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This document provides the conclusions of the pest survey card that was prepared in the context of the EFSA mandate on plant pest surveillance (M‐2020‐0114) at the request of the European Commission. The full pest survey card for Melampsora medusae f. sp. tremuloidis is published and available online in the EFSA Pest Survey Card gallery at the following link and will be updated whenever new information becomes available: https://efsa.europa.eu/plants/planthealth/monitoring/surveillance/melampsora-medusae-tremuloidis. [ABSTRACT FROM AUTHOR]

Background: Spondylitis is a spinal infection which has been increasing in susceptible populations globally. This disease is caused by various microorganisms. Fungal spondylitis is rare in clinical practice and is strongly associated with immunosuppression and diabetes. Here, we report a case of suspected ca Melampsora spondylitis. Case presentation: A patient was suspected with Melampsora spondylitis at the L3-S1 level. The patient received two surgical operations and antifungal treatments. The next-generation sequencing (NGS) analysis of the tissue specimen obtained during the two surgical procedures confirmed the diagnosis of Melampsora spondylitis. The patient was successfully treated with voriconazole, vancomycin, and meropenem following surgical debridement with pedicle screw internal fixation. Conclusion: The diagnosis of fungal spondylitis is often delayed or missed. Physicians should consider fungal spondylitis in the differential diagnosis of neck pain to facilitate early treatment and prevent spinal cord injury and disability. Although fungal infections often occur in immunocompromised patients, fungal spondylitis has also been reported in immunocompetent patients in recent years. In addition, Candida albicans is usually considered a common bacterium in fungal spondylitis. This case underscores the need to develop more advanced diagnostic and therapeutic techniques to identify the pathogenic bacteria associated with fungal spondylitis besides Candida albicans.

Variation in fitness components can be linked in some cases to variation in key traits. Metric traits that lie at the intersection of development, defense, and ecological interactions may be expected to experience environmental selection, informing our understanding of evolutionary and ecological processes. Here, we use quantitative genetic and population genomic methods to investigate disease dynamics in hybrid and non‐hybrid populations. We focus our investigation on morphological and ecophysiological traits which inform our understanding of physiology, growth, and defense against a pathogen. In particular, we investigate stomata, microscopic pores on the surface of a leaf that regulate gas exchange during photosynthesis and are sites of entry for various plant pathogens. Stomatal patterning traits were highly predictive of disease risk. Admixture mapping identified a polygenic basis of disease resistance. Candidate genes for stomatal and disease resistance map to the same genomic regions and experienced positive selection. Genes with functions to guard cell homeostasis, the plant immune system, components of constitutive defenses, and growth‐related transcription factors were identified. Our results indicate positive selection acted on candidate genes for stomatal patterning and disease resistance, potentially acting in concert to structure their variation in naturally formed backcrossing hybrid populations. [ABSTRACT FROM AUTHOR]

In June, 2021, laurel willow (Salix pentandra) near Slave Lake, Alberta, was found to be infected by a Melampsora sp. that produced bright yellow urediniospores in uredia that were present on catkins, leaves, and stems. All Melampsora species previously reported in Canada are recorded as infecting leaves; therefore, further investigation was undertaken to ascertain the identity of this pathogen. To assess the relationship between this specimen and other Melampsora spp. previously collected from Canada, samples of willow leaves infected by Melampsora spp. were sourced from mycological herbariums located at the Laurentian Forestry Centre (QFB) and the Northern Forestry Centre (CFB, WINF(M)). DNA sequence data from the internal transcribed spacer (ITS) ribosomal RNA region of the fresh specimen, herbarium specimens, and DNA sequence data deposited within GenBank, were used to conduct a phylogenetic analysis. Sequencing and BLAST analysis of the material from the sample resulted in a 99.3% sequence identity match to Melampsora epitea “Mel J” collected from Larix laricina in New York State. The ITS sequence from the herbarium sample WINF(M)7356 (described as M. abieti-capraearum from Manitoba) had 100.0% identity with the Alberta sample. Additionally, specimens WINF(M)11892 (Melampsora sp. from Manitoba) and CFB8931 (Melampsora sp. from the Yukon) had 99.0% sequence identity with the Alberta sample. From these results we applied the identity of M. epitea to the rust discovered in Slave Lake, AB. With the current emphasis on willows for bioenergy production in Canada, growers must remain vigilant for this pathogen and the damages it could cause to willow plantations.

Forests are at increasing risk from pathogen outbreak. Climate change for example enhance the risk of local disease outbreaks, and naturalization of exotic pathogens may follow human activities, warranting robust pest surveillance routines to support forest management. Melampsora pinitorqua (pine twisting rust) is of concern in Swedish forestry, and here we evaluate the use of visible rust scores (VRS) on its obligate summer host, European aspen (Populus tremula) as a tool for quantification of the pathogen. With use of species-specific primers, we could detect the native rust, but we failed to detect two exotic rusts (M. medusae and M. larici-populina). We found that aspen genotype determined the presence of fungal genetic markers (amplifying the ITS2 region of the fungal rDNA sequence) as well as DNA sequences specific to M. pinitorqua. We correlated VRS with the amount of fungal DNA in the same leaf, and we related the findings to aspen genotype-specific parameters such as the ability to synthesize and store leaf condensed tannins (CT). At the genotype level both positive and negative relationships were observed between CTs, fungal markers, and rust infestations. However, at the population level, foliar CT concentrations correlated negatively with general fungal- and rust-specific marker abundances. Our results, therefore, do not support the use of VRS to assess Melampsora infestation in Aspen. They do, however, suggest that the relationship between European aspen and rust infestation may be characterized as autochthonous in northern Sweden. [ABSTRACT FROM AUTHOR]

Forests are at increasing risk from pathogen outbreak. Climate change for example enhance the risk of local disease outbreaks, and naturalization of exotic pathogens may follow human activities, warranting robust pest surveillance routines to support forest management. Melampsora pinitorqua (pine twisting rust) is of concern in Swedish forestry, and here we evaluate the use of visible rust scores (VRS) on its obligate summer host, European aspen (Populus tremula) as a tool for quantification of the pathogen. With use of species-specific primers, we could detect the native rust, but we failed to detect two exotic rusts (M. medusae and M. larici-populina). We found that aspen genotype determined the presence of fungal genetic markers (amplifying the ITS2 region of the fungal rDNA sequence) as well as DNA sequences specific to M. pinitorqua. We correlated VRS with the amount of fungal DNA in the same leaf, and we related the findings to aspen genotype-specific parameters such as the ability to synthesize and store leaf condensed tannins (CT). At the genotype level both positive and negative relationships were observed between CTs, fungal markers, and rust infestations. However, at the population level, foliar CT concentrations correlated negatively with general fungal- and rust-specific marker abundances. Our results, therefore, do not support the use of VRS to assess Melampsora infestation in Aspen. They do, however, suggest that the relationship between European aspen and rust infestation may be characterized as autochthonous in northern Sweden.

Melampsora larici-populina (Mlp), M. medusae (Mmed), M. magnusiana (Mmag), and M. pruinosae (Mpr) are epidemic rust fungi in China. The first two are macrocyclic rust fungi distributed in temperate humid environments. The latter two are hemicyclic rusts, mainly distributed in arid and semi-arid areas. Ontogenetic variation that comes with this arid-resistance is of great interest—and may help us predict the influence of a warmer, drier, climate on fungal phylogeny. To compare the differences in the life history and ontogeny between the two types of rust, we cloned mating type genes, STE3.4 and STE3.3 using RACE-smart technology. Protein structures, functions, and mutant loci were compared across each species. We also used microscopy to compare visible cytological differences at each life stage for the fungal species, looking for variation in structure and developmental timing. Quantitative PCR technology was used to check the expression of nuclear fusion and division genes downstream of STE3.3 and STE3.4. Encoding amino acids of STE3.3 and STE3.4 in hemicyclic rusts are shorter than these in the macrocyclic rusts. Both STE3.3 and STE3.4 interact with a protein kinase superfamily member EGG12818 and an E3 ubiquitin protein ligase EGG09709 directly, and activating G-beta conformational changes. The mutation at site 74th amino acid in the conserved transmembrane domain of STE3.3 ascribes to a positive selection, in which alanine (Ala) is changed to phenylalanine (Phe) in hemicyclic rusts, and a mutation with Tyr lost at site 387th in STE3.4, where it is the binding site for β-D-Glucan. These mutants are speculated corresponding to the insensitivity of hemicyclic rust pheromone receptors to interact with MFa pheromones, and lead to Mnd1 unexpressed in teliospora, and they result in the diploid nuclei division failure and the sexual stage missing in the life cycle. A Phylogenic tree based on STE3.4 gene suggests these two rust types diverged about 14.36 million years ago. Although these rusts share a similar uredia and telia stage, they show markedly different wintering strategies. Hemicyclic rusts overwinter in the poplar buds endophytically, their urediniospores developing thicker cell walls. They form haustoria with a collar-like extrahaustorial membrane neck and induce host thickened callose cell walls, all ontogenetic adaptations to arid environments. [ABSTRACT FROM AUTHOR]

Closely related species are expected to have similar functional traits due to shared ancestry and phylogenetic inertia. However, few tests of this hypothesis are available for plant‐associated fungal symbionts. Fungal leaf endophytes occur in all land plants and can protect their host plant from disease by a variety of mechanisms, including by parasitizing pathogens (e.g., mycoparasitism). Here, we tested whether phylogenetic relatedness among species of Cladosporium, a widespread genus that includes mycoparasitic species, predicts the effect of this endophyte on the severity of leaf rust disease. First, we used congruence among different marker sequences (i.e., genealogical concordance phylogenetic species recognition criterion) to delimit species of Cladosporium. Next, in a controlled experiment, we quantified both mycoparasitism and disease modification for the selected Cladosporium species. We identified 17 species of Cladosporium; all the species reduced rust disease severity in our experiment. Cladosporium phylogeny was a significant predictor of mycoparasitism. However, we did not observe a phylogenetic effect on disease severity overall, indicating that other mechanism/s operating independently of shared ancestry also contributed to endophyte effects on disease severity. Indeed, a second experiment showed that Cladosporium endophyte exudates (no live organism) from divergent species groups equally reduced disease severity. Our results reveal that multiple mechanisms contribute to the protective effects of an endophyte against a plant pathogen, but not all traits underlying these mechanisms are phylogenetically conserved. [ABSTRACT FROM AUTHOR]

Abstract Plant defense against pathogens includes a range of mechanisms, including, but not limited to, genetic resistance, pathogen‐antagonizing endophytes, and pathogen competitors. The relative importance of each mechanism can be expressed in a hierarchical view of defense. Several recent studies have shown that pathogen antagonism is inconsistently expressed within the plant defense hierarchy. Our hypothesis is that the hierarchy is governed by contingency rules that determine when and where antagonists reduce plant disease severity. Here, we investigated whether pathogen competition influences pathogen antagonism using Populus as a model system. In three independent field experiments, we asked whether competition for leaf mesophyll cells between a Melampsora rust pathogen and a microscopic, eriophyid mite affects rust pathogen antagonism by fungal leaf endophytes. The rust pathogen has an annual, phenological disadvantage in competition with the mite because the rust pathogen must infect its secondary host in spring before infecting Populus. We varied mite–rust competition by utilizing Populus genotypes characterized by differential genetic resistance to the two organisms. We inoculated plants with endophytes and allowed mites and rust to infect plants naturally. Two contingency rules emerged from the three field experiments: (a) Pathogen antagonism by endophytes can be preempted by host genes for resistance that suppress pathogen development, and (b) pathogen antagonism by endophytes can secondarily be preempted by competitive exclusion of the rust by the mite. Synthesis: Our results point to a Populus defense hierarchy with resistance genes on top, followed by pathogen competition, and finally pathogen antagonism by endophytes. We expect these rules will help to explain the variation in pathogen antagonism that is currently attributed to context dependency.

Premise: The evolution of sex chromosomes is driven by sexual dimorphism, yet it can be challenging to document sexually dimorphic traits in dioecious plant species. At the genetic level, sexual dimorphism can be identified through sequence variation between females and males associated with sexually antagonistic traits and different fitness optima. This study aims to examine sexual dimorphism for 26 traits in three populations of Salix purpurea (a diversity panel and F1 and F2 populations) and determine the effect of the traits on biomass yield, a key trait in Salix bioenergy crops across multiple years, locations, and under manipulated growth conditions. Methods: Sexual dimorphism was evaluated for morphological, phenological, physiological, and wood composition traits in a diversity panel of unrelated S. purpurea accessions and in full‐sib F1 and F2 families produced through controlled cross pollinations and grown in replicated field trials. Results: We observed sexual dimorphism in the timing of development for several traits that were highly predictive of biomass yield across three populations of S. purpurea. Across all populations and years surveyed, males had significantly shallower branching angle. Male plants highly predictive of biomass yield across three populations of S. purpurea also accumulated more nitrogen under fertilizer amendment as measured by SPAD in the diversity panel and had greater susceptibility to the rust fungus Melampsora americana in the F2 family. Allometric modelling of biomass yield showed an effect of sex and of location on the interaction between yield and stem height. Conclusions: These results provide evidence of sexual dimorphism for certain traits in S. purpurea that may be involved in sex chromosome evolution. [ABSTRACT FROM AUTHOR]

Organisms are constantly challenged by pathogens and pests, which can drive the evolution of growth‐defense strategies. Plant stomata are essential for gas exchange during photosynthesis and conceptually lie at the intersection of the physiological demands of growth and exposure to foliar fungal pathogens. Generations of natural selection for locally adapted growth‐defense strategies can eliminate variation between traits, potentially masking trade‐offs and selection conflicts that may have existed in the past. Hybrid populations offer a unique opportunity to reset the clock on selection and to study potentially maladaptive trait variation before selection removes it. We study the interactions of growth, stomatal, ecopysiological, and disease resistance traits in poplars (Populus) after infection by the leaf rust Melampsora medusae. Phenotypes were measured in a common garden and genotyped at 227K SNPs. We isolate the effects of hybridization on trait variance, discover correlations between stomatal, ecophysiology, and disease resistance, examine trade‐offs and selection conflicts, and explore the evolution of growth‐defense strategies potentially mediated by selection for stomatal traits on the upper leaf surface. These results suggest an important role for stomata in determining growth‐defense strategies in organisms susceptible to foliar pathogens, and reinforces the contribution of hybridization studies toward our understanding of trait evolution. [ABSTRACT FROM AUTHOR]

Plant defense against pathogens includes a range of mechanisms, including, but not limited to, genetic resistance, pathogen‐antagonizing endophytes, and pathogen competitors. The relative importance of each mechanism can be expressed in a hierarchical view of defense. Several recent studies have shown that pathogen antagonism is inconsistently expressed within the plant defense hierarchy. Our hypothesis is that the hierarchy is governed by contingency rules that determine when and where antagonists reduce plant disease severity.Here, we investigated whether pathogen competition influences pathogen antagonism using Populus as a model system. In three independent field experiments, we asked whether competition for leaf mesophyll cells between a Melampsora rust pathogen and a microscopic, eriophyid mite affects rust pathogen antagonism by fungal leaf endophytes. The rust pathogen has an annual, phenological disadvantage in competition with the mite because the rust pathogen must infect its secondary host in spring before infecting Populus. We varied mite–rust competition by utilizing Populus genotypes characterized by differential genetic resistance to the two organisms. We inoculated plants with endophytes and allowed mites and rust to infect plants naturally.Two contingency rules emerged from the three field experiments: (a) Pathogen antagonism by endophytes can be preempted by host genes for resistance that suppress pathogen development, and (b) pathogen antagonism by endophytes can secondarily be preempted by competitive exclusion of the rust by the mite.Synthesis: Our results point to a Populus defense hierarchy with resistance genes on top, followed by pathogen competition, and finally pathogen antagonism by endophytes. We expect these rules will help to explain the variation in pathogen antagonism that is currently attributed to context dependency. [ABSTRACT FROM AUTHOR]

Summary: Poplar trees synthesize flavan‐3‐ols (catechin and proanthocyanidins) as a defense against foliar rust fungi, but the regulation of this defense response is poorly understood. Here, we investigated the role of hormones in regulating flavan‐3‐ol accumulation in poplar during rust infection.We profiled levels of defense hormones, signaling genes, and flavan‐3‐ol metabolites in black poplar leaves at different stages of rust infection. Hormone levels were manipulated by external sprays, genetic engineering, and drought to reveal their role in rust fungal defenses.Levels of salicylic acid (SA), jasmonic acid, and abscisic acid increased in rust‐infected leaves and activated downstream signaling, with SA levels correlating closely with those of flavan‐3‐ols. Pretreatment with the SA analog benzothiadiazole increased flavan‐3‐ol accumulation by activating the MYB–bHLH–WD40 complex and reduced rust proliferation. Furthermore, transgenic poplar lines overproducing SA exhibited higher amounts of flavan‐3‐ols constitutively via the same transcriptional activation mechanism. These findings suggest a strong association among SA, flavan‐3‐ol biosynthesis, and rust resistance in poplars. Abscisic acid also promoted poplar defense against rust infection, but likely through stomatal immunity independent of flavan‐3‐ols. Jasmonic acid did not confer any apparent defense responses to the fungal pathogen.We conclude that SA activates flavan‐3‐ol biosynthesis in poplar against rust infection. [ABSTRACT FROM AUTHOR]

Rust is one of the most important biotic stress factors that affect poplars. The aims of this work were: (i) to analyze the changes in growth and nitrogen (N) accumulation in Populus deltoides W. Bartram ex Marshall plants infected with rust (Melampsora medusae Thümen.) and to determine how internal N stores are affected by the disease, in plants growing under two N availabilities in the soil; and (ii) to evaluate the impact of rust in the early sprout in the following growing season and the cumulative effect of the disease after repeated infections. Two clones with different susceptibility to rust were analyzed. At leaf level, rust reduced gas exchange capacity, water conductance in liquid phase and photosynthetic rate in both clones. At plant level, rust reduced plant growth, accelerated leaf senescence and abscission occurred with a higher concentration of leaf N. Even though N concentration in stems and roots were not significantly reduced by rust, total N accumulation in perennial tissues was reduced in infected plants. The vigor of the early sprout of plants infected by rust in the previous season was lower than that of non-infected plants. Therefore, rust affects plant growth by reducing the photosynthetic capacity and leaf area duration, and by decreasing internal nutrient recycling. As nutrient reserves in perennial tissues are lower, rust infection reduces not only the growth of the current season, but also has a cumulative effect on the following years. The reduction of growth was similar in both clones. High availability of N in the soil had no effect on leaf physiology but increased plant growth, delayed leaf senescence and abscission, and increased total N accumulation. If fertilization increases plant growth (stem and root dry mass) it can mitigate the negative effect of the pathogen in the reduction of nutrient storages and future growth. [ABSTRACT FROM AUTHOR]

Summary: Plants release complex volatile blends after separate attack by herbivores and pathogens, which play many roles in interactions with other organisms. Large perennials are often attacked by multiple enemies, but the effect of combined attacks on volatile emission is rarely studied, particularly in trees.We infested Populus nigra trees with a pathogen, the rust fungus Melampsora larici‐populina, and Lymantria dispar caterpillars alone and in combination. We investigated poplar volatile emission and its regulation, as well as the behavior of the caterpillars towards volatiles from rust‐infected and uninfected trees.Both the rust fungus and the caterpillars alone induced volatile emission from poplar trees. However, the herbivore‐induced volatile emission was significantly reduced when trees were under combined attack by the herbivore and the fungus. Herbivory induced terpene synthase transcripts as well as jasmonate concentrations, but these increases were suppressed when the tree was additionally infected with rust. Caterpillars preferred volatiles from rust‐infected over uninfected trees.Our results suggest a defense hormone crosstalk upon combined herbivore–pathogen attack in poplar trees which results in lowered emission of herbivore‐induced volatiles. This influences the preference of herbivores, and might have other far‐reaching consequences for the insect and pathogen communities in natural poplar forests. See also the Editorial by Kessler, 220: 655–658. [ABSTRACT FROM AUTHOR]

Melampsora larici-populina Kleb. is a Eurasian species that causes leaf rust of many species of Populus from sections Tacamahaca and Aigeiros. This rust has been inadvertently introduced to many parts around the world affecting poplar species that grow naturally, which may be susceptible to this fungus. In Chile, early European settlers introduced poplars; rust, attributed to M. larici-populina, has been reported in these trees since 1918. However, a modern confirmation has been lacking, and pathogenic variation of the Chilean population of M. larici-populina has not been investigated. Using a morphological analysis of urediniospores and sequencing of internal transcribed spacer (ITS) regions, we confirmed the presence of M. larici-populina in Chile. ITS regions exhibited 100% homology with M. larici-populina. Scanning electron microscopy showed that spores were 26 to 47 pm in length. 13 to 16 pm in width, and echinulate except for apices, which are smooth, characteristics described for this species of rust. The variability of M. larici-populina is characterized by the presence of pathotypes, which allows the fungus to infect despite the resistance of certain poplar hybrids. We concluded that the identified spores belong to M. larici-populina, with virulences 1.2, 3. 4, 5, and 6. These results describe variation in virulence of M. larici-populina, which suggests the presence of a sexual stage in Chile. [ABSTRACT FROM AUTHOR]

A rust fungus attack was observed on Tree Spurge (Euphorbia dendroides) in two regions of southern Italy. Uredinia and telia of a Melampsora species were detected on the leaves, bracts of the cyathia and capsules of E. dendroides. On the basis of the morphological characteristics and rDNA ITS sequences of the above mentioned structures, the fungus was identified as M. gelmii. This report suggests that M. gelmii seems to be a specific taxon in the M. euphorbiae complex and to be more widespread than previously detected in Italy. Further investigations within the M. euphorbiae complex would be desirable in order to elucidate the identity of M. gelmii as a peculiar form of a still poorly explored species. [ABSTRACT FROM AUTHOR]

Following a request from the European Commission, the EFSA Plant Health Panel performed a pest categorisation of Melampsora medusae, a well-defined and distinguishable fungal species of the family Melampsoraceae. The pathogen is regulated in Annex IAI of Council Directive 2000/29/EC as a harmful organism whose introduction into the EU is banned. M. medusae is a heteroecious rust fungus with Populus spp. as primary telial hosts and various conifers (Larix, Pinus, Pseudotsuga, Abies, Picea and Tsuga spp.) as secondary aecial hosts. M. medusae is native to North America and has spread to South America, Africa, Asia, Oceania, as well as the EU, where M. medusae f. sp. deltoidae has been reported with a restricted distribution and low impacts from Belgium, south-west France and southern Portugal. The pest could spread to other EU countries, via dissemination of spores, movement of host plants for planting and cut branches. Climate is assumed not to be a limiting factor for the establishment of the pathogen in the EU. M. medusae is the most widespread and important Melampsora rust in North America. In western Canada, extensive damage has been reported to conifers and Populus spp. in nurseries and plantations as well as in woodlands. M. medusae is damaging in both Australia and New Zealand. The pest could have economic and environmental impacts in the EU if aggressive isolates of M. medusae were introduced into the EU. Import prohibition of host plants for planting is an available measure to reduce the risk of further introductions. Some resistant Populus cultivars are available. Moreover, increasing the genetic diversity of poplar plantations can prevent disease impacts. The main uncertainty concerns the factors explaining the low pathogenicity of the populations of M. medusae present in the EU. The criteria assessed by the Panel for consideration as a potential quarantine pest are met (the pest is present, but with a restricted distribution, and is officially under control). Given that plants for planting are not the main pathway of spread, not all criteria for consideration as a regulated non-quarantine pest are met. [ABSTRACT FROM AUTHOR]

In boreal woody plants, concentrations of defensive phenolic compounds are expected to be at a high level during the juvenile phase and decrease in maturity, although there is variation between plant species. Females of dioecious species, like most of the Salicaceae, are expected to invest their resources in defense and reproduction, while males are expected to be more growth-oriented. We studied age- and sex-dependent changes in leaf and stem phenolics, and in height and diameter growth in a dioecious Salix myrsinifolia plants over a seven-year time period. In addition, we registered flowering as well as rust damage in the leaves. From the first year and throughout ontogenetic development from juvenile to adult phases, there was no significant change in the concentrations of any of the studied compounds in the leaves of S. myrsinifolia. In the stems, the concentrations of six out of 43 identified compounds decreased slightly with age, which may be partly explained by dilution caused by the increment in stem diameter with age. The fairly steady chemistry level over seven years, accompanied by moderate genotypic phenolic variation, indicates important roles of chemical defenses against herbivory for this early-successional species. [ABSTRACT FROM AUTHOR]

Melampsora epitea(Pucciniales, Basidiomycota) has been widely known as the main rust pathogen on willows, but its species boundary has been extremely confusing.Melampsora epiteawas often confused with several morphologically similar species, such asM. arctica, M. humilis, andM. epiphylla. Recent DNA-based studies using specimens from North America and Europe suggested thatM. epiteawas a complex of several phylogenetic species. In this study, we examined the species boundary ofM. epiteabased on rDNA phylogeny and the general mixed Yule coalescent (GMYC) model from 534 specimens. Twelve distinct species corresponding to 10 known species and 2 new species were recognized from East Asian specimens. We also evaluated the association of morphological characteristics, geographic origins, and host ranges with phylogenetic relationships. Our results revealed that several previously overlooked morphological characters in uredinial and telial stages were informative in delimitingM. epiteaand allied species. Two novel species,M. salicis-futuraeandM. salicis-triandrae, are described and compared with closely related species. A lectotype forM. epiteaand epitypes forM. epitea, M. epiphylla, andM. humilisare designated to stabilize the use of names. [ABSTRACT FROM PUBLISHER]

WRKY transcription factors play important roles in response to diverse environmental stresses, but exact functions of these proteins in poplar defense are still largely unknown. In a previous study, we have shown that poplar WRKY89 is induced by salicylic acid (SA) treatment and plays an important role in resistance against fungi in transgenic poplars. Here, we determined an increase in transcript levels of Group IIa WRKY members in transgenic poplars overexpressing WRKY89 using quantitative real-time polymerase chain reaction analysis. Yeast one-hybrid assay showed that PtrWRKY18 and PtrWRKY35 were potential target genes of WRKY89. Furthermore, we demonstrated that PtrWRKY18 and PtrWRKY35 were localized in the nucleus, and exhibited no transcription activation activity. Constitutive overexpression of PtrWRKY18 and PtrWRKY35 in poplars activated pathogenesisrelated genes, and increased resistance to the biotrophic pathogen Melampsora. The results also provided support for the involvement of SA-mediated signaling in Melampsora resistance. [ABSTRACT FROM AUTHOR]

The TIFY domain contains approximately 36 conserved amino acids that form the core motif TIF[F/Y]XG, and they were reported to play important roles in plant growth, tissue development and defense regulation. Moreover, more and more evidence has shown that some members of the TIFY gene family perform their functions by modulating plant hormone signaling pathways. Poplar trees are found worldwide, and they comprise approximately 30 species. Benefit from the importance of poplar and its advanced platform, this tree is considered to be the model perennial plant. Here, we conducted a genome-wide identification of TIFY genes in poplar, and 24 TIFY genes were found. These 24 TIFY genes were assigned to different subfamilies according to the presence or absence of domains and motifs that they harbored. Careful analyses of their locations, structures, evolution and duplication patterns revealed an overview of this gene family in poplar. The expression profiles of these 24 TIFY genes were then analyzed in different tissues using publicly available expression data; their expression profiles following different JA/SA treatments and infection with leaf rust pathogen were also carefully examined by qRT-PCR assays. Based on their expression profiles, the functions of a number of TIFY genes could be predicted. By performing this study, we have provided valuable information for further functional characterisation of TIFY genes in poplar and candidate genes for the improvement of poplar disease resistance. [ABSTRACT FROM AUTHOR]

Host-parasite systems provide convincing examples of Red Queen co-evolutionary dynamics. Yet, a key process underscored in Van Valen's theory - that arms race dynamics can result in extinction - has never been documented. One reason for this may be that most sampling designs lack the breadth needed to illuminate the rapid pace of adaptation by pathogen populations. In this study, we used a 25-year temporal sampling to decipher the demographic history of a plant pathogen: the poplar rust fungus, Melampsora larici-populina. A major adaptive event occurred in 1994 with the breakdown of R7 resistance carried by several poplar cultivars widely planted in Western Europe since 1982. The corresponding virulence rapidly spread in M. larici-populina populations and nearly reached fixation in northern France, even on susceptible hosts. Using both temporal records of virulence profiles and temporal population genetic data, our analyses revealed that (i) R7 resistance breakdown resulted in the emergence of a unique and homogeneous genetic group, the so-called cultivated population, which predominated in northern France for about 20 years, (ii) selection for Vir7 individuals brought with it multiple other virulence types via hitchhiking, resulting in an overall increase in the population-wide number of virulence types and (iii) - above all - the emergence of the cultivated population superseded the initial population which predominated at the same place before R7 resistance breakdown. Our temporal analysis illustrates how antagonistic co-evolution can lead to population extinction and replacement, hence providing direct evidence for the escalation process which is at the core of Red Queen dynamics. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) were found to widely exist in eukaryotes and play important roles in most biological processes. Trees belonging to Populus spp. are economically important because of their fast-growing characteristics and wide use for wood, pulp, paper, and fuel. However, the prevalence of leaf rust disease caused by Melampsora spp. on some species of Populus severely affects their growth and decreases wood production. Therefore, the identification and characterization of lncRNAs involved in the defense of Melampsora spp. would not only help us to understand plant-pathogen interactions but also provide genetic elements for producing disease-resistant poplars. In the present study, an RNA-Seq transcriptome analysis was performed for poplar leaves in response to early infection by M. larici-populina. The hybrid variety 'NL895' ( P. × euramericana) was used as plant material. A total of 3994 lncRNAs were identified by mining the RNA-Seq data. The identified lncRNAs had lower abundance, fewer exons, and shorter lengths when compared to protein-coding genes. In addition, 53 lncRNAs were differentially expressed between treatments and controls. Two of these 53 lncRNAs were predicted to compete to be the target of miRNAs. Moreover, the differentially expressed lncRNAs were found to be preferentially located in close proximity to the protein-coding genes they co-expressed. In this study, we were able to show that lncRNAs may play important roles in plant-pathogen interactions in the poplar. This study also improves our understanding of how plants deploy their defense system when encountering biotic stresses. [ABSTRACT FROM AUTHOR]

The biomass of Salix viminalis is the most highly valued source of green energy, followed by S. schwerinii, S. dasyclados and other species. Significant variability in productivity and leaf rust resistance are noted both within and among willow species, which creates new opportunities for improving willow yield parameters through selection of desirable recombinants supported with molecular markers. The aim of this study was to identify quantitative trait loci (QTLs) linked with biomass yield-related traits and the resistance/susceptibility of Salix mapping population to leaf rust. The experimental material comprised a mapping population developed based on S. viminalis x S. schwerinii hybrids. Phenotyping was performed on plants grown in a field experiment that had a balanced incomplete block design with 10 replications. Based on a genetic map, 11 QTLs were identified for plant height, 9 for shoot diameter, 3 for number of shoots and 11 for resistance/susceptibility to leaf rust. The QTLs identified in our study explained 3%-16% of variability in the analyzed traits. Our findings make significant contributions to the development of willow breeding programs and research into shrubby willow crops grown for energy. [ABSTRACT FROM AUTHOR]

Some quantitative consequences of poplar rust attacks (Melampsora larici-populina and M. allii-populina) were analysed, both in absence and in presence of some preventive chemical treatments. Their effectiveness was previously assessed only partially and without evaluations of plant behaviour in the following vegetative season. Three IBS systemic triazoles (tebuconazole, cyproconazole and epoxyconazole), the translaminar cytotropic mandipropamid and a mixture of cyproconazole and azoxystrobin (methoxyacrilate similar to strobilurines) were tested on one-year plantlets of the susceptible clone ’ÂÂNeva’ (Populus ×canadensis). The products were sprayed twice in August 2010, the second treatment twenty days after the first one. The leaf area covered by uredinia was assessed in several dates from the beginning of August until the end of September, as well as the percentage of still living leaves on the plant (last ten days of October). In May 2011, the average leaf surface of side shoots close to the top of the crown and the Chlorophyll Content Index (CCI) were measured. The tested fungicides dramatically reduced the infections, except mandipropamid which showed no effectiveness. Tebuconazole and cyproconazole, either alone or in mixture with azoxystrobin, have offered the best performance (at the end of September, less than 10% of leaf surface covered by uredinia vs. over 50% of the control and mandipropamid treatment), followed by epoxyconazole that is anyway satisfying (about 15% of surface covered by uredinia). The systemic properties of these fungicides allowed a preventive action on leaves sprouted after both of the treatments as well, thus uredinium eruption was limited to 20% of leaf surface instead 40% on the test thesis. The same effectiveness ranking was reiterated for the percentage of still living leaves on the plant at the end of October (about 45% vs. 4% of the control test) and, during the following season, for the average leaf surface of side shoots (over 800 cm2 vs. less than 400 cm2 of the test) and for the CCI parameter (over 21 vs. 16). Thus, the phylloptosis induced by rusts involves a seriously impaired leaf sprouting during the season following the attacks, both as regards tissue amount and its photosynthetic efficiency.

The genetic consequences of range expansions have generally been investigated at wide geographical and temporal scales, long after the colonization event. A unique ecological system enabled us to both monitor the colonization dynamics and decipher the genetic footprints of expansion over a very short time period. Each year an epidemic of the poplar rust ( Melampsora larici-populina) expands clonally and linearly along the Durance River, in the Alps. The colonization dynamics observed in 2004 showed two phases with different genetic outcomes. Upstream, fast colonization maintained high genetic diversity. Downstream, the colonization wave progressively faltered, diversity eroded, and differentiation increased, as expected under recurrent founder events. In line with the high dispersal abilities of rust pathogens, we provide evidence for leapfrog dispersal of clones. Our results thus emphasize the importance of colonization dynamics in shaping spatial genetic structure in the face of high gene flow. [ABSTRACT FROM AUTHOR]

The obligate biotrophic fungus Melampsora larici-populina ( Mlp) causes devastating poplar foliar rust disease. In pathogens, the Hog1-type mitogen-activated protein kinase (MAPK) gene is involved in both pathogenesis and the response to various stresses, including osmotic stress. However, there are few reports on its role in M. larici-populina. In the present study, we identified and characterized the Hog1-type MAPK gene, MlpHog1, in M. larici-populina. The MlpHog1 protein consists of 356 amino acids and contains a conserved TGY motif. The function of MlpHog1 was probed by mutant complementation studies. Expression of MlpHog1 could partially complement the osmoregulation, sexual reproduction and pathogenicity defects of the Fusarium graminearum Hog1 mutant and largely rescued the response of the mutant to cytoplasm membrane and oxidative stresses. These observations suggest that MlpHog1 may play a role in the infectious growth of M. larici-populina and the response of the fungus to various environmental stresses. [ABSTRACT FROM AUTHOR]

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimise parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analysed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.

Plant cells secrete a wide range of proteins in extracellular spaces in response to pathogen attack. The poplar rust-induced secreted protein (RISP) is a small cationic protein of unknown function that was identified as the most induced gene in poplar leaves during immune responses to the leaf rust pathogen Melampsora larici-populina, an obligate biotrophic parasite. Here, we combined in planta and in vitro molecular biology approaches to tackle the function of RISP. Using a RISP-mCherry fusion transiently expressed in Nicotiana benthamiana leaves, we demonstrated that RISP is secreted into the apoplast. A recombinant RISP specifically binds to M. laricipopulina urediniospores and inhibits their germination. It also arrests the growth of the fungus in vitro and on poplar leaves. Interestingly, RISP also triggers poplar cell culture alkalinisation and is cleaved at the C-terminus by a plant-encoded mechanism. Altogether our results indicate that RISP is an antifungal protein that has the ability to trigger cellular responses. [ABSTRACT FROM AUTHOR]

Background: Rust caused by Melampsora larici-populina is one of the most damaging diseases of poplars. Rust is considered to be a model pathogen for genetic studies because both pathogen and host genomes are available. The poplar 'Robusta', whose general rust resistance is defeated by virulent rust E4, provides suitable host material for studies of the gene regulation involved in rust resistance/susceptibility. In this study, we investigated the microRNA-mediated susceptible poplar gene expression regulation associated with the infection of virulent rust. We were particularly interested in delineating the host-pathogen interactions with a specific focus on microRNAs (miRNAs). Results: To study the susceptibility of poplar to M. larici-populina, small RNA (sRNA) libraries, a degradome cDNA library and digital gene expression libraries were constructed for rust-inoculated and rust-free susceptible poplar 'Robusta' leaves through high-throughput sequencing. Altogether, 12,722 regulating interactions were identified. The results delineated the framework of post-transcriptional regulation of gene expression in the susceptible poplar, which was infected by the virulent rust. The results indicated that pathogen-associated molecular patterns (PAMPs) and PAMP-triggered immunity were induced by the infection of virulent rust E4 and that miRNAs still functioned at this stage. After this stage, miRNA-regulated R genes, such as TIR-NBS-LRR and CC-NBS-LRR, were not fully functional. Additionally, the rust-responsive miRNAs did not regulate the signaling component genes related to the salicylic acid pathway or the hypersensitive response. Conclusions: We found that the defense-related post-transcriptional regulation of the susceptible poplar 'Robusta' functions normally only at the stage of PAMPs and PAMP-triggered immunity (PTI). More importantly, the miRNA-mediated post-transcriptional regulation of defense signal pathway genes were inactivated by the infection of virulent rust at the stage of effector-triggered susceptibility and during the following stages of salicylic acid and hypersensitive responses. This inactivation was the major characteristic of 'Robusta' susceptibility. [ABSTRACT FROM AUTHOR]

Needle pathogens of larch (Larix spp.) in the Nordic countries are under-studied. Their incidence in Finland tends to be low and local, and this may be a function of enemy release, since species of larch were introduced to the region. Here, the ecology and incidence of larch needle pathogens and the abiotic factors that also affect larch in northern Finland are reviewed. Field observations and related laboratory analyses during the past 35 years have mainly been obtained near the Kivalo Research Area within the Arctic Circle, Finnish Lapland. The relatively recent introduction of Hypodermella laricis is a primary focus. This pathogen is not only new to Nordic countries, but can cause severe outbreaks, defoliation and crown-thinning in the canopies of all ages of most planted larch species worldwide. Symptoms of H. laricis clearly differ from those of Mycosphaerella laricina; the latter has affected Larix sibirica at high latitudes for decades. The effects of Meria laricis, Lophodermium laricinum, various rust fungi, and wind and frost are also discussed. [ABSTRACT FROM AUTHOR]