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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-β-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.

Phytoremediation is an attractive alternative to excavating and chemically treating contaminated soils. Certain plants can directly bioremediate by sequestering and/or transforming pollutants, but plants may also enhance bioremediation by promoting contaminant-degrading microorganisms in soils. In this study, we used high-throughput sequencing of bacterial 16S rRNA genes and the fungal internal transcribed spacer (ITS) region to compare the community composition of 66 soil samples from the rhizosphere of planted willows (Salix spp.) and six unplanted control samples at the site of a former petrochemical plant. The Bray-Curtis distance between bacterial communities across willow cultivars was significantly correlated with the distance between fungal communities in uncontaminated and moderately contaminated soils but not in highly contaminated (HC) soils (>2000 mg kg-1 hydrocarbons). The mean dissimilarity between fungal, but not bacterial, communities from the rhizosphere of different cultivars increased substantially in the HC blocks. This divergence was partly related to high fungal sensitivity to hydrocarbon contaminants, as demonstrated by reduced Shannon diversity, but also to a stronger influence of willows on fungal communities. Abundance of the fungal class Pezizomycetes in HC soils was directly related to willow phylogeny, with Pezizomycetes dominating the rhizosphere of a monophyletic cluster of cultivars, while remaining in low relative abundance in other soils. This has implications for plant selection in phytoremediation, as fungal associations may affect the health of introduced plants and the success of co-inoculated microbial strains. An integrated understanding of the relationships between fungi, bacteria and plants will enable the design of treatments that specifically promote effective bioremediating communities.The ISME Journal advance online publication, 29 August 2013; doi:10.1038/ismej.2013.149.

The goal of phytoremediation is to use plants to immobilize, extract or degrade organic and inorganic pollutants. In the case of organic contaminants, plants essentially act indirectly through the stimulation of rhizosphere microorganisms. A detailed understanding of the effect plants have on the activities of rhizosphere microorganisms could help optimize phytoremediation systems and enhance their use. In this study, willows were planted in contaminated and non-contaminated soils in a greenhouse, and the active microbial communities and the expression of functional genes in the rhizosphere and bulk soil were compared. Ion Torrent sequencing of 16S rRNA and Illumina sequencing of mRNA were performed. Genes related to carbon and amino-acid uptake and utilization were upregulated in the willow rhizosphere, providing indirect evidence of the compositional content of the root exudates. Related to this increased nutrient input, several microbial taxa showed a significant increase in activity in the rhizosphere. The extent of the rhizosphere stimulation varied markedly with soil contamination levels. The combined selective pressure of contaminants and rhizosphere resulted in higher expression of genes related to competition (antibiotic resistance and biofilm formation) in the contaminated rhizosphere. Genes related to hydrocarbon degradation were generally more expressed in contaminated soils, but the exact complement of genes induced was different for bulk and rhizosphere soils. Together, these results provide an unprecedented view of microbial gene expression in the plant rhizosphere during phytoremediation.The ISME Journal advance online publication, 26 September 2013; doi:10.1038/ismej.2013.163.

Salix psammophila, C. Wang & Chang Y. Yang, a desert-adapted shrub, is recognized for its exceptional drought tolerance and plays a vital role in ecosystem maintenance. However, research on S. psammophila has been limited due to the lack of an efficient and reliable genetic transformation method, including gene functional studies. The Agrobacterium-mediated transient overexpression assay is a rapid and powerful tool for analyzing gene function in plant vivo. In this study, tissue culture seedlings of S. psammophila were utilized as the recipient materials, and the plant expression vector pCAMBIA1301, containing the GUS reporter gene, was transferred into the seedlings via an Agrobacterium-mediated method. To enhance the efficiency of the system, the effects of secondary culture time, Agrobacterium concentration, infection time, and co-culture duration on the transient transformation efficiency of S. psammophila were explored. The optimal combination for the instantaneous transformation of S. psammophila tissue culture seedlings mediated by Agrobacterium was determined as follows: a secondary culture time of 30 d, a value of OD600 of 0.8, an infection time of 3 h, and a co-culture duration of 48 h. Subsequently, the effectiveness of the transformation system was validated using the S. psammophila drought response gene SpPP2C80. To further confirm the accuracy of the system, SpPP2C80-overexpressing Arabidopsis was constructed and drought resistance analysis was performed. The results were consistent with the transient overexpression of SpPP2C80 in S. psammophila tissue culture seedlings, indicating that this system can be effectively employed for studying gene function in S. psammophila. These findings provide essential information for investigating gene function in non-model plants and pave the way for advancements in molecular biology research in S. psammophila.

Influences of soil environment and willow host species on ectomycorrhizal fungi communities was studied across an hydrologic gradient in temperate North America. Soil moisture, organic matter and pH strongly predicted changes in fungal community composition. In contrast, increased fungal richness strongly correlated with higher plant-available phosphorus. The 93 willow trees sampled for ectomycorrhizal fungi included seven willow species. Host identity did not influence fungal richness or community composition, nor was there strong evidence of willow host preference for fungal species. Network analysis suggests that these mutualist interaction networks are not significantly nested or modular. Across a strong environmental gradient, fungal abiotic niche determined the fungal species available to associate with host plants within a habitat. Soil moisture, pH and organic matter alter the ectomycorrhizal fungal species present in communities regardless of host plant identity. Graphical Abstract Figure. Soil moisture, pH and organic matter alter the ectomycorrhizal fungal species present in communities regardless of host plant identity.

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 NH 4+ 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 NO 3 - 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.

Manipulating the rhizosphere microbiome to enhance plant stress tolerance is an environmentally friendly technology and a renewable resource to restore degraded environments. Here we suggest a sustainable bioremediation strategy on the example of Stebnyk mine tailings storage. We consider Salicornia europaea rhizosphere community, and the ability of the phytoremediation plant Salix viminalis to recruit its beneficial microbiome to mediate the pollution stress at the Stebnyk mine tailings storage. The tailings contain large amounts of brine salts and heavy metals that contaminate the ground water and surrounding areas, changing soil biogeochemistry and causing increased erosion. The species richness of the endophytic bacterial community of S. viminalis roots was assessed based on observed OTUs, Shannon-InvSimpson, and evenness index. Our results obtained using the plant-based enrichment strategy show that biodiversity was decreased across the contamination zones and that S. europaea supplementation significantly increased the species richness. Our results also indicate that the number of dominating bacteria was not changed across zones in both S. europaea -treated and untreated bacterial populations, and that the decrease in richness was mainly caused by the low abundant bacterial OTUs. The importance of selecting the bioremediation strains that are likely to harbor a reservoir of genetic traits that aid in bioremediation function from the target environment is discussed

Diaporthe ampelina, causal agent of Phomopsis cane and leaf spot of grapevine (Vitis vinifera L.) is isolated frequently from grapevine wood cankers, causing Phomopsis dieback. The latter disease is associated with four other Diaporthe species, three of which also are reported from hosts other than grape. To better understand the role of this Diaporthe community in Phomopsis dieback of grapevine and the potential for infection routes among alternate hosts, 76 Diaporthe isolates were recovered from wood cankers of cultivated grape, pear, apricot, almond and the wild host willow in four California counties. Isolates were characterized morphologically and assigned to species based on multigene sequence analyses. This study identified eight Diaporthe species from grapevine and one novel taxon from willow, D. benedicti. We report the first findings of D. australafricana and D. novem in North America. Our findings also expand the host ranges of D. ambigua to apricot and willow, D. australafricana to almond and willow, D. chamaeropis to grapevine and willow, D. foeniculina to willow and D. novem to almond. The generalists D. ambigua and D. eres were the most genetically diverse species, based on high nucleotide and haplotypic diversity, followed by the grapevine specialist D. ampelina. Analyses based on multilocus linkage disequilibrium could not reject the hypothesis of random mating for D. ambigua, which is further supported by relatively high haplotypic diversity, reports of both mating types and reports of successful matings in vitro. Pathogenicity assays revealed that D. ampelina was the most pathogenic species to grapevine wood.

Background Plants have evolved various defense mechanisms against insect herbivores, including the formation of physical barriers, the synthesis of toxic metabolites, and the activation of phytohormone responses. Although plant-associated microbiota influence plant growth and health, whether they play a role in plant defense against insect pests in natural ecosystems is unknown. Results Here, we show that leaves of beetle-damaged weeping willow ( Salix babylonica ) trees are more resistant to the leaf beetle Plagiodera versicolora (Coleoptera) than those of undamaged leaves. Bacterial community transplantation experiments demonstrated that plant-associated microbiota from the beetle-damaged willow contribute to the resistance of the beetle-damaged willow to P. versicolora . Analysis of the composition and abundance of the microbiome revealed that Pseudomonas spp. is significantly enriched in the phyllosphere, roots, and rhizosphere soil of beetle-damaged willows relative to undamaged willows. From a total of 49 Pseudomonas strains isolated from willows and rhizosphere soil, we identified seven novel Pseudomonas strains that are toxic to P. versicolora . Moreover, re-inoculation of a synthetic microbial community (SynCom) with these Pseudomonas strains enhances willow resistance to P. versicolora . Conclusions Collectively, our data reveal that willows can exploit specific entomopathogenic bacteria to enhance defense against P. versicolora , suggesting that there is a complex interplay among plants, insects, and plant-associated microbiota in natural ecosystems. Graphical Abstract 5putw3esi-W9k4EYdexs12 Video Abstract