Microbotryum Violaceum Research Articles

Patterns of within population dispersal and mating of the fungus Microbotryum violaceum parasitising the plant Silene latifolia.

This study explores the patterns of dispersal and mating of the anther smut Microbotryum violaceum, a model species in genetics and evolutionary biology. A French metapopulation of the fungus collected from its caryophyllaceous host Silene latifolia was analysed using microsatellites. The genetic diversity was low, populations were strongly differentiated, and there was no pattern of isolation by distance among populations. There was a strong deficit in heterozygotes, confirming the high self-fertilisation rates suggested by previous studies. Within populations there was a strong pattern of isolation by distance, with identical genotypes being highly clustered. This indicates that fungal spores are dispersed mostly between adjacent plants, and such local dispersal is important for understanding the dynamics and evolution of this disease. Local clusters of identical heterozygous genotypes did not contain significantly fewer individuals than did clusters of homozygous genotypes. As selfing between products of independent meiotic events (intertetrad selfing) rapidly reduces heterozygosity, this suggests that intratetrad matings are frequent, which helps to explain the puzzling maintenance of a sex-ratio distortion in M. violaceum.

The Effect of the Sterilising Anther-Smut Fungus Microbotryum Violaceum on the Genetic Composition of Silene Dioica Populations

Populations of the outbreeding perennial herb Silene dioica, growing on the islands of an archipelago in the Baltic Sea, are often diseased by the sterilising anther-smut fungus Microbotryum violaceum (synonym Ustilago violaceu). Since diseased host plants are sterilised, island populations with high disease incidences experience large reductions in the number of reproductive individuals. Here, we compared the genetic composition of diseased plants at six allozyme loci with that of the remaining reproductive individuals to determine whether this reduction changes the genetic composition of S. dioica island populations. We found a significant but small difference in genetic composition between diseased and healthy samples using analyses of multiple allelic types. We discuss our results in relation to changes in genetic differentiation among island populations and the impact of disease in the archipelago.

No Evidence of Sex‐Differential Pollen Limitation at the Flower Level in the Gynodioecious Gypsophila repens Infected by Microbotryum violaceum

Because in gynodioecious species females are expected to be more pollen limited than hermaphrodites, we tested for sex-differential pollen limitation at the flower level, comparing fruit production and fecundity between open-pollinated and hand-pollinated flowers of Gypsophila repens (Caryophyllaceae) over two years. No sex difference in pollen limitation was found. Hand-pollination increased fruit and seed production in the first year of the study but not in the second, and it actually decreased fruit set in the latter year, although overall fecundity was higher in that year. Ovule number differed between years, with perfect flowers containing more ovules in the second year. Gypsophila repens harbors the anther smut fungus Microbotryum violaceum, which infects and sterilizes many species of Caryophyllaceae. In G. repens, however, some infected plants are capable of seed production, although their fecundity is far lower than that of healthy plants. Seed production was not pollen limited even with the high percentage of infected individuals that could reduce the quantity of pollen available at the population level.

Morphological development of anthers induced by the dimorphic smut fungus Microbotryum violaceum in female flowers of the dioecious plant Silene latifolia.

When inoculated with the dimorphic smut fungus Microbotryum violaceum (Pers.) G. Deml and Oberwinkler, the female flower of the dioecious plant Silene latifolia (Miller) E.H.L. Krause develops anther-like structures filled with spores instead of pollen grains. Using natural scanning electron microscopy, Nomarski interference microscopy, and fluorescence microscopy, we investigated the morphological modifications of the host plant resulting from this parasitism and the localization of smut hyphae in the flower bud. Flowers of infected plants lasted significantly longer than those of healthy plants, probably because the infection strengthened floral organs, such as the flower base and the anther filaments. Smut hyphae were observed throughout all organs of the young flower buds of infected plants, including sepals, petals, stamens, and pistil primordia. In healthy female flowers, anthers initiated sporogenous cell formation, but lacked parietal cell layers. By contrast, the parietal cell layers of infected female flowers differentiated into tapetal tissue, middle cell layers, and endothecial layers, as in the anthers of healthy male flowers. Smut spore formation in the infected anther was initiated in intercellular regions between the sporogenous cells, resulting in degeneration of premature sporogenous cells, tapetal tissue, and middle cell layers. The development of the endothecial layers and epidermis in the infected anther were morphologically normal.

Herbarium studies on the distribution of anther‐smut fungus (Microbotryum violaceum) and Silene species (Caryophyllaceae) in the eastern United States

We used herbarium specimens of Silene virginica, S. caroliniana, S. rotundifolia, and S. latifolia to survey the incidence of anther-smut disease (caused by Microbotryum violaceum sensu lato) in the eastern USA. We found no evidence of a collector bias against diseased specimens. Diseased specimens were frequently found in collections of S. virginica and S. caroliniana, but not in those of S. rotundifolia or S. latifolia. Disease incidence in S. virginica and S. caroliniana increased significantly over the past century and was higher in marginal populations. The absence of disease in specimens of S. rotundifolia is consistent with field observations, but its presence in natural populations of S. latifolia (especially in Virginia) suggests that the disease is recently introduced. Changes in the host distributions were also evident. The relative abundance of S. caroliniana declined over time (especially further north), while the relative abundance of S. virginica increased. Silene latifolia was absent or rare south of Pennsylvania before ca. 1920, indicating that S. latifolia and its anther smut are likely to be recent introductions in Virginia. Methods are also presented that quantify the completeness of coverage provided by herbarium specimens.

Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism.

Nutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast-like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high-affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site-directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.

Sex‐Ratio Bias in Populations of the Phytopathogenic FungusMicrobotryum violaceumfrom Several Host Species

The present study explores the maintenance of a segregation distortion in the phytopathogenic fungus Microbotryum violaceum (anther smut). Some individuals of this species produce spores with completely biased sex ratio because of the presence of haplo‐lethal alleles linked to the mating‐type gene. To test the different hypotheses proposed to explain the maintenance of these haplo‐lethal alleles (diploid advantage to counterbalance haploid deleterious effects, neutrality of haplo‐lethal alleles only in populations with high interstrain competition, and complete neutrality), sex‐ratio bias was investigated in natural European populations of M. violaceum from Silene latifolia and other species of Caryophyllaceae. The negative relationship observed between proportions of strains expressing a sex‐ratio bias and disease prevalence was opposite the one predicted by the second hypothesis. On the contrary, the pattern of the variances, with proportions of biased strains more extreme in smaller populations, fitted perfectly with the prediction of the third hypothesis. Experimental studies are now needed to confirm the complete neutrality of the haplo‐lethal alleles linked to the mating‐type gene in M. violaceum.

Dynamics of Multiple Infection and Within‐Host Competition by the Anther‐Smut Pathogen

Infection of one host by multiple pathogen genotypes represents an important area of pathogen ecology and evolution that lacks a broad empirical foundation. Multiple infection of Silene latifolia by Microbotryum violaceum was studied under field and greenhouse conditions using the natural polymorphism for mating-type bias as a marker. Field transmission resulted in frequent multiple infection, and each stem of the host was infected independently. Within-host diversity of infections equaled that of nearby inoculum sources by the end of the growing season. The number of diseased stems per plant was positively correlated with multiple infection and with overwintering mortality. As a result, multiply infected plants were largely purged from the population, and there was lower within-host pathogen diversity in the second season. However, among plants with a given number of diseased stems, multiply infected plants had a lower risk of overwintering mortality. Following simultaneous and sequential inoculation, strong competitive exclusion was demonstrated, and the first infection had a significant advantage. Dynamics of multiple infection initially included components of coinfection models for virulence evolution and then components of superinfection models after systemic colonization. Furthermore, there was evidence for an advantage of genotypes with mating-type bias, which may contribute to maintenance of this polymorphism in natural populations.

INTRASPECIFIC COMPETITION AND MATING BETWEEN FUNGAL STRAINS OF THE ANTHER SMUT MICROBOTRYUM VIOLACEUM FROM THE HOST PLANTS SILENE LATIFOLIA AND S. DIOICA

We studied intraspecific competition and assortative mating between strains of the anther smut fungus Microbotryum violaceum from two of its host species, Silene latifolia and S. dioica. Specifically, we investigated whether strains from allopatric host populations have higher competitive ability on their native host species and show positive assortative mating. In general, strains isolated from S. latifolia outcompeted strains isolated from S. dioica on both host species, but in female hosts, heterotypic dikaryons (i.e., dikaryons composed of a haploid strain originating from S. latifolia and a haploid strain originating from S. dioica) were most successful in competition. Furthermore, the latency period was significantly shorter for heterokaryons that contained at least one strain originating from S. latifolia, compared to heterokaryons that only contained strains originating from S. dioica. The frequencies of conjugations between strains originating from S. latifolia were much higher than conjugation frequencies between strains originating from S. dioica. A significant positive correlation was detected between the relative success of strains in competition and in conjugation, suggesting that success of a strain in competition might be partly determined by its swiftness of mating. In addition, reciprocal differences within heterotypic crosses revealed a significant effect of fungal mating type, with mating type a1 being the main determinant of mating pace. The observed differences in infection success, conjugation rate, and latency period in favor of strains from S. latifolia relative to strains from S. dioica on both host species are discussed in an evolutionary context of opportunities for the maintenance of differentiation between different formae speciales upon secondary contact.

The concept of sexually transmitted diseases in plants: definition and applicability

A number of recent studies have proposed that plants as well as human and animal hosts can be infected by sexually transmitted diseases (STDs) (Kaltz and Schmid 1995, Lockhart et al. 1996). The term has been previously applied to all those diseases affecting animals and transmitted as a result of sexual contact (Lockhart et al. 1996), in contrast to other infectious diseases (OlDs) that are spread in a variety of media such as air and contacts other than sexual. Sexually transmitted diseases are very common among animals, and have been extensively studied in humans and animals of economic importance to us. During the last couple of years the term STD has been used to describe the relationship between the flower smut Microbotryum violaceum and several species of Silene. However, until now there is no clear definition for a plant STD. With this review we aim to define STDs and OlDs in plants, to determine if STDs are common in nature, and if the terms are enough to describe the complexity of diseases in plants. In addition, we will discuss if the terms may be used to increase our understanding of the dynamic of plant-pathogen interactions.

Karyotypic similarity identifies multiple host-shifts of a pathogenic fungus in natural populations

The detection of incipient host-shifts is important to the study of emergent diseases because it allows the examination of ecological and genetic conditions that favor novel inter-species transmission. Mixed populations of Silene latifolia and Silene vulgaris were investigated for the putative occurrence of host-shifts by the fungal plant pathogen Microbotryum violaceum (the cause of anther-smut disease) between S. latifolia (a common host for the pathogen) and S. vulgaris (a rare host). Samples of the fungus from mixed and pure host populations were studied for variation in their electrophoretic karyotypes. A karyotype distance matrix showed that fungal samples clustered by locality, but not by host species. Fungal samples from S. vulgaris were indistinguishable from sympatric samples from S. latifolia in multiple cases. The results indicated at least two independent host-shifts, one in the US and perhaps two in Italy. The karyotype and ecological data indicate that the direction of the host-shifts is from S. latifolia to S. vulgaris.

INTRASPECIFIC COMPETITION AND MATING BETWEEN FUNGAL STRAINS OF THE ANTHER SMUT MICROBOTRYUM VIOLACEUM FROM THE HOST PLANTS SILENE LATIFOLIA AND S. DIOICA

We studied intraspecific competition and assortative mating between strains of the anther smut fungus Microbotryum violaceum from two of its host species, Silene latifolia and S. dioica. Specifically, we investigated whether strains from allopatric host populations have higher competitive ability on their native host species and show positive assortative mating. In general, strains isolated from S. latifolia outcompeted strains isolated from S. dioica on both host species, but in female hosts, heterotypic dikaryons (i.e., dikaryons composed of a haploid strain originating from S. latifolia and a haploid strain originating from S. dioica) were most successful in competition. Furthermore, the latency period was significantly shorter for heterokaryons that contained at least one strain originating from S. latifolia, compared to heterokaryons that only contained strains originating from S. dioica. The frequencies of conjugations between strains originating from S. latifolia were much higher than conjugation frequencies between strains originating from S. dioica. A significant positive correlation was detected between the relative success of strains in competition and in conjugation, suggesting that success of a strain in competition might be partly determined by its swiftness of mating. In addition, reciprocal differences within heterotypic crosses revealed a significant effect of fungal mating type, with mating type a1 being the main determinant of mating pace. The observed differences in infection success, conjugation rate, and latency period in favor of strains from S. latifolia relative to strains from S. dioica on both host species are discussed in an evolutionary context of opportunities for the maintenance of differentiation between different formae speciales upon secondary contact.

A plant pathogen reduces the enemy–free space of an insect herbivore on a shared host plant

An important mechanism in stabilizing tightly linked host-parasitoid and prey-predator interactions is the presence of refuges that protect organisms from their natural enemies. However, the presence and quality of refuges can be strongly affected by the environment. We show that infection of the host plant Silene latifolia by its specialist fungal plant pathogen Microbotryum violaceum dramatically alters the enemy-free space of a herbivore, the specialist noctuid seed predator Hadena bicruris, on their shared host plant. The pathogen arrests the development of seed capsules that serve as refuges for the herbivore's offspring against the specialist parasitoid Microplitis tristis, a major source of mortality of H. bicruris in the field. Pathogen infection resulted both in lower host-plant food quality, causing reduced adult emergence, and in twofold higher rates of parasitism of the herbivore. We interpret the strong oviposition preference of H. bicruris for uninfected plants in the field as an adaptive response, positioning offspring on refuge-rich, high-quality hosts. To our knowledge, this is the first demonstration that plant-inhabiting micro-organisms can affect higher trophic interactions through alteration of host refuge quality. We speculate that such interference can potentially destabilize tightly linked multitrophic interactions.

The Ecology and Genetics of a Host Shift:Microbotryumas a Model System

The need to prevent and cure emerging diseases often precludes their continuing study in situ. We present studies on the process of disease emergence by host shifts using the model system of anther-smut disease (Microbotryum violaceum) on the plant genus Silene (Caryophyllaceae). This system has little direct social impact, and it is readily amenable to experimental manipulation. Our microevolutionary studies have focused on the host shift of Microbotryum from Silene alba (=latifolia; white campion) onto Silene vulgaris (bladder campion) in a population in Virginia. Karyotypic variation shows that the host shift is recent and originates from the disease on sympatric S. alba. Analysis of the spatial pattern of disease shows that the host shift has been contingent on the co-occurrence of the two species at a local scale. Cross-inoculation studies show that families of the new host differ greatly in their susceptibility to the pathogen, indicating the potential for rapid evolution of resistance. Disease expression on the new host is frequently abnormal, suggesting that the pathogen is imperfectly adapted to its new host. In experimental populations, disease transmission within populations of the old host is greater than within populations of the new host. However, there is also a high transmission rate of the disease from the new host back to the old host, suggesting a feedback effect that increases disease prevalence in the community as a whole. Continuing studies of these populations are designed to determine whether this new host-pathogen system is likely to be self-sustaining and to quantify evolutionary changes in both the host and the pathogen.

Within- and among-population variation in infectivity, latency and spore production in a host–pathogen system

Abstract In spatially structured populations, host–parasite coevolutionary potential depends on the distribution of genetic variation within and among populations. Inoculation experiments using the plant, Silene latifolia, and its fungal pathogen, Microbotryum violaceum, revealed little overall differentiation in infectivity/resistance, latency or spore production among host or pathogen populations. Within populations, fungal strains had similar means, but varied in performance across plant populations. Variation in resistance among seed families indicates the potential for parasite-mediated selection, whereas there was little evidence for local pathogen genotype × plant genotype interactions assumed by most theoretical coevolution models. Lower spore production on sympatric than allopatric hosts confirmed local fungal maladaptation already observed for infectivity. Correlations between infectivity and latency or spore production suggest a common mechanism for variation in these traits. Our results suggest low variation available to this pathogen for tracking its coevolving host. This may be caused by random drift, breeding system or migration characteristic of metapopulation dynamics.

Isolation of 44 polymorphic microsatellite loci in three host races of the phytopathogenic fungus Microbotryum violaceum

AbstractWe report the development of 44 microsatellite markers in three host races of the fungus Microbotryum violaceum, a sexually transmitted disease of the Caryophyllaceae. An enrichment protocol was used to isolate microsatellite loci from three strains, collected, respectively, from the plant species Gypsophila repens, Dianthus sylvestris, and Silene vulgaris. Polymorphism and cross‐amplification were explored with 32 isolates of M. violaceum, collected on 12 different plant species in natural populations.

Isolates of Microbotryum violaceum from North American host species are phylogenetically distinct from their European host-derived counterparts

Microbotryum violaceum is a basidiomycete that infects the anthers of its Caryophyllaceae host species. Individual fungal isolates are host limited, though they are not morphologically distinct. This study used variable regions of the highly conserved gamma-tubulin, beta-tubulin, and ribosomal RNA-encoding genes to determine the relationships among M. violaceum fungal isolates from different host species and different geographical locations. Phylogenetic trees from intron nucleotide sequences in two protein-coding genes were compared to trees produced from internal transcribed spacers (ITS) of rDNA. Phylogenetic analyses suggested that there are two clades, one from North America and one from Europe. Isolates from both clades grouped according to host species, although in some analyses isolates from closely related host species were placed together. These results are consistent with the view that M. violaceum has experienced cospeciation with its hosts.

The spatial distribution of plant populations, disease dynamics and evolution of resistance

Empirical studies of the interaction between the anther smut fungus Microbotryum violaceum and its host plant Lychnis alpina were combined with modelling approaches to investigate how variation in the spatial distribution of host populations influences disease dynamics and variation in resistance. Patterns of disease incidence and prevalence were surveyed in three contrasting systems of natural L. alpina populations where there is substantial variation in spatial structure, ranging from large continuous populations through to small isolated patches. Disease incidence (fraction of populations where disease was present) was highest in the continuous situation, and lowest in the most isolated populations. The reverse was true for prevalence (fraction of individuals diseased). To better understand the long‐term ecological and evolutionary consequences of differences in among population spatial structure, we developed a two‐dimensional spatially explicit simulation model in which host‐population spacing was modelled by varying the percentage of sites suitable for the host. The general patterns of disease incidence and prevalence generated in the simulations corresponded well with the patterns observed in natural populations of L. alpina and M. violaceum; i.e. the fraction of sites with disease increased while the average disease prevalence in diseased populations decreased when host populations became more connected. One likely explanation for the differences in disease incidence and prevalence seen in natural populations is that the evolution of host resistance varies as a function of the degree of fragmentation. This is supported by simulation results that were qualitatively similar to the survey data when resistance was allowed to vary, but not when hosts were assumed to be uniformly susceptible. In the former, the frequency of resistance increased markedly as host populations became more connected.

Natural enemies and sex: how seed predators and pathogens contribute to sex-differential reproductive success in a gynodioecious plant.

In insect-pollinated plants flowers must balance the benefits of attracting pollinators with the cost of attracting natural enemies, when these respond to floral traits. This dilemma can have important evolutionary consequences for mating-system evolution and polymorphisms for floral traits. We investigate the benefits and risks associated with flower size and sex morph variation in Dianthus sylvestris, a gynodioecious species with pistillate flowers that are much smaller than perfect flowers. We found that this species is mainly pollinated by nocturnal pollinators, probably moths of the genus Hadena, that also oviposit in flowers and whose caterpillars feed on developing fruits and seeds. Hadena preferred larger flowers as oviposition sites, and flowers in which Hadena had deposited eggs bore more pollen on their stigmas, suggesting that Hadena is indeed the principle pollinator, or that pollinators and these seed predators employ the same choice criteria for flowers. Globally, perfect flowers suffered more predation by seed predators than did pistillate flowers, suggesting that seed predators play an important role in reproductive system dynamics in this species. On the other hand, female flowers were more likely to be contaminated with spores of another natural enemy, the pathogenic fungus Microbotryum violaceum, that are transmitted by pollinating insects. This complex interplay between two natural enemies, one of which, Hadena, is also a pollinator and vector of fungal spores, may contribute to the maintenance of the polymorphic reproductive system of the host plant.

Dimorphic mating-type chromosomes in the fungus Microbotryum violaceum.

Fungi often mate as haploids, and sex chromosomes (i.e., mating-type chromosomes) that are dimorphic for their size or overall DNA content have never been reported in this kingdom. Using electrophoretic techniques for karyotype analysis, a highly dimorphic chromosome pair that determines mating compatibility is shown to occur in populations of the fungus Microbotryum violaceum. This substantiates the evolution of such dimorphism as a general feature associated with haploid determination of mating compatibility, which previously had been known only in haplodioecious plants (mosses and liverworts). Size-dimorphic sex chromosomes are present in a lineage of M. violaceum native to Europe, as well as a lineage native to North America. However, they are very different in size between these lineages, indicating either independent evolution of the dimorphism or a large degree of divergence since their isolation. Several DNA sequences that show sequence similarity to transposons were isolated from these sex chromosomes.