Genetic evidence for three discrete taxa of Melampsora (Pucciniales) affecting willows (Salix spp.) in New York State

Rust fungi in the genus Melampsora (Pucciniales) are the most important pathogens of shrub willows (Salix spp.) cultivated for biomass in New York State and temperate regions worldwide. The taxonomy and species identification of these fungi historically have been problematic as they are morphologically indistinguishable on willow and often have complex life histories. Melampsora of Salix in North America, therefore, have been circumscribed to the collective species Melampsora epitea Thüm. and further delineated to formae speciales by aecial host. Ribosomal DNA (rDNA) data was obtained from 75 collections/isolates of Melampsora in NY State affecting either native and cultivated Salix spp. or suspected alternate hosts. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian (BI) analyses were conducted on three data partitions (individual and concatenated): complete internal transcribed spacer (ITS) and partial large subunit (LSU) rDNA sequences for all collections. Analyses of the ITS and concatenated ITS-LSU sequences revealed that Melampsora on native and cultivated willows in NY State consisted of three phylogenetically delineable taxa (phylotaxa); monophyly for each phylotaxon was strongly supported by ML, MP, and BI credibility values. Phylotaxa were also delimited phylogenetically by aecial host: Alpine currant (Ribes alpinum), eastern larch (Larix laricina), or balsam fir (Abies balsamea).     

<Emphasis Type="Italic">Melampsora salicis-bakko</Emphasis>, a new species on willows in Japan evidenced by morphological and molecular phylogenetic analyses

Through the morphological and molecular examinations of Melampsora species on willows, we clarified the taxonomic identity of the rust specimens on Salix bakko, S. hultenii and S. leucopithecia from Japan and described the following rust fungus as a new species, Melampsora salicis-bakko. This rust fungus resembled M. caprearum in morphology of teliospores, but it differed from M. caprearum mainly in the density of spines on the urediniospores. Molecular phylogenetic analyses using the rDNA ITS region (complete ITS1, 5.8S rRNA gene and ITS2) revealed that M. salicis-bakko was monophyletic, and that this rust fungus was distinct from other Melampsora species, including M. caprearum.     

Correspondence of ectomycorrhizal diversity and colonisation of willows (Salix spp.) grown in short rotation coppice on arable sites and adjacent natural stands

Willows (Salix spp.) are mycorrhizal tree species sometimes cultivated as short rotation coppice (SRC) on arable sites for energy purposes; they are also among the earliest plants colonising primary successional sites in natural stands. The objective of this study was to analyse the degree of colonisation and diversity of ectomycorrhizal (EM) communities on willows grown as SRC in arable soils and their adjacent natural or naturalized stands. Arable sites usually lack ectomycorrhizal host plants before the establishment of SRC, and adjacent natural or naturalized willow stands were hypothesized to be a leading source of ectomycorrhizal inoculum for the SRC. Three test sites including SRC stands (Salix viminalis, Salix dasyclados, and Salix schwerinii) and adjacent natural or naturalized (Salix caprea, Salix fragilis, and Salix?×?mollissima) stands in central Sweden were investigated on EM colonisation and morphotypes, and the fungal partners of 36 of the total 49 EM fungi morphotypes were identified using molecular tools. The frequency of mycorrhizas in the natural/naturalized stands was higher (two sites) or lower (one site) than in the corresponding cultivated stands. Correspondence analysis revealed that some EM taxa (e.g. Agaricales) were mostly associated with cultivated willows, while others (e.g. Thelephorales) were mostly found in natural/naturalized stands. In conclusion, we found strong effects of sites and willow genotype on EM fungi formation, but poor correspondence between the EM fungi abundance and diversity in SRC and their adjacent natural/naturalized stands. The underlying mechanism might be selective promotion of some EM fungi species by more effective spore dispersal.     

Resistance toMelampsora larici-epitea leaf rust inSalix: analyses of quantitative trait loci

Quantitative resistance ofSalix toMelampsora larici-epitea leaf rust was studied in 2Salix mapping populations. One population was a backcross between aS. schwerinii ×S. viminalis hybrid andS. viminalis, and the other was an F₂ population betweenS. viminalis andS. dasyclados. A leaf disc bioassay was used to study the components of quantitative resistance (latent period, uredinia number, and uredinia size) to 3 isolates of the leaf rust. The analysis of quantitative trait loci (QTLs) revealed 9 genomic regions in the backcross population and 7 genomic regions in the F₂ population that were important for rust resistance, with QTLs explaining 8–26% of the phenotypic variation. An important genomic region was identified for the backcross population in linkage group 2, where QTLs were identified for all resistance components for 2 of the rust isolates. Four of the QTLs had overlapping mapping intervals, demonstrating a common genetic background for latent period, uredinia diameter, and uredinia number. QTLs specific to some rust isolates and to some resistance components were also found, indicating a combination of common and specific mechanisms involved in the various resistance components. Breeding implications in relation to these findings are discussed.     

Growth, herbivory and disease in relation to gender in Salix viminalis L.

Many species of dioecious plants show sex-related differences in growth rate and rates of attack by various herbivores and diseases. The common pattern is for males to grow faster than females and to be less well defended against herbivores. In willows (Salix spp.), the predominance of female-biased sex ratios has been ascribed in part to differential feeding by herbivores. In this study of Salix viminalis, seven families grown on agricultural land showed no gender-related variation in shoot biomass or rates of herbivory by insects (lepidopterans and cecidomyiids). However, Melampsora rust disease was found to be more severe on females than on males when the plants were in a non-reproductive stage. After flowering and seed-set females tended to be more affected in some families but less affected in others. Although, on average, there was a female bias in the sex ratio of S. viminalis, sex ratios differed significantly between families. These ratios were not related to any of the recorded biotic agents, but rather to relationships between families. These results are interpreted in terms of resource allocation between reproduction, growth and defence, and causes for divergence from the expected patterns are discussed. The results may have implications for S. viminalis breeding strategies where the aim is to produce biofuel. For instance, these findings suggest that gender can be ignored when selecting for a high growth rate and resistance to Melampsora and certain insect pests. Received: 2 November 1996 / Accepted: 8 February 1997     

Plant-mediated effects of different Salix species on the performance of the braconid parasitoid Perilitus brevicollis

The blue willow beetle, Phratora vulgatissima, is considered to be the most damaging herbivorous pest in Salix short-rotation coppices throughout Europe. The braconid parasitoid Perilitus brevicollis is an important natural enemy of Phratora. As several different Salix species are used in coppices, I investigated the bottom-up (tritrophic) effects of Salix on the parasitoid. Three host plants were studied: the introduced fast-growing S. viminalis, which is highly susceptible to the beetle; S. dasyclados, which is introduced and moderately-resistant to the beetle; and the native slow-growing Salix cinerea, which is not currently used in coppices. The identity of the host-plant species had significant effects on parasitoid larval development time; parasitoids developed rapidly on the susceptible S. viminalis and slowly on the moderately resistant S. dasyclados. Increased development time resulted in reduced adult longevity. Host-plant species identity also affected larval survival; 57%, 64%, and 49% of the parasitoids successfully completed larval development in beetles fed S. viminalis, S. cinerea, and S. dasyclados, respectively. Parasitoid development was also correlated with the body size of their beetle host, but this effect was independent of the identity of the host-plant species. The results of this study suggest that the parasitoid has higher survival and growth rates when it parasitizes beetles feeding on the common coppice species S. viminalis, but the performance of the parasite is reduced when the beetle feeds on the moderately-resistant S. dasyclados. Conversely, the omnivorous biocontrol agents sometimes used in these systems appear to perform better on S. dasyclados compared to S. viminalis. The results of this study suggest that Perilitus parasitoids and omnivorous beetle predators may provide complementary protection to Salix and therefore be useful in coppice management.     

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.     

QTL mapping of resistance to leaf rust in Salix

Genetic mapping of quantitative trait loci (QTL) for resistance to Melampsora leaf rust was performed in two willow families: the progeny from a backcross between Salix viminalis and a hybrid S. viminalis × Salix schwerinii (population S₁), and the F₁ progeny of a cross between two S. viminalis (population S₃). Disease levels were scored in the field for three consecutive years. In the laboratory, five different rust strains were sprayed onto leaf disks and the following resistance components were scored: latent period, diameter and number of uredinia, and flecking. One major QTL and 14 smaller were identified in the S₁ host population. One rust strain, that represents a Melampsora form with limited incidence on S. viminalis, showed lower aggressiveness to the S₁ host population together with a different pattern in QTLs. In the S₃ host population, we detected 13 QTLs for rust resistance, of which two were located to the same genomic regions as those found for the S₁ population. We showed that the strongest QTL co-segregated with a gene homologous to a known Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat resistance gene in poplar. The importance of the identified QTLs is discussed in relation to breeding for durable resistance.     

Biomass Harvest from Natural Willow Rings around Prairie Wetlands

The study followed the harvest of natural willow from three wetlands using a prototype modified agricultural round baler nicknamed a Bio-Baler. The study reports fuel characteristics and combustion testing of biomass harvested from natural willow rings. Composition of native willow species in the harvested willow rings was determined. We specifically measured regrowth of the biomass and number of regenerated stems per stump 1 year after harvest to determine how different willow species responded to mechanical biomass cutting with the Bio-Baler. The results of combustion testing for the natural willow were essentially similar to those with “conventional” wood chips or planted willows. The ash content was approximately 1.65%, slightly lower than for planted willow plantations. The calorific value of the natural willow was 19.6 MJ kg^?1 (dry basis) similar to what is expected for wood and planted willow. Four Salix species (Salix bebbiana Sarg., Salix petiolaris Sm., Salix eriocephala Michx., and Salix discolor Muhl) were identified in the willow rings. Stem biomass increased for all species except S. bebbiana after willow was harvested with the Bio-Baler. Overall, willow regeneration was not affected by mechanical harvesting compared to hand cutting. Regenerated stem density was 93 stems per square meter for mechanically harvested stumps compared to 105 stems per square meter for hand-pruned stumps. Based on the results, biomass harvested from natural willow rings has acceptable fuel characteristics when compared to purpose-grown willows and mechanical harvest with a Bio-Baler does not have a negative effect on willow regeneration.     

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.     

Variation in the uptake of Arsenic, Cadmium, Lead, and Zinc by different species of willows Salix spp. grown in contaminated soils

The experiment assessed the variability of in seven clones of willow plants of high biomass production (Salix smithiana S-218, Salix smithiana S-150, Salix viminalis S-519, Salix alba S-464, Salix ’Pyramidalis’ S-141, Salix dasyclados S-406, Salix rubens S-391). They were planted in a pots for three vegetation periods in three soils differing in the total content of risk elements. Comparing the calculated relative decrease of total metal contents in soils, the phytoextraction potential of willows was obtained for cadmium (Cd) and zinc (Zn), moderately contaminated Cambisol and uncontaminated Chernozem, where aboveground biomass removed about 30% Cd and 5% Zn of the total element content, respectively. The clones showed variability in removing Cd and Zn, depending on soil type and contamination level: S. smithiana (S-150) and S. rubens (S-391) demonstrated the highest phytoextraction effect for Cd and Zn. For lead (Pb) and arsenic (As), the ability to accumulate the aboveground biomass of willows was found to be negligible in both soils. The results confirmed that willow plants show promising results for several elements, mainly for mobile ones like cadmium and zinc in moderate levels of contamination. The differences in accumulation among the clones seemed to be affected more by the properties of clones, not by the soil element concentrations or soil properties. However, confirmation and verification of the results in field conditions as well as more detailed investigation of the mechanisms of cadmium uptake in rhizosphere of willow plants will be determined by further research.     

Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae

Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.     

Phylogenetic species recognition reveals host-specific lineages among poplar rust fungi

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.     

Morphological and biomechanical responses of floodplain willows to tidal flooding and salinity

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Molecular cytogenetic characterisation of Salix viminalis L. using repetitive DNA sequences

Salix viminalis L. (2n?=?38) is a diploid dicot species belonging to the Salix genus of the Salicaceae family. This short-rotation woody crop is one of the most important renewable bioenergy resources worldwide. In breeding for high biomass productivity, limited knowledge is available on the molecular cytogenetics of willow, which could be combined with genetic linkage mapping. The present paper describes the adaptation of a fluorescence in situ hybridisation (FISH) protocol as a new approach to analyse the genomic constitution of Salix viminalis using the heterologous DNA clones pSc119.2, pTa71, pTa794, pAs1, Afa-family, pAl1, HT100.3, ZCF1 and the GAA microsatellite marker. Three of the nine probes showed unambiguous signals on the metaphase chromosomes. FISH analysis with the pTa71 probe detected one major 18S-5.8S-26S rDNA locus on the short arm of one chromosome pair; however, the pTa794 rDNA site was not visible. One chromosome pair showed a distinct signal around the centromeric region after FISH with the telomere-specific DNA clone HT100.3. Two chromosome pairs were found to have pAs1 FISH signals, which represent a D-genome-specific insert from Aegilops tauschii. Based on the FISH study, a set of chromosomes with characteristic patterns is presented, which could be used to establish the karyotype of willow species.     

Phenotypic plasticity in host plant preference of the willow leaf beetle Phratora vulgatissima: the impact of experience made by adults

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

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.     

Isozyme Variation and Genetic Distances of Erysiphe graminis DC. Formae Speciales

Abstract Isozymes of ten different enzymes and unspecific stained proteins were used as biochemical genetic markers to study genetic variation within and between E. graminis ff. sp. hordei, avenae, secalis and tritici. In addition, grainproteins of the corresponding host species were examined. In each forma specialis, one genotype proved to be predominant. 131 distinct isozyme and 93 protein bands were distinguishable in these genotypes. However, divergent banding patterns differed only in 8 bands from the predominant banding patterns found within the formae speciales avena, secalis and tritici. The genetic relationships between powdery mildew formae speciales and host species were computed by cluster analysis from similarity (F) and dissimilarity (D) coefficients and illustrated by phylogenetic trees. Marked correspondence was found between E. graminis ff. sp. secalis and tritici (F: 82–90%). Lower homologies were obtained from the comparison ofthese formae speciales respectively with E. graminis ff. sp. hordei (F: 28–34%) and avenae (F: 24–32%). All phylogenetic trees constructed revealed the same arrangement classification of the formae speciales with similar graduation. The comparison of the host species revealed the highest similarity between S. cereale and T. aestivum (F: 74%). Regression analysis confirmed significant correlation between the genetic relationships within host species and powdery mildew formae speciales (r²= 0.81).     

Biotechnological methods of <Emphasis Type="Italic">in vitro</Emphasis> propagation in willows (<Emphasis Type="Italic">Salix</Emphasis> spp.)

Many populations of high-mountainous relic dioecious willows in Central Europe only consist of female individuals and are thus limited in their reproductive potential. We completed micropropagation experiments with shoot apexes and nodal segments of common and endangered willow (Salix) species, which can help to reintroduce autochthonous genotypes to their natural sites. Until recently, cultivation of green young shoot apexes of S. alba and S. lapponum showed the highest percentage of regeneration. We successfully applied the two-times-sterilisation due to high contamination of natural explants. The OK medium was the most efficient culture medium. In vitro propagation of willows with unisexual catkins, anther and ovule cultures were tested and optimised. Isolated anthers were cultivated on selected media and then microcallus and calluses of S. caprea and calluses of S. viminalis were formed on the A medium. Among various tested and optimised media for the ovule culture, the CP medium was the most efficient one. In this case, only the microcalluses of S. viminalis were observed. We developed biotechnological procedures that can be useful in conserving fragmented populations of high-mountainous willows.     

Integrating a Numerical Taxonomic Method and Molecular Phylogeny for Species Delimitation of Melampsora Species (Melampsoraceae,Pucciniales) on Willows in China

The species in genus Melampsora are the causal agents of leaf rust diseases on willows in natural habitats and plantations. However, the classification and recognition of species diversity are challenging because morphological characteristics are scant and morphological variation in Melampsora on willows has not been thoroughly evaluated. Thus, the taxonomy of Melampsora species on willows remains confused, especially in China where 31 species were reported based on either European or Japanese taxonomic systems. To clarify the species boundaries of Melampsora species on willows in China, we tested two approaches for species delimitation inferred from morphological and molecular variations. Morphological species boundaries were determined based on numerical taxonomic analyses of morphological characteristics in the uredinial and telial stages by cluster analysis and one-way analysis of variance. Phylogenetic species boundaries were delineated based on the generalized mixed Yule-coalescent (GMYC) model analysis of the sequences of the internal transcribed spacer (ITS1 and ITS2) regions including the 5.8S and D1/D2 regions of the large nuclear subunit of the ribosomal RNA gene. Numerical taxonomic analyses of 14 morphological characteristics recognized in the uredinial-telial stages revealed 22 morphological species, whereas the GMYC results recovered 29 phylogenetic species. In total, 17 morphological species were in concordance with the phylogenetic species and 5 morphological species were in concordance with 12 phylogenetic species. Both the morphological and molecular data supported 14 morphological characteristics, including 5 newly recognized characteristics and 9 traditionally emphasized characteristics, as effective for the differentiation of Melampsora species on willows in China. Based on the concordance and discordance of the two species delimitation approaches, we concluded that integrative taxonomy by using both morphological and molecular variations was an effective approach for delimitating Melampsora species on willows in China.