Melampsora Species Research Articles

Molecular and pathogenic variation within Melampsora on Salix in western North America reveals numerous cryptic species

In North America Melampsora rusts that parasitize willows (Salix species) have never been adequately studied and mostly have been referred to a collective species, Melampsora epitea (Kunze & Schm.) Thüm, of European origin. Even taxa that are nominally distinct from M. epitea, such as M. abieti-caprearum and M. paradoxa, currently are considered to be “races” of M. epitea. Within the range of our field surveys and collections in the Pacific Northwest and the Southwest only two species of Melampsora thus were expected: M. epitea (including its races) and M. ribesii-purpureae. In this study of Melampsora on 19 species of Salix in the western United States 14 phylogenetic species, or phylotypes, were apparent from nuclear rDNA sequencing of 140 collections or isolates. Our collections of the races of M. epitea, M. abieti-caprearum and M. epitea f. sp. tsugae belonged to one phylotype, termed lineage ‘N’. Assuming that M. ribesii-purpureae represents one other phylotype, 12 phylotypes still are unaccounted for by current taxonomy. Moreover Eurasian M. ribesii-purpureae was not closely related to any of the phylotypes reported here. Even more problematic was the resistance of Eurasian species of Salix, including the type host of M. epitea, S. alba, to North American Melampsora, including phylotype ‘N’, in both the field and in inoculation experiments. These results suggest the need for the description of many new species of Melampsora on Salix in western North America. Additional analyses presented here might guide further research in this direction.

Poplar rust systematics and refinement of Melampsora species delineation

We present a review of previous taxonomic treatments of the Melampsora species occurring on poplar and describe the features associated with each spore stage in a typical poplar rust life cycle. The morphological, biological and ecological characters traditionally used for taxonomy are summarized for all Melampsora taxa, including 17 accepted species, 2 formae speciales and 2 hybrids, currently listed in the literature as pathogenic on Populus spp. We discuss the historical taxonomic decisions that led to nomenclatural and classification complications for this group of rusts, and highlight the lack of type material for three species known only from the original collections: M. cumminsii, M. multa, and M. osmaniensis. Even when all the traditional features are considered, the taxonomy of Melampsora poplar rust species remains uncertain and incomplete. An overview of recent molecular studies suggests the need for a comprehensive revision of species concepts, based on phylogenetic relationships. To that end, we propose a polyphasic approach, including the Genealogical Concordance Phylogenetic Species Recognition method, be used to build robust and meaningful systematic framework for the Melampsora poplar rusts.

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

BackgroundRust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology.ResultsTo support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs.ConclusionsThe current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.

Specificity of SSR loci for Melampsora species on poplars

Two rust fungi, Melampsora larici populina and Melampsora medusae are common in all poplar growing regions worlwide. M. larici populina is native to Eurasia, while M. medusae is endemic to eastern Unated States on Populus deltoides. Poplar leaf rust (Melampsora sp.) is widely spread disease in our country, and can cause significant growing problems. Race that prevails in our growing region is M. Larici populina. This work initiated the research of population composition, i.e. qualitative participation of Melampsora races in popoluation of black poplars hybrid progenies using molecular methods. When five SSR markers were used the results showed that M. larici populina was found in three tested clones, while only M. medusae was determined in clone I-214.

Fungal pathogen (mis-) identifications: A case study with DNA barcodes on Melampsora rusts of aspen and white poplar

Wide variation and overlap in morphological characters have led to confusion in species identification within the fungal rust genus Melampsora. The Melampsora species with uredinial–telial stages on white poplar and aspens are especially prone to misidentification. This group includes the Melampsora populnea species complex and the highly destructive pine twisting rust, Melampsora pinitorqua, which alternates between hosts in Populus section Populus and Pinus. Our objective was to compare morphologically based identification to genetic material extracted from Melampsora species pathogenic to aspen and white poplar. We compared morphometric traits and DNA barcodes obtained from internal transcribed spacer ( ITS), large ribosomal RNA subunit ( 28S), and mitochondrial cytochrome oxidase 1 ( CO1) sequences to delimit within this taxonomically difficult group. Eight different Melampsora species were initially defined based on host specificity and morphometric data. DNA barcodes were then overlaid on these initial species definitions. The DNA barcodes, specifically those defined on ITS and 28S sequences, provided a highly accurate means of identifying and resolving Melampsora taxa. We highlighted species misidentification in specimens from Canadian herbaria related to either Melampsora medusae f. sp. tremuloidae or Melampsora aecidioides. Finally, we evidenced that the north-American species found on Populus alba, M. aecidioides is closely related but distinct from the four species of the M. populnea complex ( Melampsora larici-tremulae, Melampsora magnusiana, Melampsora pinitorqua, and Melampsora rostrupii) found in Eurasia.

Generation of a wheat leaf rust, Puccinia triticina, EST database from stage‐specific cDNA libraries

Thirteen cDNA libraries constructed from small amounts of leaf rust mRNA using optimized methods served as the source for the generation of 25 558 high-quality DNA sequence reads. Five life-cycle stages were sampled: resting urediniospores, urediniospores germinated over water or plant extract, compatible, interactive stages during appressorium or haustorium formation just before sporulation, and an incompatible interaction. mRNA populations were subjected to treatments such as full-length cDNA production, subtractive and normalizing hybridizations, and size selection methods combined with PCR amplification. Pathogen and host sequences from interactive libraries were differentiated in silico using cereal and fungal sequences, codon usage analyses, and by means of a partial prototype cDNA microarray hybridized with genomic DNAs. This yielded a non-redundant unigene set of 9760 putative fungal sequences consisting of 6616 singlets and 3144 contigs, representing 4.7 Mbp. At an E-value 10(-5), 3670 unigenes (38%) matched sequences in various databases and collections but only 694 unigenes (7%) were similar to genes with known functions. In total, 296 unigenes were identified as most probably wheat and ten as rRNA sequences. Annotation rates were low for germinated urediniospores (4%) and appressoria (2%). Gene sets obtained from the various life-cycle stages appear to be remarkably different, suggesting drastic reprogramming of the transcriptome during these major differentiation processes. Redundancy within contigs yielded information about possible expression levels of certain genes among stages. Many sequences were similar to genes from other rusts such as Uromyces and Melampsora species; some of these genes have been implicated in pathogenicity and virulence.

A new species of Melampsora on Populus yunnanensis from China

Melampsora nujiangensis, a rust fungus found on Populus yunnanensis in China, is described as a new species. Light and scanning electron microscopy with herbarium specimens of the rust fungus show that the shape of its urediniospores differs from that of other known species of Melampsora, and its urediniospore walls are thinner than the other species. Furthermore, in phylogenetic trees based on the DNA sequences (28S and ITS) the rust fungus is phylogenetically separated by high bootstrap values. These results indicate that the fungus is an isolated species among the genus Melampsora.

Isolation and characterization of 15 microsatellite loci in the poplar rust fungus, Melampsora larici‐populina, and cross‐amplification in related species

AbstractWe developed 15 microsatellite loci in the poplar rust fungus, Melampsora larici‐populina, using two enrichment protocols. Polymorphism of each locus was assessed on a panel of 30 isolates, comprising three subpanels (world, regional and local scales). Thirteen loci were polymorphic with three to eight alleles detected. The 15 loci were also tested on five related Melampsora species, M. allii‐populina, M. medusae f. sp. deltoidae, M. larici‐tremulae, M. rostrupii and M. pinitorqua, and partial or global cross‐amplification events were detected.

First Report of the Aecial State of Melampsora larici-populina on Larix spp. in North America

Melampsora larici-populina Kleb. was reported for the first time in eastern North America during 2002, on Populus spp., its telial host (1). M. larici-populina, a heteroecious rust, alternates between species of Populus and Larix. Since M. larici-populina was observed again in 2003, we investigated the possibility that its basidiospores may infect larch (Larix spp.) resulting in spermogonia and aecia. Identification of Melampsora species from aeciospore morphology is difficult but urediniospores are distinctive. This is important since the native M. medusae also alternates between Populus and Larix spp. During the spring of 2004, aecia were observed on needles of exotic (Larix decidua Mill. and L. leptolepis (Siebold and Zucc.) Gordon) and indigenous (L. laricina (K. Koch)) larch in an arboretum in Lotbinière (Quebec, Canada) where M. larici-populina has previously been found. Larch needles with yellow blister-like fructifications were collected in May 2004 and fixed on top of petri plates to allow aeciospore release onto leaves of Jackii poplar (Populus balsamifera L. × P. deltoides Marsh.). After approximately 10 days, uredinia appeared on the abaxial surface of the poplar leaves. Some of the many needles collected yielded uredinia cultures on Jackii poplars. The majority of these cultures were identified as being M. larici-populina; one was M. medusae. M. larici-populina urediniospores were 32 to 48 μm long and possessed a characteristic apical bald spot. DNA was extracted from aecia and uredinia, and the internal transcribed spacer (ITS) of the ribosomal RNA gene was amplified in real-time polymerase chain reaction (PCR) by specific primers for M. medusae or M. larici-populina created from sequences (GenBank Accession Nos. AY429656 and AY429657). The 120 base pairs target fragments amplified only with the M. larici-populina specific primers with the 14 samples that were identified as M. larici-populina by morphological characteristics of the urediniospores. No PCR amplification was obtained with M. medusae primers. These results were not unexpected since larch has been previously reported as an aecial host of M. larici-populina elsewhere (2). The ability of M. larici-populina to overwinter and complete its life cycle has important consequences since it proves that it is established and can go through sexual reproduction. A complete life cycle in eastern North America may allow M. larici-populina to generate pathogenic variation that will challenge poplar breeders in this region.

Development and characterization of microsatellite markers for the poplar rust fungi Melampsora medusae and Melampsora larici‐populina

Abstract Melampsora species and their hybrids are obligate parasites of Populus and Salix species worldwide. The increasing interest in Populus and Salix for biomass and fibre production necessitates genetic markers for population studies of Melampsora spp. Libraries enriched for simple sequence repeats were used to develop five microsatellite markers for Melampsora medusae and Melampsora larici‐populina. The variation detected by these markers will be valuable for phylogenetic and population genetic studies, substantiate putative hybrids, and deployment of resistant poplar clones.

Molecular and Morphological Characterization of the Willow Rust Fungus, Melampsora epitea, from Arctic and Temperate Hosts in North America

Current taxonomy places all rust fungi that occur on willow (Salix spp.) in North America in one species complex, Melampsora epitea Thüm. Characteristics of M. epitea isolates from the Canadian arctic were compared to M. epitea isolates from temperate regions of North America. Sequences from internal transcribed spacer (ITS) regions of rDNA were obtained from urediniospores from rust-infected Salix leaves collected in the Canadian arctic and in Minnesota and compared. Phylogenetic analysis of nuclear ribosomal ITS regions indicated that arctic M. epitea samples were divergent from temperate M. epitea isolates, perhaps in part because all rusts examined diverged according to host species. Four urediniospore characteristics were examined: area, circularity (shape factor), major axis length and spine density. Statistically significant (P < 0.05) differences were observed for spine density among all host species except S. nigra and S. bebbiana. However major axis length differed between these species. These results represent the first evidence that arctic and temperate Melampsora species on Salix hosts in North America have evolved distinct molecular and morphological characters.

Molecular and morphological characterization of the willow rust fungus, Melampsora epitea, from arctic and temperate hosts in North America

Current taxonomy places all rust fungi that occur on willow (Salix spp.) in North America in one species complex, Melampsora epitea Thüm. Characteristics of M. epitea isolates from the Canadian arctic were compared to M. epitea isolates from temperate regions of North America. Sequences from internal transcribed spacer (ITS) regions of rDNA were obtained from urediniospores from rust-infected Salix leaves collected in the Canadian arctic and in Minnesota and compared. Phylogenetic analysis of nuclear ribosomal ITS regions indicated that arctic M. epitea samples were divergent from temperate M. epitea isolates, perhaps in part because all rusts examined diverged according to host species. Four urediniospore characteristics were examined: area, circularity (shape factor), major axis length and spine density. Statistically significant (P < 0.05) differences were observed for spine density among all host species except S. nigra and S. bebbiana. However major axis length differed between these species. These results represent the first evidence that arctic and temperate Melampsora species on Salix hosts in North America have evolved distinct molecular and morphological characters.

Morphological and molecular phylogenetic analysis of Melampsora species on poplars in China

Many species of Melampsora on Populus have been reported in China, based on morphological characteristics of both uredial and telial states, and on host species, but their morphology and taxonomy are still poorly defined. In this study, 196 specimens representing Melampsora species on poplars and collected from various areas of China were used for morphological observations. The morphological characteristics of urediniospores and teliospores were examined with light and scanning electron microscopy. The specimens could be classified into five groups based on their morphology. For the sequencing of the nuclear large subunit rDNA (D1/D2), 5.8S rDNA and their internal transcribed spacers, ITS1 and ITS2 region, 54 specimens were selected from the specimens used in morphological observations. These specimens were separated into six clades by phylogenetic analyses of the D1/D2 and ITS regions. Correlations among morphological groups and phylogenetic clades based on these results suggest a revision of these species. In particular, no evidence to discriminate specimens of M. acedioides, M. magnusiana, and M.rostrupii was found from either morphological characteristics or sequence analysis.

P17 The role of clinico-genetic monitoring of risk groups for early diagnostics of female reproductive system tumors

Fungal species belonging to the genus Melampsora (Basidiomycota, Pucciniales) comprise rust pathogens that alternate between Salicaceae and other plant hosts. Species delineation and identification are difficult within this group due to the paucity of observable morphological features. Several Melampsora rusts are highly host-specific and this feature has been used for identification at the species level. However, this criterion is not always reliable since different Melampsora rust species can overlap on one host but specialize on a different one. To date, two different species recognition methods are used to recognize and define species within the Melampsora genus: (i) morphological species recognition, which is based solely on morphological criteria; and (ii) ecological species recognition, which combines morphological criteria with host range to recognize and define species. In order to clarify species recognition within the Melampsora genus, we applied phylogenetic species recognition to Melampsora poplar rusts by conducting molecular phylogenetic analyses on 15 Melampsora taxa using six nuclear and mitochondrial loci. By assessing the genealogical concordance between phylogenies, we identified 12 lineages that evolved independently, corresponding to distinct phylogenetic species. All 12 lineages were concordant with host specialization, but only three belonged to strictly defined morphological species. The estimation of the species tree obtained with Bayesian concordance analysis highlighted a potential co-evolutionary history between Melampsora species and their reciprocal aecial host plants. Within the Melampsora speciation process, aecial host may have had a strong effect on ancestral evolution, whereas telial host specificity seems to have evolved more recently. The morphological characters initially used to define species boundaries in the Melampsora genus are not reflective of the evolutionary and genetic relationships among poplar rusts. In order to construct a more meaningful taxonomy, host specificity must be considered an important criterion for delineating and describing species within the genus Melampsora as previously suggested by ecological species recognition.

The most frequent 'rusts' on trees and shrubs in Serbia

This paper describes 15 species of fungi in the order Uredinales, causing the diseases known as "rusts". These fungi develop as obligate parasites and cause very dangerous diseases on cultivated plants. Among the "rusts" occurring on the trees, the following rusts are of special economic significance Melampsorella caryophyllacearum (rust on fir), Cronartium flaccidum (rust on two-needle pines) and Melampsora species (rusts on poplars). Fungi Chrysomyxa pirolata, Pucciniastrum epilobii, Gymnosporangium clavariiforme and G. tremelloides have been identified for the first time in Serbia.

Melampsora × columbiana, a natural hybrid of M. medusae and M. occidentalis

Hybrids of Melampsora medusae and M. occidentalis are herein described as M. xcolumbiana. This hybrid taxon is characterized by isolates that generally exhibit morphological intermediacy in uredinial and telial traits, and mixed virulence/avirulence on Populus trichocarpa and P. deltoides, the natural hosts of M. occidentalis and M. medusae, respectively. Hybrids frequently contain ribosomal ITS sequences from both Melampsora species, but discordant isolates were also found, like M. medusae in having a urediniospore equatorial smooth spot, and like M. occidentalis in spine density, and intermediate in urediniospore length. Isolates with hybrid urediniospore morphology but homozygous for ITS sequences from M. medusae or M. occidentalis were also found, suggesting that the primary F1 hybrids have produced F2 and/or backcross progeny. In a 1997 survey of the leaf rust population on hybrid poplar in the Pacific Northwest, this ‘new population’ of Melampsora xcolumbiana was the only taxon found. Specimens from nearly a century ago indicate that an older hybrid population is of wide distribution in North America. This ‘older population’ of Melampsora xcolumbiana was found on specimens of all species of Populus in sections Aigeiros and Tacamahaca, and on many of their hybrids, in areas outside the type localities of M. occidentalis and M. medusae. These three Melampsora taxa, M. medusae, M. occidentalis, and M. xcolumbiana, are the only ones that occur naturally on sections Aigeiros and Tacamahaca of Populus in North America.

Differentiating characteristics between Melampsoridium rusts infecting birch and alder leaves

Melampsoridium rust epidemics on alder occurred in Estonia in 1996–1998, and in Finland in 1997–1998. In this study we show that the length and roundness of urediniospores and the PCR-amplified internal transcribed spacer (ITS) sequences of rusts occurring on Alnus and Betula are different. Morphologically the rust on alder belongs to M. hiratsukanum, and based on our results is not conspecific with M. betulinum. The sequence comparisons indicated that 5.8S rDNA was similar in both Melampsoridium species and identical to several Cronartium sequences. The 5.8S rDNA of Melampsoridium was also more similar to Pucciniastrum, Chrysomyxa and Puccinia than to Melampsora species. The ITS sequences were distinct from all other rusts, but relatively similar to Cronartium, Pucciniastrum and Chrysomyxa sequences.

Differentiating Characteristics between Melampsoridium Rusts Infecting Birch and Alder Leaves

Melampsoridium rust epidemics on alder occurred in Estonia in 1996–1998, and in Finland in 1997–1998. In this study we show that the length and roundness of urediniospores and the PCR-amplified internal transcribed spacer (ITS) sequences of rusts occurring on Alnus and Betula are different. Morphologically the rust on alder belongs to M. hiratsukanum, and based on our results is not conspecific with M. betulinum. The sequence comparisons indicated that 5.8S rDNA was similar in both Melampsoridium species and identical to several Cronartium sequences. The 5.8S rDNA of Melampsoridium was also more similar to Pucciniastrum, Chrysomyxa and Puccinia than to Melampsora species. The ITS sequences were distinct from all other rusts, but relatively similar to Cronartium, Pucciniastrum and Chrysomyxa sequences.

Melampsora and Marssonina pathogens of poplars and willows in New Zealand

SummaryNew Zealand has hosted three Melampsora species attacking poplars (Populus spp.), namely, Melampsora larici‐populina, Melampsora medusae and for a single season, Melampsora medusae‐populina, a unique interspecific hybrid. The predominant species, M. larici‐populina, over‐winters via larch and each year new races appear causing defoliation of previously rust‐resistant cultivars. Melampsora medusae also occurs in some seasons following over‐wintering on certain hosts or by aerial transport from Australia. Willows (Salix spp.) host four Melampsora species, Melampsora coleosporioides, Melampsora epitea var. epitea and two unidentified species attacking Salix viminalis and Salix daphnoides/Salix incana× open pollinated hybrids, respectively. The host range and pathogenicity of these species have remained static as no breakdown in host resistance has been observed. The anthracnose fungi, Marssonina brunnea and Marssonina castagnei on poplar, and Marssonina salicicola on tree willows occur throughout New Zealand. All three species can cause severe defoliation of susceptible cultivars in moist districts where annual rainfall exceeds 1000 mm. Although all form abundant microconidia during autumn, the Drepanopeziza states have never been observed in New Zealand. During an unusually wet summer (199–93), a new strain of Marssonina salicicoa appeared which defoliated the previously resistant cultivar, salix matsudana × salix alba cv. 1002 The importance of monitoring the morphological (conidial dimensions, DNA fingerprints) and physiological (host range and pathogenicity) attributes of local populations of Melampsora and Marssonina species is discussed.

PCR-SSCP Analysis of the Ribosomal DNA ITS Regions of the Willow Rust Fungi in Japan.

Single-strand DNA conformation polymorphism (SSCP) analysis using internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) amplified by polymerase chain reaction (PCR) was performed to detect DNA polymorphism of nine species of Melampsora rust fungi on willows. The rDNA ITS 1 region (approximately 300-320bp in size), was amplified from 49 collections. The SSCP patterns in the amplified ITS 1 region of five species, M. capraearum, M. epiphylla, M. larici-urbaniana, M. microsora and M. yezoensis were species-specific. On the other hand, no distinction was found in the SSCP patterns between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. In improved PCR-SSCP analysis using restriction fragments of the amplified ITS 1 and ITS 2 regions, no difference was detected between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. These results suggest the respective two species have a close genetic and taxonomical relationship. We revealed the PCR-SSCP analysis may be beneficial as a tool for distinguishing species of Melampsora because the analysis is simple and sensitive.