Molecular and phenotypic analyses reveal association of diverse Colletotrichum acutatum groups and a low level of C. gloeosporioides with olive anthracnose

Anthracnose (Colletotrichum spp.) is an important disease causing major yield losses and poor oil quality in olives. The objectives were to determine the diversity and distribution pattern of Colletotrichum spp. populations prevalent in olives and their relatedness to anthracnose pathogens in other hosts, assess their pathogenic variability and host preference, and develop diagnostic tools. A total of 128 Colletotrichum spp. isolates representing all olive-growing areas in Portugal and a few isolates from other countries were characterized by molecular and phenotypic assays and compared with reference isolates. Arbitrarily primed PCR data, internal transcribed spacer of rRNA gene and beta-tubulin 2 nucleotide sequences, colony characteristics, and benomyl sensitivity showed Colletotrichum acutatum to be dominant (>97%) with limited occurrence of Colletotrichum gloeosporioides (<3%). Among C. acutatum populations, five molecular groups, A2 to A6, were identified. A2 was widely prevalent (89%), coinciding with a high incidence of anthracnose and environmental conditions suitable to disease spread. A4 was dominant in a particular region, while other C. acutatum groups and C. gloeosporioides were sporadic in their occurrence, mostly related to marginal areas of olive cultivation. C. gloeosporioides, isolated from olive fruits with symptoms indistinguishable from those of C. acutatum, showed same virulence rating as the most virulent C. acutatum isolate from group A2. C. acutatum and C. gloeosporioides isolates tested in infected strawberry fruits and strawberry and lupin plants revealed their cross-infection potential. Diagnostic tools were developed from beta-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of C. acutatum groups.     

The distinctive population structure of Colletotrichum species associated with olive anthracnose in the Algarve region of Portugal reflects a host–pathogen diversity hot spot

Anthracnose ( Colletotrichum spp.) is an important disease of olive fruits. Diversity and biogeographic relationships of the olive anthracnose pathogens in the Algarve (Portugal) were investigated, along with host association patterns and disease levels during 2004–2007, to test the hypothesis that this region is a host–pathogen diversity hot spot. Diverse Colletotrichum acutatum and Colletotrichum gloeosporioides populations were identified based on rRNA-internal transcribed spacer and partial β-tubulin 2 gene sequences of 95 isolates. Spatial and temporal variations in the occurrence of the eight genetic entities of the pathogens were linked to olive biogeography. Disease occurrence patterns suggest that C. acutatum populations are more stable pathogens, while C. gloeosporioides populations appear to be more influenced by favourable conditions. Three unique C. acutatum populations were identified, but none of the eight populations were dominant, with the most frequent type representing only 27%. Thus, the population structure of olive anthracnose pathogens in the Algarve is distinct from other parts of Portugal and other world locations, where only one or two genetic entities are dominant. This pattern and level of genetic diversity in a restricted area, where oleaster (wild olive tree), ancient landraces and modern cultivars of olive occur in close proximity, suggests the Algarve as a centre of diversity of the anthracnose pathogens and corroborates recent work suggesting western Mediterranean as an important centre of olive diversity and domestication.     

Molecular assays reveal the presence and diversity of genes encoding pea footrot pathogenicity determinants in Nectria haematococca and in agricultural soils

Aim:  The aim of this study was to develop molecular assays for investigating the presence and diversity of pathogenicity genes from the pea footrot pathogen Nectria haematococca (anamorph Fusarium solani f.sp. pisi ) in soils.
Methods and Results:  Polymerase chain reaction (PCR) assays were developed to amplify four N. haematococca pathogenicity genes ( PDA , PEP1 , PEP3 and PEP5 ) from isolates and soil-DNA from five agricultural fields with a prior footrot history. A collection of 15 fungi isolated on medium selective for Fusarium spp. exhibited variation in their virulence to peas as assessed via a disease index (DI: 0–5; no virulence to the highest virulence). PCR analyses showed that three isolates in which all four pathogenicity genes were detected resulted in the highest DI (>3·88). All four pathogenicity genes were detected in soil-DNA obtained from all five fields with a footrot disease history, but were not amplified from soils, which had no footrot history. Denaturing gradient gel electrophoresis and/or sequence analysis revealed diversity amongst the pathogenicity genes.
Conclusion:  The PCR assays developed herein enable the specific detection of pathogenic N. haematococca in soils without recourse to culture.
Significance and Impact of the Study:  Molecular assays that specifically target pathogenicity genes have the capacity to assess the presence of the footrot-causing pathogen in agricultural soils.     

Genetic and Phenotypic Diversity among Botrytis cinerea Isolates in Iran

Forty-four Botrytis cinerea isolates from different hosts and geographical regions were studied for colony morphology, mycelial growth rate at different temperatures, pathogenicity and molecular diversity. Botrytis cinerea isolates had temperature optima of 20–25°C and isolates showed variation in growth rate at different temperatures. Two morphological types were identified among tested isolates: mycelial and sclerotial. The pathogenicity of isolates was tested on grapevine leaves, and it was revealed that nine of 44 isolates were non-pathogenic and among them seven were of mycelial type. There was no correlation between mycelium growth rate and pathogenicity. Genetic diversity was investigated using nine arbitrary decaprimers. No relationship was found between molecular clusters and geographical region or sampling time; whereas isolates from the same plant host tended to cluster with each other. Seven of nine non-pathogenic isolates were separated from pathogenic ones.     

A Reservoir of Drug-Resistant Pathogenic Bacteria in Asymptomatic Hosts

The population genetics of pathogenic bacteria has been intensively studied in order to understand the spread of disease and the evolution of virulence and drug resistance. However, much less attention has been paid to bacterial carriage populations, which inhabit hosts without producing disease. Since new virulent strains that cause disease can be recruited from the carriage population of bacteria, our understanding of infectious disease is seriously incomplete without knowledge on the population structure of pathogenic bacteria living in an asymptomatic host. We report the first extensive survey of the abundance and diversity of a human pathogen in asymptomatic animal hosts. We have found that asymptomatic swine from livestock productions frequently carry populations of Salmonella enterica with a broad range of drug-resistant strains and genetic diversity greatly exceeding that previously described. This study shows how agricultural practice and human intervention may lead and influence the evolution of a hidden reservoir of pathogens, with important implications for human health.     

<Emphasis Type="Italic">Ganoderma </Emphasis>diseases of perennial crops in India – an overview

The species of Ganoderma recorded from India as causing diseases of perennial crops are listed, and their host range and taxonomy discussed. Four new hosts of G. lucidum are also reported. A decline in productivity and the death of trees are the main economic impacts due to Ganoderma diseases, and the fungus is identified as a serious pathogen of cash crops, forest plantations and trees in natural forests in the country. Ganoderma diseases have been recorded on 144 hosts in India, the major pathogens being G. lucidum and G. applanatum. G. lucidum has been recorded on 91 hosts, and appears to cause the most widespread diseases. Identification has largely been made from morphological and cultural characters, and the names currently in use should therefore be treated with caution. Cultural methods of disease control are largely inefficient in minimising inoculum pressure and in reducing the disease incidence. Chemical methods in combination with soil amendments form short-term solutions for managing the disease and improving productivity. The immediate priorities for developing an efficient management system for Ganoderma diseases in India are: (1) a thorough understanding of the etiology and epidemiology of the diseases on different hosts, (2) clarifying current ambiguity in species names, (3) assessing the inter-relationships between populations of Ganoderma on different hosts and (4) developing tools for early detection of diseases in important crops.     

Cladosporium fulvum (syn. Passalora fulva), a highly specialized plant pathogen as a model for functional studies on plant pathogenic Mycosphaerellaceae

Taxonomy:  Cladosporium fulvum is an asexual fungus for which no sexual stage is currently known. Molecular data, however, support C. fulvum as a member of the Mycosphaerellaceae, clustering with other taxa having Mycosphaerella teleomorphs . C. fulvum has recently been placed in the anamorph genus Passalora as P. fulva . Its taxonomic disposition is supported by its DNA phylogeny, as well as the distinct scars on its conidial hila, which are typical of Passalora , and unlike Cladosporium s.s. , which has teleomorphs that reside in Davidiella , and not Mycosphaerella .
Host range and disease symptoms:  The presently known sole host of C. fulvum is tomato (members of the genus Lycopersicon ). C. fulvum is mainly a foliar pathogen. Disease symptoms are most obvious on the abaxial side of the leaf and include patches of white mould that turn brown upon sporulation. Due to stomatal clogging, curling of leaves and wilting can occur, leading to defoliation.
C. fulvum as a model pathogen:  The interaction between C. fulvum and tomato is governed by a gene-for-gene relationship. A total of eight Avr and Ecp genes, and for four of these also the corresponding plant Cf genes, have been cloned. Obtaining conclusive evidence for gene-for-gene relationships is complicated by the poor availability of genetic tools for most Mycosphaerellaceae – plant interactions. Newly developed tools, including Agrobacterium -mediated transformation and RNAi, added to the genome sequence of its host tomato, which will be available within a few years, render C. fulvum attractive as a model species for plant pathogenic Mycosphaerellaceae.
Useful websites:  http://www.sgn.cornell.edu/help/about/index.html ; http://cogeme.ex.ac.uk     

Context-dependent transmission of a generalist plant pathogen: host species and pathogen strain mediate insect vector competence

The specificity of pathogen–vector–host interactions is an important element of disease epidemiology. For generalist pathogens, different pathogen strains, vector species, or host species may all contribute to variability in disease incidence. One such pathogen is Xylella fastidiosa Wells et al., a xylem-limited bacterium that infects dozens of crop, ornamental, and native plants in the USA. This pathogen also has a diverse vector complex and multiple biologically distinct strains. We studied the implications of diversity in this pathogen–vector–host system, by quantifying variability in transmission efficiency of different X. fastidiosa strains (isolates from almond and grape genetic groups) for different host plants (grape, almond, and alfalfa) by two of the most important vectors in California: glassy-winged sharpshooter [ Homalodisca vitripennis (Germar)] and green sharpshooter ( Draeculacephala minerva Ball) (both Hemiptera: Cicadellidae). Transmission of isolates of the almond strain by H. vitripennis did not differ significantly, whereas transmission varied significantly among isolates from the grape strain (15–90%). Host plant species did not affect H. vitripennis transmission. Conversely, D. minerva efficiency was mediated by both host plant species and pathogen strain. No acquisition of an almond isolate occurred regardless of plant type (0/122), whereas acquisition of a grape isolate from alfalfa was 10-fold higher than from grape or almond plants. These results suggest that pathogen, vector, and host diversity impose contingencies on the transmission ecology of this complex disease system. Studies aimed at the development of management strategies for X. fastidiosa diseases should consider the complexity of these interactions as they relate to disease spread.     

Climate change accelerates local disease extinction rates in a long‐term wild host–pathogen association

Pathogens are a significant component of all plant communities. In recent years, the potential for existing and emerging pathogens of agricultural crops to cause increased yield losses as a consequence of changing climatic patterns has raised considerable concern. In contrast, the response of naturally occurring, endemic pathogens to a warming climate has received little attention. Here, we report on the impact of a signature variable of global climate change – increasing temperature – on the long‐term epidemiology of a natural host–pathogen association involving the rust pathogen Triphragmium ulmariae and its host plant Filipendula ulmaria. In a host–pathogen metapopulation involving approximately 230 host populations growing on an archipelago of islands in the Gulf of Bothnia we assessed changes in host population size and pathogen epidemiological measures over a 25‐year period. We show how the incidence of disease and its severity declines over that period and most importantly demonstrate a positive association between a long‐term trend of increasing extinction rates in individual pathogen populations of the metapopulation and increasing temperature. Our results are highly suggestive that changing climatic patterns, particularly mean monthly growing season (April‐November) temperature, are markedly influencing the epidemiology of plant disease in this host–pathogen association. Given the important role plant pathogens have in shaping the structure of communities, changes in the epidemiology of pathogens have potentially far‐reaching impacts on ecological and evolutionary processes. For these reasons, it is essential to increase understanding of pathogen epidemiology, its response to warming, and to invoke these responses in forecasts for the future.     

Clonality in South African isolates and evidence for a European origin of the root pathogen Thielaviopsis basicola

Thielaviopsis basicola is a soil-borne fungal pathogen with a wide host range and a cosmopolitan distribution. It causes disease on many agricultural crops, and in South Africa is the causal agent of black pod rot of groundnuts and black root rot on chicory. Knowledge of the population diversity of T. basicola could provide valuable information regarding management strategies, the possible movement, origin, and reproductive strategies of the fungus. The objective of this study was to determine the population diversity of T. basicola isolates from groundnuts and chicory in South Africa using co-dominant polymorphic markers. These markers were also used to compare isolates from South Africa with those from other hosts and geographic regions. Seven loci revealed nine alleles and two genotypes, one on groundnut and one on chicory, differing at only two loci. T. basicola isolates from eight different countries and ten different hosts revealed 17 genotypes across the seven loci with 39 different alleles. The lack of diversity for the two South African host-related populations of isolates suggests that T. basicola was introduced into South Africa. Some evidence is provided for a European origin of the pathogen, possibly linked to trade in root crops.     

The Evolution of Resistance to Simian Immunodeficiency Virus (SIV): A Review

Examining how pathogens cross species boundaries, spread within species, and persist within their hosts is an essential part of understanding the factors that underpin the evolution of virulence and host resistance. Here, we review current knowledge about the genetic diversity, molecular epidemiology, prevalence, and pathogenicity of simian immunodeficiency viruses (SIVs). SIVs have crossed species boundaries from simian hosts to humans on at least 12 separate occasions, one of which led to the global HIV–AIDS crisis. Though SIVs infect a wide range of primates, scientists have only recently begun to describe the natural history of SIV infection in their natural hosts. Several new studies reveal how both viral and host factors are responsible for the transmission to, and adaptation in, new hosts. These studies also suggest that the spread of the virus may be affected by host-specific traits, including social structure, mating system and demographic history. These studies challenge the traditional view that SIV is relatively benign in its natural host, and instead suggest that a highly dynamic relationship exists between SIV and its simian hosts.     

A Single,Plastic Population of Mycosphaerella pinodes Causes Ascochyta Blight on Winter and Spring Peas (Pisum sativum) in France

Plant diseases are caused by pathogen populations continuously subjected to evolutionary forces (genetic flow, selection, and recombination). Ascochyta blight, caused by Mycosphaerella pinodes, is one of the most damaging necrotrophic pathogens of field peas worldwide. In France, both winter and spring peas are cultivated. Although these crops overlap by about 4 months (March to June), primary Ascochyta blight infections are not synchronous on the two crops. This suggests that the disease could be due to two different M. pinodes populations, specialized on either winter or spring pea. To test this hypothesis, 144 pathogen isolates were collected in the field during the winter and spring growing seasons in Rennes (western France), and all the isolates were genotyped using amplified fragment length polymorphism (AFLP) markers. Furthermore, the pathogenicities of 33 isolates randomly chosen within the collection were tested on four pea genotypes (2 winter and 2 spring types) grown under three climatic regimes, simulating winter, late winter, and spring conditions. M. pinodes isolates from winter and spring peas were genetically polymorphic but not differentiated according to the type of cultivars. Isolates from winter pea were more pathogenic than isolates from spring pea on hosts raised under winter conditions, while isolates from spring pea were more pathogenic than those from winter pea on plants raised under spring conditions. These results show that disease developed on winter and spring peas was initiated by a single population of M. pinodes whose pathogenicity is a plastic trait modulated by the physiological status of the host plant.     

Pseudomonas viridiflava, a multi host plant pathogen with significant genetic variation at the molecular level

The pectinolytic species Pseudomonas viridiflava has a wide host range among plants, causing foliar and stem necrotic lesions and basal stem and root rots. However, little is known about the molecular evolution of this species. In this study we investigated the intraspecies genetic variation of P. viridiflava amongst local (Cretan), as well as international isolates of the pathogen. The genetic and phenotypic variability were investigated by molecular fingerprinting (rep-PCR) and partial sequencing of three housekeeping genes (gyrB, rpoD and rpoB), and by biochemical and pathogenicity profiling. The biochemical tests and pathogenicity profiling did not reveal any variability among the isolates studied. However, the molecular fingerprinting patterns and housekeeping gene sequences clearly differentiated them. In a broader phylogenetic comparison of housekeeping gene sequences deposited in GenBank, significant genetic variability at the molecular level was found between isolates of P. viridiflava originated from different host species as well as among isolates from the same host. Our results provide a basis for more comprehensive understanding of the biology, sources and shifts in genetic diversity and evolution of P. viridiflava populations and should support the development of molecular identification tools and epidemiological studies in diseases caused by this species.     

Forest species diversity reduces disease risk in a generalist plant pathogen invasion

Empirical evidence suggests that biodiversity loss can increase disease transmission, yet our understanding of the 'diversity-disease hypothesis' for generalist pathogens in natural ecosystems is limited. We used a landscape epidemiological approach to examine two scenarios regarding diversity effects on the emerging plant pathogen Phytophthora ramorum across a broad, heterogeneous ecoregion: (1) an amplification effect exists where disease risk is greater in areas with higher plant diversity due to the pathogen's wide host range, or (2) a dilution effect where risk is reduced with increasing diversity due to lower competency of alternative hosts. We found evidence for pathogen dilution, whereby disease risk was lower in sites with higher species diversity, after accounting for potentially confounding effects of host density and landscape heterogeneity. Our results suggest that although nearly all plants in the ecosystem are hosts, alternative hosts may dilute disease transmission by competent hosts, thereby buffering forest health from infectious disease.     

Intercontinental variation of Sclerospora graminicola

Collections of S. graminicola were made from local pearl millet crops in West Africa and India. Asexual inoculum was derived from the collections in Polythene tunnels and used to infect pearl millet cultivars from both continents. Analysis of deviance of the incidence of disease was performed on the logit scale using GLIM. The host × pathogen interactions were interpreted by use of the Finlay-Wilkinson regression technique widely used in the study of genotype × environment interactions. While the West African collections were consistently more pathogenic than the Indian ones, West African hosts were potentially more susceptible to Indian than to West African pathogen isolates; conversely some Indian hosts were more vulnerable to West African than to Indian isolates. There was a fairly stable relationship between incidence of disease and relative pathogenicity of West African isolates but the Indian isolates were more variable in their susceptibility to changing background levels of disease. These results could influence resistance breeding strategies in pearl millet improvement programmes. The methods of analysis could be valuable for application to other complex and ill-defined pathosystems.     

Host Specificity of Pathogenic Pythium Species: Implications for Tree Species Diversity

In the Janzen–Connell hypothesis, host-specific natural enemies enhance species diversity and influence the structure of plant communities. This study tests the explicit assumption of host specificity for soil pathogens of the genus Pythium that cause damping-off disease of germinating seeds and seedlings. We isolated Pythium spp. from soil of a tropical forest in Panama. Then, in an inoculation experiment, we determined the pathogenicity of 75 tropical isolates of unknown pathogenicity and seven pathogenic temperate isolates of Pythium on seeds and/or seedlings of eight tropical tree species. Only three tropical isolates, one identified as P. ultimum and two as P. aphanidermatum , were pathogenic. Tropical pathogenic isolates were pathogenic on 4–6 of eight tree species. Temperate isolates were pathogenic on 0–4 of eight species, indicating that some tropical tree species are susceptible to novel isolates of Pythium . No tree species was susceptible to all isolates and two species were not susceptible to any isolate. Collectively, these results indicate that these Pythium isolates vary widely in their pathogenicity, causing differential mortality of potential host species; likewise, the tree species vary in their susceptibility to a given Pythium isolate. These differences in pathogenicity and susceptibility indicate some support for the Janzen–Connell assumption of host specificity. While they are not restricted to a single species, their intermediate level of specificity suggests that Pythium spp. have the potential to have some effect on forest community structure and diversity.     

Intraspecies diversity of Macrophomina phaseolina in Iran

To study the pathogenic and genetic diversity of the Macrophomina phaseolina in Iran, 52 isolates of the fungus were isolated from 24 host plants across the 14 Iranian provinces. All isolates were confirmed to the species based on the species-specific primers. The aggressiveness of M. phaseolina isolates was evaluated on the common bean. Based on the pathogenicity tests, M. phaseolina isolates from the different hosts displayed different levels of aggressiveness on the common beans. The results showed that there was significant variation in the aggressiveness of the pathogen; however, there was no distinct pattern of differentiation based on the host or geographical origin linked to the virulence of the isolates, as frequently theisolates from the same host or geographical origin had different levels of aggressiveness. Inter-simple sequence repeat (ISSR) markers were used to assess the genetic diversity of the fungus. The unweighted pair-group method, using arithmetic mean clustering of data, showed that isolates did not clearly differentiate to the specific group according to the host or geographical origins; however, usually the isolates from the same host or the same geographical origin tend to group nearly. Our results did not show a correlation between the genetic diversity based on the ISSR and pathogenic patterns on common bean in the greenhouse. Similar to the M. phaseolina populations in the other countries, the Iranian isolates were highly diverse based on the pathogenic and genotypic characteristics.     

Agrobacterium-Mediated Transformation and Insertional Mutagenesis in <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> for Investigating Varied Pathogenicity Lifestyles

Colletotrichum acutatum is a cosmopolitan pathogen causing economically important diseases known as anthracnose on a wide range of hosts. This fungus exhibits varied pathogenicity lifestyles and the tools essential to understand the molecular mechanisms are still being developed. The transformation methods currently available for this species for gene discovery and functional analysis involve protoplast transformation and are laborious and inefficient. We have developed a protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of C. acutatum. Using this protocol we were able to transform C. acutatum isolates belonging to different genetic groups and originating from different hosts. The transformation efficiency was up to 156 transformants per 10⁴ conidia, with >70% transformants showing single location/single copy integration of T-DNA. Binary vector pBHt2-GFP was constructed, enabling green fluorescence protein tagging of C. acutatum strains, which will be a useful tool for epidemiology and histopathology studies. The ATMT protocol developed was used to identify putative pathogenicity mutants, suggesting the applicability of this technique for rapid generation of a large panel of insertional mutants of C. acutatum leading to the identification of the genes associated with the varied lifestyles.     

Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range

Plant pathogenic fungi adapt quickly to changing environments including overcoming plant disease resistance genes. This is usually achieved by mutations in single effector genes of the pathogens, enabling them to avoid recognition by the host plant. In addition, horizontal gene transfer (HGT) and horizontal chromosome transfer (HCT) provide a means for pathogens to broaden their host range. Recently, several reports have appeared in the literature on HGT, HCT and hybridization between plant pathogenic fungi that affect their host range, including species of Stagonospora/Pyrenophora, Fusarium and Alternaria. Evidence is given that HGT of the ToxA gene from Stagonospora nodorum to Pyrenophora tritici-repentis enabled the latter fungus to cause a serious disease in wheat. A nonpathogenic Fusarium species can become pathogenic on tomato by HCT of a pathogenicity chromosome from Fusarium oxysporum f.sp lycopersici, a well-known pathogen of tomato. Similarly, Alternaria species can broaden their host range by HCT of a single chromosome carrying a cluster of genes encoding host-specific toxins that enabled them to become pathogenic on new hosts such as apple, Japanese pear, strawberry and tomato, respectively. The mechanisms HGT and HCT and their impact on potential emergence of fungal plant pathogens adapted to new host plants will be discussed.     

Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility

A diverse collection of isolates identified as Colletotrichum acutatum, including a range of fruit-rot and foliar pathogens, was examined for mtDNA RFLPs and RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase (GS) gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene. RFLPs of mtDNA, RFLPs of the 900-bp GS intron and sequence analysis of each intron identified the same seven distinct molecular groups, or clades, within C. acutatum sensu lato. Sequence analysis produced highly concordant tree topologies with definitive phylogenetic relationships within and between the clades. The clades might represent phylogenetically distinct species within C. acutatum sensu lato. Mating tests also were conducted to assess sexual compatibility with tester isolates known to outcross to form the teleomorph Glomerella acutata. Mating compatibility was identified within one clade, C, and between two phylogenetically distinct clades, C and J4. The C clade represented isolates from a wide range of hosts and geographic origins. J4 clade contained isolates from Australia or New Zealand recovered from fruit rot and pine seedlings with terminal crook disease. That isolates in two phylogenetically distinct clades were capable of mating suggests that genetic isolation occurred before reproductive isolation. No other isolates were sexually compatible with the mating testers, which also were in groups C and J4. Certain clades identified by mtDNA and intron analysis (D1, J3 and J6) appeared to represent relatively host-limited populations. Other clades (C1, F1 and J4) contained isolates from a wide range of hosts. Isolates described as C. acutatum f. sp. pineum were clearly polyphyletic.