Melampsora rust species on biomass willows in central and north-eastern Germany

Melampsora willow rusts are the most important fungal pathogens in short rotation coppices of biomass willows. In the past, breeding programmes for rust resistant biomass willows concentrated on the distinction of races within the forma specialis Melampsora larici-epitea f. sp. larici-epitea typica that colonized Salix viminalis and related clones. In a new breeding program that is based on a wider range of willow species it is necessary to identify further Melampsora species and formae specialis that are pathogens of willow species other than S. viminalis. Therefore, three stock collections with Salix daphnoides, Salix purpurea, and other shrub willow species (including S. viminalis) species were sampled in north-eastern Germany. A fourth stock collection in central Germany contributed rusts of tree willows (Salix fragilis and Salix alba) and the large shrub Salix caprea. Out of 156 rust samples, 149 were successfully sequenced for ITS rDNA. A phylogenetic analysis combining Neighbour-Joining, Maximum-Likelihood and Bayesian analysis revealed six species: Melampsora ribesii-purpureae, Melampsora allii-salicis-albae, Melampsora sp. aff. allii-fragilis, Melampsora larici-pentandrae, Melampsora larici-caprearum, and Melampsora larici-epitea. The first four species were found exclusively on the expected hosts. Melampsora larici-caprearum had a wider host range comprising S. caprea and S. viminalis hybrids. Melampsora larici-epitea can be further differentiated into two formae speciales. The forma specialis larici-epitea typica (59 samples) colonized Salix viminalis clones, Salix purpurea, Salix×dasyclados, and Salix×aquatica. In contrast to this relatively broad host range, f. sp. larici-daphnoides (65 samples) was found exclusively on Salix daphnoides. With the distinction and identification of the rust species/formae speciales it is now possible to test for race-specific resistances in a more targeted manner within the determined pairings of rust and willow species.     

Identification of AFLP molecular markers for resistance against Melampsora larici-populina in Populus

We have identified AFLP markers tightly linked to the locus conferring resistance to the leaf rust Melampsora larici-populina in Populus. The study was carried out using a hybrid progeny derived from an inter-specific, controlled cross between a resistant Populus deltoides female and a susceptible P. nigra male. The segregation ratio of resistant to susceptible plants suggested that a single, dominant locus defined this resistance. This locus, which we have designated Melampsora resistance (Mer), confers resistance against E1, E2, and E3, three different races of Melampsora larici-populina. In order to identify molecular markers linked to the Mer locus we decided to combine two different techniques: (1) the high-density marker technology, AFLP, which allows the analysis of thousands of markers in a relatively short time, and (2) the Bulked Segregant Analysis (BSA), a method which facilitates the identification of markers that are tightly linked to the locus of interest. We analyzed approximately 11,500 selectively amplified DNA fragments using 144 primer combinations and identified three markers tightly linked to the Mer locus. The markers can be useful in current breeding programs and are the basis for future cloning of the resistance gene.     

A new species of Melampsora on Populus yunnanensis from China

Melampsora nujiangensis, a rust fungus found on Populus yunnanensis in China, is described as a new species. Light and scanning electron microscopy with herbarium specimens of the rust fungus show that the shape of its urediniospores differs from that of other known species of Melampsora, and its urediniospore walls are thinner than the other species. Furthermore, in phylogenetic trees based on the DNA sequences (28S and ITS) the rust fungus is phylogenetically separated by high bootstrap values. These results indicate that the fungus is an isolated species among the genus Melampsora. Contribution no. 202 from the Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

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).     

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.     

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

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

Correlation between pathogenicity and molecular characteristics in the willow leaf rusts <Emphasis Type="Italic">Melampsora epitea</Emphasis> and <Emphasis Type="Italic">M. humilis</Emphasis> in Japan

 We studied the correlation between pathogenicity and restriction fragment length polymorphism (RFLP) type, which was determined by polymerase chain reaction-based RFLP analysis of the internal transcribed spacer regions of ribosomal DNA, in the willow leaf rust fungi Melampsora epitea and M. humilis. Eighteen clones of eight Salix species were inoculated with urediniospores from seven collections of the two rust species. M. epitea and M. humilis (RFLP type-5 collections) were pathogenic to six to eight Salix species. RFLP type-7 collections of M. epitea were pathogenic to only two Salix species. The taxonomic relationships of the two rust species are discussed. Received: December 11, 2002 / Accepted: February 17, 2003 RID="*" ID="*" Contribution no. 179, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan Acknowledgments We thank K. Katsuya, former professor at the University of Tsukuba, for his encouragement in this study. We are also grateful to M. Yashima, Botanical Garden, University of Tohoku, for his assistance in collecting materials and to R. Suzuki, University of Tsukuba, for providing a rust isolate.     

Genetic linkage mapping in aspen (Populus tremula L. and Populus tremuloides Michx.)

A large number of simple sequence repeat (SSR) marker-containing genetic maps are available for several Populus species. For aspen however, no SSR-containing map has been published so far. In this study, genetic linkage mapping was carried out with an interspecific mapping pedigree of 61 full-sib hybrids of European × quaking aspen (Populus tremula L. × Populus tremuloides Michx.), using the two-way pseudo-testcross strategy. Amplified fragment-length polymorphism (AFLP) and SSR markers were used for mapping, resulting in the first SSR-containing genetic linkage maps for aspen. The maps allow comparisons with a Populus consensus map and other published genetic maps of the genus Populus. The maps showed good collinearity to each other and to the Populus consensus map and provide a direct link to the Populus trichocarpa genomic sequence. Sex as a morphological trait was assessed in the mapping population and mapped on a non-terminal position of linkage group XIX on the male P. tremuloides map.     

Rust disease (Melampsora epited) of willow (Salix spp.) grown as short rotation coppice (SRC) in inter- and intra-species mixtures

Twenty inter‐ and intra‐species genotypes of willows (Salix spp.) were grown in large mono‐plots and incorporated into five, 10,15 and 20–way mixtures. In each growing season from 1996 to 2001 the level of rust disease caused by Melampsora epitea was recorded on each genotype where it was growing as a mono‐plot or as part of a mixture. Three genotypes, S. schwerinii×viminalis×dasyclados’V7531′, S. schwerinii×aquatica‘V7533’ and S. viminalis‘Gigantea’ remained virtually rust free through the six yr of the trial. It is, however, argued that there are benefits of including such genotypes in a mixture in order to reduce the risk of them developing rust susceptibility. S. burjatica‘Germany’, S. dasyclados×aquatica‘V7511’ and S. dasyclados×caprea‘V794’ were severely affected by rust, although levels tended to be less when included in mixtures. The levels of rust on S. mollissima‐undulata‘SQ83’ were much higher in the first three‐year harvest cycle than during the second cycle. On a number of genotypes, e.g. S. burjatica‘Germany’ and S. dasyclados×aquatica‘V7511’ rust was more severe on the regrowth from freshly coppiced stools.     

An experimental test of hybrid resistance to insects and pathogens using Salix caprea, S. repens and their F1 hybrids

The aim of this study was to assess the responses of herbivores and pathogens to hybrid plants under controlled conditions. F1 hybrids and parental species, produced by hand-pollinating willows in the field, were potted and kept in an experimental field under controlled conditions. In 1997, plant growth and survival were measured along with densities of insects and the degree of pathogen infection on the willows. The survival rate was higher for S. repens than for the hybrids and lowest for S. caprea. Densities of the sawflies Pontania pedunculi and P. brigmanii and the leaf-galling midge Iteomyia capreae were higher on hybrids and on S. caprea than on S. repens. The densities of Crepidodera fulvicornis (Chrysomelidae), chrysomelid larvae and the bud-galling midge Dasineura rosaria did not differ between any of the plant categories. Hybrids were more severely infected by rust (Melampsora sp.) than S. caprea and the totally resistant S. repens. Densities of herbivores on hybrid willows were consistent with the dominance hypothesis (i.e. herbivore densities were similar to densities on one of the parental species) or supported the no-difference hypothesis. Furthermore, herbivore densities on hybrid plants were most similar to densities on the more susceptible parent. The breakdown in rust resistance in hybrid plants suggests that resistance traits are severely disrupted by the genetic re-arrangement in hybrids and that this increased susceptibility could select against hybridisation. Received: 17 February 1998 / Accepted: 15 June 1998     

松杨栅锈菌遗传多样性初步分析

采用ITS-nrDNA-RELP、测序技术、RAPD(随机扩增多态性DNA)分子标记技术,对我国松杨栅锈菌不同地域的5个生理小种11个菌系进行了遗传多样性分化研究.结果表明,该菌在我国的遗传分化与地理来源相关,可分为西部地理群和北方地理群.西部地理群又可分为高山森林生态型(HMF)和平原生态型(WPL).小种遗传分化不一定与致病性分化一致.t检验表明,各生理小种RAPD遗传多样性指数无明显差异,高山森林小种遗传多样性指数(0.5172)略高于平原小种遗传多样性指数(0.5089).核糖体基因转录间隔区高度保守,不适合该菌种内群体遗传多样性分化研究.     

Isolation and characterization of microsatellite loci from the rust pathogen,Melampsora lini

We developed and characterized primers for 11 variable microsatellite loci present in the genome of the flax rust, Melampsora lini. The microsatellite loci were identified by sequencing clones from a library of EcoRI DNA fragments enriched for four simple sequence repeat motifs (AAG, AAT, TC and TG). All 11 primer pairs successfully amplified DNA fragments from a sample of 102 M. lini isolates (98 isolated from Linum marginale and four from Linum usitatissimum), revealing a total of 32 alleles. Allelic diversity at the 11 loci ranged from 0.030 to 0.449.     

Plastome phylogeny and lineage diversification of Salicaceae with focus on poplars and willows

Phylogenetic relationships and lineage diversification of the family Salicaceae sensu lato (s.l.) remain poorly understood. In this study, we examined phylogenetic relationships between 42 species from six genera based on the complete plastomes. Phylogenetic analyses of 77 protein coding genes of the plastomes produced good resolution of the interrelationships among most sampled species and the recovered clades. Of the sampled genera from the family, Flacourtia was identified as the most basal and the successive clades comprised both Itoa and Poliothyrsis, Idesia, two genera of the Salicaceae sensu stricto (s.s.) (Populus and Salix). Five major subclades were recovered within the Populus clade. These subclades and their interrelationships are largely inconsistent with morphological classifications and molecular phylogeny based on nuclear internal transcribed spacer sequence variations. Two major subclades were identified for the Salix clade. Molecular dating suggested that species diversification of the major subclades in the Populus and Salix clades occurred mainly within the recent Pliocene. In addition, we found that the rpl32 gene was lost and the rps7 gene evolved into a pseudogene multiple times in the sampled genera of the Salicaceae s.l. Compared with previous studies, our results provide a well‐resolved phylogeny from the perspective of the plastomes.     

Phylogeny of Populus-Salix (Salicaceae) and their relative genera using molecular datasets

Although extensive studies have demonstrated that several genera formerly placed in the family Flacourtiaceae are the closest relatives of Populus-Salix. However, the closest relative or relatives remain uncertain due to the relatively low phylogenetic resolution and insufficient phylogenetic information. In addition, the question as to whether Populus and Salix are truly monophyletic remains open because of the absence of comprehensive studies with large-scale samples of both genera. In the present study, 54 species representing 11 genera of Salicaceae sensu lato (now including the genera of Flacourtiaceae) were studied using a combined rbcL and matK gene sequence dataset with a total length of 2134 bp, to explore the closest genera that are relatives of Populus-Salix and to determine the relationships between Populus and Salix. The resulting phylogenetic trees showed high resolution in each of the main clades. The results confirmed that (1) Idesia and Bennettiodendron are the closest relatives to Populus-Salix, followed by Poliothyrsis, and (2) Populus and Salix are a truly monophyletic lineage in which the most recent ancestor is shared by the two genera. This finding is significant for a wide variety of evolutionary studies and for understanding the classification and biosystematics of Salicaceae.     

Potentials and limitations of the cross-species transfer of nuclear microsatellite marker in six species belonging to three sections of the genus Populus L.

The genus Populus is classified into six different sections, and depending on the declaration of hybrids, the number of species varies between 22 and 85. Species within one section, and sometimes between sections, are crossable to each other, resulting in many naturally but also artificially produced hybrids. Morphological attributes for a clone characterisation are often difficult to evaluate when different poplar species or even hybrids are crossed; thus, molecular markers are needed to characterise the different species. Taking advantage of the large microsatellite resource developed for Populus trichocarpa, however, amplification of these microsatellite markers in other Populus species either often fails, or in the case of amplification, unrelated genomic regions are amplified. To meet this obvious problem of the species transferability of microsatellite markers, in total, 305 microsatellite loci, mainly from P. trichocarpa but also few from Populus tremuloides and Populus nigra, were tested for their transferability to diverse genotypes of six species belonging to three sections of the genus Populus. Ultimately, 209 microsatellite loci could be amplified with varying sizes in the different species. The PCR products of selected loci were separated in a polyacrylamide gel and sequenced to assure that the expected loci were derived from the database genome of P. trichocarpa. The present results constitute a large study for microsatellite transferability for Populus species. The documented microsatellite loci can be applied to species-, hybrid- and clone-specific diagnostic approaches or as universal markers for comprehensive ecological studies.     

Detection and validation of EST‐derived SNPs for poplar leaf rust Melampsora medusae f. sp. deltoidae

We report the discovery, characterization and validation of 118 single nucleotide polymorphisms (SNPs) for poplar leaf rust Melampsora medusae f. sp. deltoidae identified using a gene‐targeted approach in an expressed sequence tag (EST) library. We developed a genotyping assay using the iPLEX? primer extension method for two multiplex assays of 28 and 22 SNPs.     

Multilayered structure of tension wood cell walls in Salicaceae sensu lato and its taxonomic significance

Salicaceae have been enlarged to include a majority of the species formerly placed in the polyphyletic tropical Flacourtiaceae. Several studies have reported a peculiar and infrequently formed multilayered structure of tension wood in four of the tropical genera. Tension wood is a tissue produced by trees to restore their vertical orientation and most studies have focused on trees developing tension wood by means of cellulose‐rich, gelatinous fibres, as in Populus and Salix (Salicaceae s.s.). This study aims to determine if the multilayered structure of tension wood is an anatomical characteristic common in other Salicaceae and, if so, how its distribution correlates to phylogenetic relationships. Therefore, we studied the tension wood of 14 genera of Salicaceae and two genera of Achariaceae, one genus of Goupiaceae and one genus of Lacistemataceae, families closely related to Salicaceae or formerly placed in Flacourtiaceae. Opposite wood and tension wood were compared with light microscopy and three‐dimensional laser scanning confocal microscopy. The results indicate that a multilayered structure of tension wood is common in the family except in Salix, Populus and one of their closest relatives, Idesia polycarpa. We suggest that tension wood may be a useful anatomical character in understanding phylogenetic relationships in Salicaceae. Further investigation is still needed on the tension wood of several other putatively close relatives of Salix and Populus, in particular Bennettiodendron, Macrohasseltia and Itoa.     

Life cycle and morphology ofMelampsora yezoensis onSalix serissaefolia

Uredinial and telial states of aMelampsora species occurring on the leaves ofSalix serissaefolia were for the first time recorded. Field observations and inoculation experiments showed that the spermogonial and aecial states of the fungus were formed onChelidonium majus var.asiaticum andCorydalis incisa. The fungus was identified asMelampsora yezoensis based on the morphological observation of all the spore states. Urediniospores were able to infectS. erocarpa andS. pierotii as well asS. serissaefolia andS. jessoensis. Salix serissaefolia, S. eriocarpa, andS. pierotii are new uredinial and telial hosts ofM. yezoensis, andC. majus var.asiaticum andC. incisa are new spermogonial and aecial hosts of the fungus. Contribution No. 124, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry University of Tsukuba.     

An integrated genetic map of Populus deltoides based on amplified fragment length polymorphisms

Amplified fragment length polymorphism (AFLP) is an efficient molecular technique for generating a large number of DNA-based genetic markers in Populus. We have constructed an integrated genetic map for a Populus backcross population derived from two selected P. deltoides clones using AFLP markers. A traditional strategy for genetic mapping in outcrossing species, such as forest trees, is based on two-way pseudo-testcross configurations of the markers (testcross markers) heterozygous in one parent and null in the other. By using the markers segregating in both parents (intercross markers) as bridges, the two parent-specific genetic maps can be aligned. In this study, we detected a number of non-parental heteroduplex markers resulting from the PCR amplification of two DNA segments that have a high degree of homology to one another but differ in their nucleotide sequences. These heteroduplex markers detected have served as bridges to generate an integrated map which includes 19 major linkage groups equal to the Populus haploid chromosome number and 24 minor groups. The 19 major linkage groups cover a total of 2,927 cM, with an average spacing between two markers of 23. 3 cM. The map developed in this study provides a first step in producing a highly saturated linkage map of the Populus deltoides genome. Received: 10 September 1999 / Accepted: 3 November 1999