Melampsora Research Articles

Molecular evidence indicates that subarctic willow communities in Scotland support a diversity of host-associated Melampsora rust taxa

Rare and threatened subarctic willow scrub communities in the UK are the subject of ongoing conservation programmes, yet little is known about the diversity of fungal taxa that they support. Isolates of the rust genus Melampsora were sampled from 112 leaves of eight subarctic willow (Salix) taxa and their hybrids from twelve sites in the UK. In order to determine the number of Melampsora taxa present in the samples, isolates were sequenced for the Internal Transcribed Spacer (ITS) region of rDNA and data were subject to phylogenetic analysis. Maximum likelihood and Bayesian analysis indicated that the isolates fell into three strongly supported host-associated clades. Clade I contained only isolates from Salix herbacea and was distinguished morphologically by dense urediniospore echinulation and thin cell walls. Clade II contained isolates from Salix arbuscula and Salix reticulata only. These could not be distinguished morphologically from isolates in Clade III which were found on Salix lapponum, Salix myrsinites, Salix myrsinifolia, Salix aurita, Salix lanata, and their hybrids. Clade II was most distinct in ITS sequence, differing by 50 bases from Clades I and III, while the latter clades differed in sequence by only 24 bases on average. Clades I and III are likely to represent the previously recognised taxa Melampsora alpina Juel 1894 and Melampsora epitea Thüm. 1879 respectively, but Clade II has not apparently been described before. Significant differences in the intensity of infection by isolates of Clade III were found among different Salix species at a single site, suggesting either differences in resistance among Salix taxa, or the presence of further cryptic taxa within Clade III. The study illustrates the power of molecular phylogenetic analysis to reveal cryptic biodiversity within Melampsora, and suggests that conserving Salix host diversity within subarctic willow communities will ensure that a diversity of associated Melampsora taxa is maintained.

A Comprehensive Analysis of Genes Encoding Small Secreted Proteins Identifies Candidate Effectors in Melampsora larici-populina (Poplar Leaf Rust)

The obligate biotrophic rust fungus Melampsora larici-populina is the most devastating and widespread pathogen of poplars. Studies over recent years have identified various small secreted proteins (SSP) from plant biotrophic filamentous pathogens and have highlighted their role as effectors in host-pathogen interactions. The recent analysis of the M. larici-populina genome sequence has revealed the presence of 1,184 SSP-encoding genes in this rust fungus. In the present study, the expression and evolutionary dynamics of these SSP were investigated to pinpoint the arsenal of putative effectors that could be involved in the interaction between the rust fungus and poplar. Similarity with effectors previously described in Melampsora spp., richness in cysteines, and organization in large families were extensively detailed and discussed. Positive selection analyses conducted over clusters of paralogous genes revealed fast-evolving candidate effectors. Transcript profiling of selected M. laricipopulina SSP showed a timely coordinated expression during leaf infection, and the accumulation of four candidate effectors in distinct rust infection structures was demonstrated by immunolocalization. This integrated and multifaceted approach helps to prioritize candidate effector genes for functional studies.

Using hierarchical clustering of secreted protein families to classify and rank candidate effectors of rust fungi.

Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components.

Infection dynamic of some cultivars of shrubby willow (Salix spp.) by rust (Melampsora spp.) and willow beetle (Phratora vulgatissima L.).

Summary The aim of the study was to asses of population dynamic of Melampsora spp. and Phratora vulgatissima, the most dangerous bioenergy willows agrophags. Investigation was conducted in Research Centre for Cultivar Testing collection in years 2010–2011. First symptoms of rust infection were observed on leaves cultivars Dobkowska, Marie and Christina in first decade of July. On leaves cultivars Marcel, Marie and Christina the increase of infection at following observations was observed. On Dobkowska and Wodtur uredinia were observed in July and August. The further development of the disease and formation of new uredinia was not recognized. Depending of the willow cultivar the greatest damages caused by P. vulgatissima foraging were observed in August and September. The biggest leaf area loss was noted on Wodtur.

Effect of the preparation Silwet Gold on the efficacy of the fungicydes used for the protection of willow against Melampsora epitea Wpływ preparatu Silwet Gold na skutecznośc fungicydów stosowanych do ochrony wierzby przed Melampsora epitea

Summary Willow plants with the symptoms of rust (Melampsora epitea) were sprayed with the fungicides: Discus 500 WG, Dithane NeoTec 75 WG, Domark 100 EC and Signum 33 WG (without a surfactant), and in a mixture with the preparation Silwet Gold 0.015% as a surfactant. After 2 spray treatments at 7-day intervals, the mean number of uredinia patches per leaf was recorded. It was stated that surfactant Silwet Gold caused the increase in efficacy of the used fungicides by 13–80% depending on the experiment. Also on treated with surfactant plants the percentage of dried up uredinia was higher and ranged from 1.5 to almost 67%. The percentage of diseased leaves was by 1 to 35% lower as compared to willow protected without a surfactant. All the tested fungicides with the addition of Silwet Gold completely covered the top and bottom sides of the leaf blades.

Evaluation of the yield potential and physicochemical properties of the biomass of Salix viminalis × Populus tremula hybrids

This study presents the characteristics of four Salix viminalis×Populus tremula hybrids, produced for the first time in the world grown in a three-year field experiment. Shoot weight per plant and major biomass yield components, including plant height, number of shoots per rootstock and shoot diameter, were determined. The infection severity caused by leaf rust (Melampsora sp.) was also evaluated. The biomass of three-year-old hybrid plants was subjected to chemical analyses and calorimetric tests to determine the energy value of biomass as solid fuel. Among the studied genotypes the highest yield was achieved by one of the studied hybrids. Its biometric parameters did not differ significantly from the standard genotype, and they were superior to the parameters of the maternal form. All Salix×Populus hybrids were more susceptible to rust infections than their maternal form and one hybrid was more resistant to infections caused by fungi of the genus Melampsora. Two hybrids have optimal biomass parameters as regards both calorific value and amount of carbon, hydrogen, sulfur and nitrogen.

DNA barcoding in the rust genus Chrysomyxa and its implications for the phylogeny of the genus

Chrysomyxa rusts are fungal pathogens widely present in the boreal forest. Taxonomic delimitation and precise species identification are difficult within this genus because several species display similar morphological features. We applied a DNA barcode system based on the ribosomal internal transcribed spacer region (ITS), large subunit (28S) ribosomal RNA gene, mitochondrial cytochrome oxidase 1 (CO1) and mitochondrial NADH dehydrogenase subunit 6 (NAD6) in 86 strains from 16 different Chrysomyxa species, including members of the Chrysomyxa ledi species complex. The nuclear ITS and 28S loci revealed higher resolving power than the mitochondrial genes. Amplification of the full CO1 barcode region failed due to the presence of introns limiting the dataset obtained with this barcode. In most cases the ITS barcodes were in agreement with taxonomic species based on phenotypic characters. Nevertheless we observed genetically distinct (different DNA barcodes) lineages within Chrysomyxa pyrolae and Chrysomyxa rhododendri, providing some evidence for allopatric speciation within these morphologically defined species. This finding, together with the observed pattern of host specificities of the studied rust fungi, suggest that species diversification within the C. ledi species complex might be governed by a set of factors such as specialisation to certain Ericaceae species as telial hosts and to a lesser extent specialization to different spruce species as aecial hosts. Moreover allopatric speciation by geographic disruption of species also seems to take place. When our data were integrated into a broader phylogenetic framework the Chrysomyxa genus unexpectedly was not resolved as a monophyletic group. Indeed the spruce cone rusts C. pyrolae and C. monesis coalesced with the pine needle rusts belonging to the genus Coleosporium, whereas the microcyclic species Chrysomyxa weirii was embedded within a clade comprising the genus Melampsora.

Infection Behaviour of Melampsora larici-populina on the Leaf Surface of Populus purdomii

Abstract Behaviours of urediospore germtube in Melampsora larici-populina on the leaf surface of Populus purdomii were studied by light microscope, scanning electron microscope (SEM), transmission electron microscope (TEM), and fluorescence microscope. Crab-like fusion cells on leaf surface, intercellular hyphal cells in leaf tissues, as well as nucleus states, were observed and counted up in this study. Under unsaturated humidity, 32% of germinated tubes fused into a distinguishable swollen crab-shaped cell at the merging site, and 10.5% of observed crab-like cells had more than three nuclei. Wedge-shaped mycelia developed and then penetrated the leaf surface directly, or indirectly through stomata. Tips of germtube passed through the intercellular cells of poplar leaves directly were found in TEM. Aniline blue dyeing also showed that the infecting hyphae could invade into the cuticle and epidemic cell wall directly. For the case of infection through stomata, there were two different situations. Short branches and wedge hyphae usually penetrated the leaf surface via opened stomata, whereas, some germtube branches and wedge hyphae penetrated leaves through the guard cell walls or stoma lips. In the latter case, the stomata were always closed. The samples from wild forestlands had the same fused cells and wedge hyphae, but the occurrence rate was much higher than that in the chamber. Even under the saturated air humidity, germtubes could roll back and formed fusion structure, or merged together with their tips. The fusion cells might centralize the plasma of merged germtubes and have a strong survival capacity to protect germtubes from dying under arid circumstances, and provide a chance of genetic variation as well.

Melampsora larici-populina Transcript Profiling During Germination and Timecourse Infection of Poplar Leaves Reveals Dynamic Expression Patterns Associated with Virulence and Biotrophy

Melampsora larici-populina is responsible for poplar leaf rust disease and causes severe epidemics in poplar plantations in Europe. The poplar rust genome has been recently sequenced and, in order to find the genetic determinants associated with its biotrophic lifestyle, we generated a whole-genome custom oligoarray and analyzed transcript profiles of M. larici-populina during the infection timecourse in poplar leaves. Different stages were investigated during the asexual development of the rust fungus, including resting and germinating urediniospores and seven in planta stages in the telial host. In total, 76% of the transcripts were detected during leaf infection as well as in urediniospores, whereas 20% were only detected in planta, including several transporters and many small secreted proteins (SSP). We focused our analysis on gene categories known to be related to plant colonization and biotrophic growth in rust pathogens, such as SSP, carbohydrate active enzymes (CAZymes), transporters, lipases, and proteases. Distinct sets of SSP transcripts were expressed all along the infection process, suggesting highly dynamic expression of candidate rust effectors. In contrast, transcripts encoding transporters and proteases were mostly expressed after 48 h postinoculation, when numerous haustoria are already formed in the leaf mesophyll until uredinia formation, supporting their role in nutrient acquisition during biotrophic growth. Finally, CAZymes and lipase transcripts were predominantly expressed at late stages of infection, highlighting their importance during sporulation.

The genetic structure of the plant pathogenic fungus Melampsora larici-populina on its wild host is extensively impacted by host domestication

Wild and cultivated plants represent very different habitats for pathogens, especially when cultivated plants bear qualitative resistance genes. Here, we investigated to what extent the population genetic structure of a plant pathogenic fungus collected on its wild host can be impacted by the deployment of resistant cultivars. We studied one of the main poplar diseases, poplar rust, caused by the fungus Melampsora larici-populina. A thousand and fifty individuals sampled from several locations in France were phenotyped for their virulence profile (ability to infect or not the most deployed resistant cultivar 'Beaupré'), and a subset of these was genotyped using 25 microsatellite markers. Bayesian assignment tests on genetic data clustered the 476 genotyped individuals into three genetic groups. Group 1 gathered most virulent individuals and displayed evidence for selection and drastic demographic changes resulting from breakdown of the poplar cultivar 'Beaupré'. Group 2 comprised individuals corresponding to ancestral populations of M.larici-populina naturally occurring in the native range. Group 3 displayed the hallmarks of strict asexual reproduction, which has never previously been demonstrated in this species. We discuss how poplar cultivation has influenced the spatial and genetic structure of this plant pathogenic fungus, and has led to the spread of virulence alleles (gene swamping) in M.larici-populina populations evolving on the wild host.

Sequencing the fungal tree of life

Sequencing the Fungal Tree of Life F. Martin 1 , D. Cullen 2 , D. Hibbett 3 , A. Pisabarro 4 , J. W. Spatafora 5 , S. E. Baker 6,7, I. V. Grigoriev 7 UMR INRA/UHP 1136, Interactions Arbres/Micro-Organismes, INRA-Nancy, 54280 Champenoux, France University of Wisconsin-Madison, Madison, WI, USA Clark University, Worcester, MA, USA Department of Agrarian Production, Public University of Navarre, 31006 Pamplona, Spain Dept. Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland, Washington, USA, 99352 US DOE Joint Genome Institute, Walnut Creek, California, USA March 2011 The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02- 05CH11231 DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California.

Effect of Melampsora larici-populina on growthand biomass yield of eight clones of Populus nigra

This study evaluates the effect of the identified pathogenic races E1, E2 and E3 of the leaf rust Melampsora larici-populina on some growth traits and biomass yield in the species Populus nigra. A field trial was conducted with 8 clones of P. nigra using fungicide-sprayed and unsprayed treatments in 3 replications of 4 plants. In the course of three years the occurrence of the rust was evaluated on a six-point scale. The plant height and stem diameter were measured during the trial. In the last year the plants were harvested and the dry weight was determined. In the untreated plants a significant negative correlation was found between the intensity of rust occurrence and the values of stem diameter and dry matter yield (P < 0.05). A decline in dry matter yield caused by the rust was low (below 9%) or zero in a half of the clones while it ranged between 19% and 28% in the other half of clones. In some clones the yield decline was relatively low although the expression of rust symptoms was rather high which could be attributed to a tolerance to the given pathogen.

Comportement des cultivars de Peuplier envers la rouille

Entre 1987 et 2010, plus d’une centaine de cultivars de Peuplier ont été soumis à des tests d’évaluation de leur comportement envers la rouille du mélèze (Melampsora larici-populina ). Pour la plupart des clones, il a été recherché en laboratoire si une ou plusieurs virulences étaient nécessaires pour les infecter. Puis tous ont été soumis à l’infection dans trois pépinières sous un inoculum connu et reflétant la diversité des races de l’agent pathogène. Les sensibilités moyennes sont fournies pour deux périodes ainsi que les virulences nécessaires à l’infection. Presque tous les clones sélectionnés pour une résistance complète ont subi un contournement de celle-ci avec développement d’infections souvent sévères, notamment sur les clones interaméricains.

Validation of Melampsora larici-populina reference genes for in planta RT-quantitative PCR expression profiling during time-course infection of poplar leaves

The foliar rust caused by Melampsora larici-populina ( Mlp) is the main disease affecting poplar plantations in Europe. The biotrophic status of this fungus is a major limitation to address in planta transcripts profiling. Thus, identification of reference rust genes steadily expressed during plant tissue colonization is a crucial point. A quantitative PCR approach to assess fungal ITS amplification profile and Reverse Transcription quantitative-PCR was set to compare candidate reference genes amplification profiles in poplar infected tissues. We selected two M. larici-populina genes encoding an alpha-tubulin and the elongation factor-1-alpha that showed the highest expression stability across biological samples and for which transcript levels were correlated with fungal ITS amplification during time-course infection of poplar leaves. We report the use of these reference genes to assess in planta expression profiles of two genes involved in thiamine biosynthesis ( THI1 and THI2) for which specific haustorium expression was previously described in the bean rust fungus Uromyces fabae. Mlp- THI1 and Mlp-THI2 showed similar expression profiles. Trancripts were barely detectable in urediniospores as well as during the early stages of infection compared to those reported in the bean rust, whereas a strong induction was observed after haustorial formation after 24 hpi. These data are in frame with the results obtained in U. fabae and consistent with a metabolic reorientation that likely occurs after the fungus derived nutrients from its host in the haustorial structure essential for fungal biotrophy.

Laser Capture Microdissection of Uredinia Formed by Melampsora larici-populina Revealed a Transcriptional Switch Between Biotrophy and Sporulation

The foliar rust caused by the basidiomycete Melampsora larici-populina is the main disease affecting poplar plantations in Europe. The biotrophic status of rust fungi is a major limitation to study gene expression of cell or tissue types during host infection. At the uredinial stage, infected poplar leaves contain distinct rust tissues such as haustoria, infection hyphae, and uredinia with sporogenous hyphae and newly formed asexual urediniospores. Laser capture microdissection (LCM) was used to isolate three areas corresponding to uredinia and subjacent zones in the host mesophyll for expression analysis with M. larici-populina whole-genome exon oligoarrays. Optimization of tissue preparation prior to LCM allowed isolation of RNA of good integrity for genome-wide expression profiling. Our results indicate that the poplar rust uredinial stage is marked by distinct genetic programs related to biotrophy in the host palisade mesophyll and to sporulation in the uredinium. A strong induction of transcripts encoding small secreted proteins, likely containing rust effectors, is observed in the mesophyll, suggesting a late maintenance of suppression of host defense in the tissue containing haustoria and infection hyphae. On the other hand, cell cycle and cell defense rescue transcripts are strongly accumulated in the sporulation area. This combined LCM-transcriptomic approach brings new insights on the molecular mechanisms underlying urediniospore formation in rust fungi.

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.

Changes in antioxidant enzyme activities and isozyme profiles in leaves of male and female Populus cathayana infected with Melampsora larici-populina

Populus cathayana Rehd., a dioecious tree species, is widely distributed in the northern, central and southwestern regions of China. In poplars, Melampsora larici-populina Kleb. is mainly responsible for rust disease, which is considered to be the world's most important disease of poplars. Yet, little is known about sex-related responses to rust disease in poplars. The purpose of this work was to determine whether sexually different responses occur in the antioxidant system of poplars after infection by rust disease. Three-month-old male and female P. cathayana individuals were inoculated with M. larici-populina in a greenhouse. After 12 days of incubation, we investigated the changes in physiology, biochemistry, enzyme activities and isozyme profiles. It was discovered that (i) leaf rust disease inhibited photosynthesis, caused oxidative stress and cellular membrane damage and changed antioxidant enzyme activities and isozyme profiles in poplar leaves; (ii) male poplars suffered from lower infection levels and less negative effects of leaf rust disease than did females; (iii) males showed higher antioxidant activities and less H(2)O(2) accumulation than did females after being infected by leaf rust. Thus, our results indicate that leaf rust disease is more severe in female poplars, and they suffer from greater negative effects than do males. This is the first report about sexually different responses of poplars in the antioxidant reactions to rust disease. It offers some useful information about the effect of leaf rust on dioecious plants, especially on dioecious woody plants.

Poplar and Pathogen Interactions: Insights from Populus Genome-Wide Analyses of Resistance and Defense Gene Families and Gene Expression Profiling

Our understanding of the molecular basis of plant-pathogen interactions is derived mostly from studies of model annual plant species, and until recently, few addressed disease resistance and defense responses in long-lived species such as trees. The release of the Populus genome sequence has permitted extensive genome-wide surveys of gene families and comparative analyses of other sequenced plant genomes. These have revealed striking features for gene families that play key roles in the plant defense response. For example, the NBS-LRR resistance ( R )-gene family is expanded compared with other plant genomes, including R -gene subfamilies not previously reported in plants. Some of these genes are clustered on the subtelomeric part of a chromosome that shows segregation distortion in genetic studies. Similar expansion is observed for other genes playing key roles in plant defense such as pathogenesis-related proteins. Among the many pathogens that infect poplar trees, Melampsora spp. fungi, which cause rust diseases in plants, are responsible for considerable damage in poplar plantations. This biotrophic pathogen has attracted recent attention and here we describe molecular insights into the Populusdefense response against rust infection. Transcript profiles derived from compatible (susceptible) and incompatible (specific host -resistance) Populus-Melampsora interactions were leveraged to describe molecular changes occurring during defense responses against rust fungi. This highlighted responses that are similar to defense responses of annual plant species, such as up-regulation of transcripts encoding pathogenesis-related proteins. The molecular evidence gathered for the poplar-rust pathosystem indicates a temporal delay in the activation of defense responses between susceptibility and partial or full resistance that is consistent with the signal conversion model described for Arabidopsis. The genome of Melampsora larici-populina, which infects several species of Populus, was recently sequenced and provides a model pathosystem for forest pathology and offers unprecedented opportunities to understand how tree species cope with disease. Ultimately, understanding the molecular basis of Populus rust resistance will greatly improve our understanding of other major diseases of poplar.

Characterization of 41 microsatellite loci developed from the genome sequence of the poplar rust fungus, Melampsora larici-populina

Forty-one microsatellite markers were developed for the poplar rust fungus Melampsora larici-populina. The genome sequence of M. larici-populina strain 98AG31 was screened in silico for microsatellite loci using Magellan software. Thirty-nine loci, evaluated on a sexual population of 35 M. larici-populina isolates sampled on larch trees in the French Alps, were identified to be highly polymorphic. These markers revealed two to 34 alleles, with an average of 7.23 alleles per locus. The estimated heterozygosity ranged from 0.029 to 0.958 across the 41 microsatellite loci. These markers will be highly useful for population genetics studies of M. larici-populina.

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.