Responses of soil and rhizosphere microbial communities to Cd-hyperaccumulating willows and Cd contamination.

BACKGROUND: The pollution of soil by heavy metals, particularly Cd, is constitutes a critical international environmental concern. Willow species are renowned for their efficacy in the phytoremediation of heavy metals owing to their high Cd absorption rate and rapid growth. However, the mechanisms underlying microbial regulation for high- and low-accumulating willow species remain poorly understood. Therefore, we investigated the responses of soil and rhizosphere microbial communities to high- and low-Cd-accumulating willows and Cd contamination. We analyzed soil properties were analyzed in bulk soil (SM) and rhizosphere soil (RM) planted with high-accumulating (H) and low-accumulating (L) willow species. RESULTS: Rhizosphere soil for different willow species had more NH4+ than that of bulk soil, and RM-H soil had more than RM-L had. The available phosphorus content was greater in hyper-accumulated species than it was in lower-accumulated species, especially in RM-H. Genome sequencing of bacterial and fungal communities showed that RM-L exhibited the highest bacterial diversity, whereas RM-H displayed the greatest richness than the other groups. SM-L exhibited the highest diversity and richness of fungal communities. Ralstonia emerged as the predominant bacterium in RM-H, whereas Basidiomycota and Cercozoa were the most enriched fungi in SM-H. Annotation of the N and C metabolism pathways revealed differential patterns: expression levels of NRT2, NarB, nirA, nirD, nrfA, and nosZ were highest in RM-H, demonstrating the effects of NO3-and N on the high accumulation of Cd in RM-H. The annotated genes associated with C metabolism indicated a preference for the tricarboxylic pathway in RM-H, whereas the hydroxypropionate-hydroxybutyrate cycle was implicated in C sequestration in SM-L. CONCLUSIONS: These contribute to elucidation of the mechanism underlying high Cd accumulation in willows, particularly in respect of the roles of microbes and N and C utilization. This will provide valuable insights for repairing polluted soil using N and employing organic acids to improve heavy metal remediation efficiency.

Dual inoculation with rhizosphere-promoting bacterium Bacillus cereus and beneficial fungus Peniophora cinerea improves salt stress tolerance and productivity in willow.

Utilization of rhizosphere microorganisms to improve plant growth and salt tolerance has recently attracted widespread attention. The growth and salt tolerance of willows inoculated with Bacillus cereus JYZ-SD2 and Peniophora cinerea XC were studied under different salt stress conditions. The results showed that the chlorophyll content of willow cuttings inoculated with the XC strain increased significantly by 51.27%. After salt stress of willow cuttings inoculated with B. cereus JYZ-SD2 and P. cinerea XC (solely or in combination), the amount of sodium in the roots from the epidermis to the pericycle decreased and the content of sodium in the pericycle was significantly lower than that of the uninoculated willow, while the proportion of potassium increased. Willow cuttings inoculated with microorganisms showed increased activity of SOD and POD. At the salt concentration of 100 mmol/L, the highest SOD activity was found in B. cereus JYZ-SD2-inoculated willows, with 59.88% increase compared to uninoculated willows; the highest POD activity was found in P. cinerea XC and B. cereus JYZ-SD2 co-inoculated willows, with 51.05% increase compared to uninoculated willows. The Na-K-ATPase and Ca-Mg-ATPase activities of inoculated P. cinerea XC willow cuttings were also 59.38% and 60% higher than that of uninoculated willows, respectively. The qPCR analysis showed that the expression of vp2 gene in the microorganism-inoculated willow leaves was always higher than that in willow alone. The expression of vp2 gene in P. cinerea XC-inoculated willow cuttings was 270.81% higher than that in uninoculated willows. Further observation of the ultrastructure of root cells under salt stress revealed that most of the vesicles in the root tip cells of willow were intact and secreted phagocytic vesicles to absorb sodium ions in the cytoplasm. This study shows that the combined beneficial fungi and rhizosphere-promoting bacteria inoculation technology as a practical biotechnological approach to enhance the growth of willows in salt-affected soils.

Use of ensiled green willow biomass in biogas fermentation.

The biggest challenges of our era include climate change and the global fossil energy problem. Extensive utilization of renewable energy sources should be a part of the solution for both these problems. Biogas is a versatile renewable energy carrier that has the potential to substitute fossil fuels. The most frequently utilized substrates for the anaerobic digestion (AD) process include maize silage today, but there is an increasing demand for second-generation biomass sources, which are cheaper and do not interfere with the cultivation of food production. Green biomass from short rotation coppice willow (GWB) may be a promising alternative. However, to ensure feedstock quantity and quality all year round, a preservation method has to be developed. We attempted to ensilage the biomass and subsequently utilized the resulting willow-silage in batch fermenters. Various mixtures of lactic acid bacteria were employed to facilitate ensiling by inoculation of the substrate in anaerobic jars for 60 days. During the ensiling analytical investigations, (HPLC, pH, oTS/TS%) were carried out in order to follow the build-up of fermentation products. AD fermentations were assembled from the ensilaged biomass and the methane production was measured for 56 days. The total methane yields of the ensilaged biomass were 8-15% higher than that of the fresh biomass and methane production rates were also improved. Our findings suggest that ensiling is not only an excellent preservation method for willow biomass, but also stimulates its AD.

Affinibrenneria salicis gen. nov. sp. nov. isolated from Salix matsudana bark canker.

L3-3HAT, a Gram-negative-staining, facultatively anaerobic, motile bacterial strain, was isolated from the symptomatic bark of Salix matsudana canker in China. 16S rRNA gene analysis revealed that the novel strain shares the highest sequence similarity with Brenneria goodwinii FRB141T (95.5%). In phylogenetic trees based on four housekeeping genes (gyrB, rpoB, atpD, and infB) and the 16S rRNA gene sequence, the novel strain formed a separate branch from the five genera of the family Pectobacteriaceae (Lonsdalea, Brenneria, Dickeya, Pectobacterium, and Sodalis), suggesting that the novel strain should belong to a novel species of a novel genus within the family Pectobacteriaceae. The result was also supported by phylogenomics, amino acid identity and average nucleotide identity. The major fatty acids were C14:0, C16:0, C17:0 cyclo, and C19:0 cyclo É·8c. Genome analysis showed that the novel strain has a large genome (5.89 Mb) with 5,052 coding genes, including 181 virulence genes by searching the pathogen-host interactions database (PHI-base), indicating that the novel strain is a potential pathogen of plants and animals. Based on phenotypic and genotypic characteristics, the L3-3HAT strain represents a novel species of a novel genus in the Pectobacteriaceae family, for which the name Affinibrenneria salicis gen nov. sp. nov. is proposed. The strain type is L3-3HAT (= CFCC 15588T = LMG 31209T).

Microbiomes of willow-galling sawflies: effects of host plant, gall type, and phylogeny on community structure and function.

While free-living herbivorous insects are thought to harbor microbial communities composed of transient bacteria derived from their diet, recent studies indicate that insects that induce galls on plants may be involved in more intimate host-microbe relationships. We used 16S rDNA metabarcoding to survey larval microbiomes of 20 nematine sawfly species that induce bud or leaf galls on 13 Salix species. The 391 amplicon sequence variants (ASVs) detected represented 69 bacterial genera in six phyla. Multi-variate statistical analyses showed that the structure of larval microbiomes is influenced by willow host species as well as by gall type. Nevertheless, a "core" microbiome composed of 58 ASVs is shared widely across the focal galler species. Within the core community, the presence of many abundant, related ASVs representing multiple distantly related bacterial taxa is reflected as a statistically significant effect of bacterial phylogeny on galler-microbe associations. Members of the core community have a variety of inferred functions, including degradation of phenolic compounds, nutrient supplementation, and production of plant hormones. Hence, our results support suggestions of intimate and diverse interactions between galling insects and microbes and add to a growing body of evidence that microbes may play a role in the induction of insect galls on plants.

Azacoccones F-H, new flavipin-derived alkaloids from an endophytic fungus Epicoccum nigrum MK214079.

Three new flavipin-derived alkaloids, azacoccones F-H (1-3), along with six known compounds (4-9) were isolated from the endophytic fungus Epicoccum nigrum MK214079 associated with leaves of Salix sp. The structures of the new compounds were established by analysis of their 1D/2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS) data. The absolute configuration of azacoccones F-H (1-3) was determined by comparison of experimental electronic circular dichroism (ECD) data with reported ones and biogenetic considerations. Epicocconigrone A (4), epipyrone A (5), and epicoccolide B (6) exhibited moderate antibacterial activity against Staphylococcus aureus ATCC 29213 with minimal inhibitory concentration (MIC) values ranging from 25 to 50 µM. Furthermore, epipyrone A (5) and epicoccamide A (7) displayed mild antifungal activity against Ustilago maydis AB33 with MIC values of 1.6 and 1.8 mM, respectively. Epicorazine A (8) showed pronounced cytotoxicity against the L5178Y mouse lymphoma cell line with an IC50 value of 1.3 µM.

Genome comparison and transcriptome analysis of the invasive brown root rot pathogen, Phellinus noxius, from different geographic regions reveals potential enzymes associated with degradation of different wood substrates.

Phellinus noxius is a root-decay pathogen with a pan-tropical/subtropical distribution that attacks a wide range of tree hosts. For this study, genomic sequencing was conducted on P. noxius isolate P919-02W.7 from Federated States of Micronesia (Pohnpei), and its gene expression profile was analyzed using different host wood (Acer, Pinus, Prunus, and Salix) substrates. The assembled genome was 33.92 Mbp with 2954 contigs and 9389 predicted genes. Only small differences were observed in size and gene content in comparison with two other P. noxius genome assemblies (isolates OVT-YTM/97 from Hong Kong, China and FFPRI411160 from Japan, respectively). Genome analysis of P. noxius isolate P919-02W.7 revealed 488 genes encoding proteins related to carbohydrate and lignin metabolism, many of these enzymes are associated with degradation of plant cell wall components. Most of the transcripts expressed by P. noxius isolate P919-02W.7 were similar regardless of wood substrates. This study highlights the vast suite of decomposing enzymes produced by P. noxius, which suggests potential for degrading diverse wood substrates, even from temperate host trees. This information contributes to our understanding of pathogen ecology, mechanisms of wood decomposition, and pathogenic/saprophytic lifestyle.

Copper stress in flooded soil: Impact on enzyme activities, microbial community composition and diversity in the rhizosphere of Salix integra.

Climate change has increased flooding frequency, making the heavy metal polluted areas more vulnerable, and led to increased global land degradation. Information about the alteration of soil microbiota under heavy metal pollution and flooding is still rather limited. Fast-growing trees are candidates for phytoremediation of heavy metal polluted soils. Therefore, the impact of Cu pollution on microbiota in soil used for cultivating Salix integra Thunb. was investigated with and without flooding for 60 d. Bacterial and fungal communities were accessed via partial 16S rRNA (V3-V4) and internal transcribed spacer (ITS) genes. The activity of invertase, urease and cellulase were markedly decreased by 28.5-59%, 55.0-76.7% and 17.3-34.1%, respectively, with increasing Cu levels. Flooding significantly increased the activity of polyphenol oxidase and peroxidase by 56.3% and 41.4% at the highest Cu level compared to its respective non-flooded condition. High Cu concentration significantly decreased the richness and diversity of the bacterial community, and fungi were more sensitive than bacteria under flooding conditions. Redundancy analysis suggests that Cu, Fe and soil organic matter are the key determinants affecting the composition of microbial communities. Our findings provide new insight into the responses of soil microbes to Cu-contamination and contribute to our understanding of metal toxicity in soil-woody plant systems under flooded conditions.

Salt Tolerance Mechanism and Species Identification of the Plant Rhizosphere Bacterium JYZ-SD2.

A salt-tolerant microbe strain JYZ-SD2 was investigated to develop biological soil amendments to stimulate salix growth and acclimation in costal salt-affected soils. The salt tolerance mechanism of strain JYZ-SD2 was investigated by detecting the salt-tolerant growth characteristics, biofilm formation, ion distribution, secondary metabolites, and zymogram profiling. The strain was identified by physiological and biochemical characteristics (Biolog), 16S rDNA sequencing, and cry1/7/9 gene expressing. With increasing of NaCl concentration, strain JYZ-SD2 adapted to the increased osmotic pressure by prolonging the retardation period, slowing down the growth rate of the logarithmic phase, increasing spo0A gene expression, increasing biofilm formation, reducing Na+ uptake, and changing the expression of metabolites and intracellular soluble proteins. The results showed that strain JYZ-SD2 could be assigned to Bacillus cereus.

Different twig litter (Salix caprea) diameter does affect microbial community activity and composition but not decay rate.

Small twigs represent a substantial input of organic carbon into forest soils, but potential influencing factors on their decomposition have rarely been investigated. Here, we studied potential effects of twig size on decomposition and associated composition and activity of microbial communities during decomposition. Because the surface area for microbial colonization and the volume of accessible substrate increases with decreasing twig size, we hypothesized that twig size affects both microbial community and decomposition rate. Litterbags with twigs (Salix caprea) of two different diameters were placed within the litter layer and consecutively collected over a seven-year period. We determined the mass loss and microbial measures after each sampling event. The observed microbial parameters suggested a faster microbial colonization of thin twigs, where the proportion of bacteria was higher than in thick twigs. The development of the microbial community in thick twigs was more gradual and the proportion of fungi was higher. Despite this differential and successional development of microbial communities (and against our hypothesis), the mass loss among different twig diameters did not differ after our seven-year experiment, indicating that surface-to-volume ratios, though a primary control on microbial succession, may have limited predictive power for twig decomposition rates.

Trees, fungi and bacteria: tripartite metatranscriptomics of a root microbiome responding to soil contamination.

BACKGROUND: One method for rejuvenating land polluted with anthropogenic contaminants is through phytoremediation, the reclamation of land through the cultivation of specific crops. The capacity for phytoremediation crops, such as Salix spp., to tolerate and even flourish in contaminated soils relies on a highly complex and predominantly cryptic interacting community of microbial life. METHODS: Here, Illumina HiSeq 2500 sequencing and de novo transcriptome assembly were used to observe gene expression in washed Salix purpurea cv. 'Fish Creek' roots from trees pot grown in petroleum hydrocarbon-contaminated or non-contaminated soil. All 189,849 assembled contigs were annotated without a priori assumption as to sequence origin and differential expression was assessed. RESULTS: The 839 contigs differentially expressed (DE) and annotated from S. purpurea revealed substantial increases in transcripts encoding abiotic stress response equipment, such as glutathione S-transferases, in roots of contaminated trees as well as the hallmarks of fungal interaction, such as SWEET2 (Sugars Will Eventually Be Exported Transporter). A total of 8252 DE transcripts were fungal in origin, with contamination conditions resulting in a community shift from Ascomycota to Basidiomycota genera. In response to contamination, 1745 Basidiomycota transcripts increased in abundance (the majority uniquely expressed in contaminated soil) including major monosaccharide transporter MST1, primary cell wall and lamella CAZy enzymes, and an ectomycorrhiza-upregulated exo-ß-1,3-glucanase (GH5). Additionally, 639 DE polycistronic transcripts from an uncharacterised Enterobacteriaceae species were uniformly in higher abundance in contamination conditions and comprised a wide spectrum of genes cryptic under laboratory conditions but considered putatively involved in eukaryotic interaction, biofilm formation and dioxygenase hydrocarbon degradation. CONCLUSIONS: Fungal gene expression, representing the majority of contigs assembled, suggests out-competition of white rot Ascomycota genera (dominated by Pyronema), a sometimes ectomycorrhizal (ECM) Ascomycota (Tuber) and ECM Basidiomycota (Hebeloma) by a poorly characterised putative ECM Basidiomycota due to contamination. Root and fungal expression involved transcripts encoding carbohydrate/amino acid (C/N) dialogue whereas bacterial gene expression included the apparatus necessary for biofilm interaction and direct reduction of contamination stress, a potential bacterial currency for a role in tripartite mutualism. Unmistakable within the metatranscriptome is the degree to which the landscape of rhizospheric biology, particularly the important but predominantly uncharacterised fungal genetics, is yet to be discovered.

Soil contamination alters the willow root and rhizosphere metatranscriptome and the root-rhizosphere interactome.

Phytoremediation using willows is thought to be a sustainable alternative to traditional remediation techniques involving excavation, transport, and landfilling. However, the complexity of the interaction between the willow and its associated highly diverse microbial communities makes the optimization of phytoremediation very difficult. Here, we have sequenced the rhizosphere metatranscriptome of four willow species and the plant root metatranscriptome for two willow species growing in petroleum hydrocarbon-contaminated and non-contaminated soils on a former petroleum refinery site. Significant differences in the abundance of transcripts related to different bacterial and fungal taxa were observed between willow species, mostly in contaminated soils. When comparing transcript abundance in contaminated vs. non-contaminated soil for each willow species individually, transcripts for many microbial taxa and functions were significantly more abundant in contaminated rhizosphere soil for Salix eriocephala, S. miyabeana and S. purpurea, in contrast to what was observed in the rhizosphere of S. caprea. This agrees with the previously reported sensitivity of S. caprea to contamination, and the superior tolerance of S. miyabeana and S. purpurea to soil contamination at that site. The root metatranscriptomes of two species were compared and revealed that plants transcripts are mainly influenced by willow species, while microbial transcripts mainly responded to contamination. A comparison of the rhizosphere and root metatranscriptomes in the S. purpurea species revealed a complete reorganization of the linkages between root and rhizosphere pathways when comparing willows growing in contaminated and non-contaminated soils, mainly because of large shifts in the rhizosphere metatranscriptome.

Asymmetric response of root-associated fungal communities of an arbuscular mycorrhizal grass and an ectomycorrhizal tree to their coexistence in primary succession.

The arbuscular mycorrhizal (AM) grass Calamagrostis epigejos and predominantly ectomycorrhizal (EcM) tree Salix caprea co-occur at post-mining sites spontaneously colonized by vegetation. During succession, AM herbaceous vegetation is replaced by predominantly EcM woody species. To better understand the interaction of AM and EcM plants during vegetation transition, we studied the reciprocal effects of these species' coexistence on their root-associated fungi (RAF). We collected root and soil samples from three different microenvironments: stand of C. epigejos, under S. caprea canopy, and contact zone where roots of the two species interacted. RAF communities and mycorrhizal colonization were determined in sampled roots, and the soil was tested for EcM and AM inoculation potentials. Although the microenvironment significantly affected composition of the RAF communities in both plant species, the effect was greater in the case of C. epigejos RAF communities than in that of S. caprea RAF communities. The presence of S. caprea also significantly decreased AM fungal abundance in soil as well as AM colonization and richness of AM fungi in C. epigejos roots. Changes observed in the abundance and community composition of AM fungi might constitute an important factor in transition from AM-dominated to EcM-dominated vegetation during succession.

Test of aerobic TCE degradation by willows (Salix viminalis) and willows inoculated with TCE-cometabolizing strains of Burkholderia cepacia.

Trichloroethylene (TCE) is a widespread soil and groundwater pollutant and clean-up is often problematic and expensive. Phytoremediation may be a cost-effective solution at some sites. This study investigates TCE degradation by willows (S. viminalis) and willows inoculated with three strains of B. cepacia (301C, PR1-31 and VM1330-pTOM), using chloride formation as an indicator of dehalogenation. Willows were grown in non-sterile, hydroponic conditions for 3 weeks in chloride-free nutrient solution spiked with TCE. TCE was added weekly due to rapid loss by volatilization. Chloride and TCE in solution were measured every 2-3 days and chloride and metabolite concentrations in plants were measured at test termination. Based on transpiration, no tree toxicity of TCE exposure was observed. However, trees grown in chloride-free solution showed severely inhibited transpiration. No or very little chloride was formed during the test, and levels of chloride in TCE-exposed trees were not elevated. Chloride concentrations in chloride containing TCE-free nutrient solution doubled within 23 days, indicating active exclusion of chloride by root cell membranes. Only traces of TCE-metabolites were detected in plant tissue. We conclude that TCE is not, or to a limited extent (less than 3%), aerobically degraded by the willow trees. The three strains of B. cepacia did not enhance TCE mineralization. Future successful application of rhizo- and phytodegradation of TCE requires measures to be taken to improve the degradation rates.

Resolving the Melampsora epitea complex.

Melampsora epitea (Pucciniales, Basidiomycota) has been widely known as the main rust pathogen on willows, but its species boundary has been extremely confusing. Melampsora epitea was often confused with several morphologically similar species, such as M. arctica, M. humilis, and M. epiphylla. Recent DNA-based studies using specimens from North America and Europe suggested that M. epitea was a complex of several phylogenetic species. In this study, we examined the species boundary of M. epitea based 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 delimiting M. epitea and allied species. Two novel species, M. salicis-futurae and M. salicis-triandrae, are described and compared with closely related species. A lectotype for M. epitea and epitypes for M. epitea, M. epiphylla, and M. humilis are designated to stabilize the use of names.

Modeling of phytoextraction efficiency of microbially stimulated Salix dasyclados L. in the soils with different speciation of heavy metals.

Bioaugmentation of soils with selected microorganisms during phytoextraction can be the key solution for successful bioremediation and should be accurately calculated for different physicochemical soil properties and heavy metal availability to guarantee the universality of this method. Equally important is the development of an accurate prediction tool to manage phytoremediation process. The main objective of this study was to evaluate the role of three metallotolerant siderophore-producing Streptomyces sp. B1-B3 strains in the phytoremediation of heavy metals with the use of S. dasyclados L. growing in four metalliferrous soils as well as modeling the efficiency of this process based on physicochemical and microbiological properties of the soils using artificial neural network (ANN) analysis. The bacterial inoculation of plants significantly stimulated plant biomass and reduced oxidative stress. Moreover, the bacteria affected the speciation of heavy metals and finally their mobility, thereby enhancing the uptake and bioaccumulation of Zn, Cd, and Pb in the biomass. The best capacity for phytoextraction was noted for strain B1, which had the highest siderophore secretion ability. Finally, ANN model permitted to predict efficiency of phytoextraction based on both the physicochemical properties of the soils and the activity of the soil microbiota with high precision.

Identification of Quantitative Trait Loci Conditioning the Main Biomass Yield Components and Resistance to Melampsora spp. in Salix viminalis × Salix schwerinii Hybrids.

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 × 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.

Identification of a Differentially Expressed TIR-NBS-LRR Gene in a Major QTL Associated to Leaf Rust Resistance in Salix.

An earlier identified major quantitative trait locus for resistance towards the willow leaf rust fungus Melampsora larici-epitea in a Salix viminalis x (S. viminalis × S. schwerinii) population was used to identify potential resistance genes to the rust pathogen. Screening a genomic bacterial artificial chromosome library with markers from the peak position of the QTL region revealed one gene with TIR-NBS-LRR (Toll Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat) domain structure indicative of a resistance gene. The resistance gene analog was denoted RGA1 and further analysis revealed a number of non-synonymous single nucleotide polymorphisms in the LRR domain between the resistant and susceptible Salix genotypes. Gene expression levels under controlled conditions showed a significantly lower constitutive expression of RGA1 in the susceptible genotype. In addition, the susceptible genotype showed a significantly reduced expression level of the RGA1 gene at 24 hours post inoculation with M. larici-epitea. This indicates that the pathogen may actively suppress RGA1 gene expression allowing a compatible plant-pathogen interaction and causing infection.

Agromyces aureus sp. nov., isolated from the rhizosphere of Salix caprea L. grown in a heavy-metal-contaminated soil.

A Gram-reaction-positive, motile, yellow-pigmented and rod-shaped bacterial strain, designated AR33T, was isolated from the rhizosphere of Salix caprea L. growing in a former zinc/lead mining and processing site in Austria. A polyphasic approach was applied to determine its taxonomic position. 16S rRNA gene sequence analysis, and morphological and chemotaxonomic properties showed that strain AR33T belongs to the genus Agromyces. Strain AR33T had peptidoglycan type B2γ and the major menaquinones were MK-11, MK-10 and MK-12. The main branched-chain fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Strain AR33T showed catalase and oxidase activity and multiple heavy metal resistances to zinc, lead and cadmium. The DNA G+C content was 70.1 mol%. Levels of 16S rRNA gene sequence similarity with closely related recognized species of the genus Agromyces ranged between 98 and 99 %. However, DNA-DNA hybridization between strain AR33T and the type strains of three Agromyces species showed values lower than 42 % relatedness. Therefore, differential phenotypic characteristics together with DNA-DNA relatedness suggested that strain AR33T can be recognized as representing a distinct Agromyces species, for which the name Agromyces aureus sp. nov. is proposed. The type strain is AR33T (=DSM 101731T=LMG 29235T).

Molecular inference, multivariate morphometrics and ecological assessment are applied in concert to delimit species in the Russula clavipes complex.

Species of Russula subsect. Xerampelinae are notoriously difficult to identify and name and have not been subject to molecular study. A group of species, referred to here as the R. clavipes complex, growing in association with Salix, Betula and Populus as well as coniferous tree species from temperate to arctic and alpine habitats, were examined. Analyses of the nuc rDNA internal transcribed spacer (ITS) region and a numerical analysis of morphological characters were used. The R. clavipes complex is a monophyletic group within Russula subsect. Xerampelinae, according to molecular results. The complex includes three species: R. nuoljae is a phylogenetically and morphologically well-supported species while the other two, R. clavipes and R. pascua, are similar based on ITS data and morphology but separate based on their ecology. Russula pseudoolivascens is conspecific with R. clavipes Several combinations of characters traditionally used in the taxonomy of R. subsect. Xerampelinae are inappropriate for species delimitation in this group and the adequacy of the ITS for species identification in this group is discussed. Detailed microscopic observations on the type collection of R. nuoljae are presented and illustrated, along with a key to the European members of R. subsect. Xerampelinae.