic DNA (RAPD), ribotyping, small-plasmid content, and sequencing of RAPD markers with widespread distribution among the strains. Collection strains representing the diversity of this species have been studied together with some new isolates, many of which were obtained from wines produced by spontaneous malolactic fermentation. The RAPD profiles were strain specific and discerned two main groups of strains coincident with clusters obtained by macrorestriction typing in a previous work. Ribotyping and the conservation of RAPD markers indicates that O. oeni is a relatively homogeneous species. Furthermore, identical DNA sequences of some RAPD markers among strains representative of the most divergent RAPD clusters indicates that O. oeni is indeed a phylogenetically tight group, probably corresponding to a single clone, or clonal line of descent, specialized to grow in the wine environment and universally spread.     

Biochemical and molecular characteristics of indigenous strains of the entomopathogenic fungus Beauveria bassiana of Central India

Forty-eight isolates of indigenous strains of Beauveria bassiana from various insect hosts collected from Central India were characterised by employing protease zymography and RAPD analysis. Results of protease zymographic profiles were reproducible and significant enough to contribute to the biochemical diversity of this species. RAPD analysis revealed the presence of high level of genetic diversity and indicated that 0-66% significant differences has evolved between these isolates. The sets of amplified bands showing identical pattern to others were grouped at 100% similarity level. A wide range in the value (0.25-1.00) of Jaccard similarity coefficient was observed among all the isolates. The grouping of the indigenous strains, obtained from numerical analysis of these data, appears to be related to the host specificity in B. bassiana. Clear groups were seen for strains isolated from Lepidopteran and Coleopteran insect hosts.     

High gene flow and outcrossing within populations of two cryptic fungal pathogens on a native and non-native host in Cameroon

In this study, we determined the genetic diversity of 126 isolates representing both Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae, collected from Theobroma cacao and Terminalia spp. in Cameroon, using simple sequence repeat (SSR) markers. SSR alleles showed clear genetic distinction between L. theobromae and L. pseudotheobromae, supporting their earlier separation as sister species. Both L. theobromae and L. pseudotheobromae populations from Cameroon had high levels of gene diversity, moderate degrees of genotypic diversity, and high levels of gene flow between isolates from T. cacao and Terminalia spp. There was no evidence for geographic substructure in these populations across the region studied, and the SSR alleles were randomly associated in both species, suggesting outcrossing. The significant levels of aggressiveness, evolutionary potential represented by high levels of diversity, outcrossing and gene flow between geographically and host defined populations, identify these fungi as high-risk pathogens for their native and non-native hosts in Cameroon.