Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues.

High-throughput sequencing has become a prominent tool to assess plant-associated microbial diversity. Still, some technical challenges remain in characterising these communities, notably due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are approaches to limit plant DNA contamination. However, a systematic comparison of these factors and their interactions, which could limit plant DNA contamination in the study of plant mycobiota, is still lacking. Here, three primers targeting the ITS2 region were evaluated alone or in combination with PNA clamps both on nettle (Urtica dioica) root DNA and a mock community. PNA clamps did not improve the richness or diversity of the fungal communities but increased the number of fungal reads. Among the tested factors, the most significant was the primer pair. Specifically, the 5.8S-Fun/ITS4-Fun pair exhibited a higher OTU richness but fewer fungal reads. Our study demonstrates that the choice of primers is critical for limiting plant and fungal DNA co-amplification. PNA clamps increase the number of fungal reads when ITS2 is targeted but do not result in higher fungal diversity recovery at high sequencing depth. At lower read depths, PNA clamps might enhance microbial diversity quantification for primer pairs lacking fungal specificity.

Rare earth elements perturb root architecture and ion homeostasis in Arabidopsis thaliana.

Rare earth elements (REEs) are crucial elements for current high-technology and renewable energy advances. In addition to their increasing usage and their low recyclability leading to their release into the environment, REEs are also used as crop fertilizers. However, little is known regarding the cellular and molecular effects of REEs in plants, which is crucial for better risk assessment, crop safety and phytoremediation. Here, we analysed the ionome and transcriptomic response of Arabidopsis thaliana exposed to a light (lanthanum, La) and a heavy (ytterbium, Yb) REE. At the transcriptome level, we observed the contribution of ROS and auxin redistribution to the modified root architecture following REE exposure. We found indications for the perturbation of Fe homeostasis by REEs in both roots and leaves of Arabidopsis suggesting competition between REEs and Fe. Furthermore, we propose putative ways of entry of REEs inside cells through transporters of microelements. Finally, similar to REE accumulating species, organic acid homeostasis (e.g. malate and citrate) appears critical as a tolerance mechanism in response to REEs. By combining ionomics and transcriptomics, we elucidated essential patterns of REE uptake and toxicity response of Arabidopsis and provide new hypotheses for a better evaluation of the impact of REEs on plant homeostasis.

Prospecting for rare earth element (hyper)accumulators in the Paris Herbarium using X-ray fluorescence spectroscopy reveals new distributional and taxon discoveries.

BACKGROUND: Rare earth elements (REEs) are increasingly crucial for modern technologies. Plants could be used as a biogeochemical pathfinder and a tool to extract REEs from deposits. However, a paucity of information on suitable plants for these tasks exists. METHODS: We aimed to discover new REE-(hyper)accumulating plant species by performing an X-ray fluorescence (XRF) survey at the Herbarium of the Muséum national d'Histoire naturelle (MNHN, Paris, France). We selected specific families based on the likelihood of containing REE-hyperaccumulating species, using known taxa that accumulate REEs. A total of 4425 specimens, taken in the two main evolutionary lineages of extant vascular plants, were analysed, including the two fern families Blechnaceae (n = 561) and Gleicheniaceae (n = 1310), and the two flowering plant families Phytolaccaceae (n = 1137) and Juglandaceae (n = 1417). KEY RESULTS: Yttrium (Y) was used as a proxy for REEs for methodological reasons, and a total of 268 specimens belonging to the genera Blechnopsis (n = 149), Dicranopteris (n = 75), Gleichenella (n = 32), Phytolacca (n = 6), Carya (n = 4), Juglans (n = 1) and Sticherus (n = 1) were identified with Y concentrations ranging from the limit of detection (LOD) >49 µg g-1 up to 1424 µg g-1. Subsequently, analysis of fragments of selected specimens by inductively coupled plasma atomic emission spectroscopy (ICP-AES) revealed that this translated to up to 6423 µg total REEs g-1 in Dicranopteris linearis and up to 4278 µg total REEs g-1 in Blechnopsis orientalis which are among the highest values ever recorded for REE hyperaccumulation in plants. It also proved the validity of Y as an indicator for REEs in XRF analysis of herbarium specimens. The presence of manganese (Mn) and zinc (Zn) was also studied by XRF in the selected specimens. Mn was detected in 1440 specimens ranging from the detection limit at 116 µg g-1 up to 3807 µg g-1 whilst Zn was detected in 345 specimens ranging from the detection limit at 77 µg g-1 up to 938 µg g-1. CONCLUSIONS AND IMPLICATIONS: This study led to the discovery of REE accumulation in a range of plant species, substantially higher concentrations in species known to be REE hyperaccumulators, and records of REE hyperaccumulators outside of the well-studied populations in China.

Differential effects of tree species identity on rhizospheric bacterial and fungal community richness and composition across multiple trace element-contaminated sites.

Soil microbial communities play a key role in plant nutrition and stress tolerance. This is particularly true in sites contaminated by trace metals, which often have low fertility and stressful conditions for woody plants in particular. However, we have limited knowledge of the abiotic and biotic factors affecting the richness and composition of microbial communities inhabiting the rhizosphere of plants in contaminated sites. Using high-throughput amplicon sequencing, we studied the rhizospheric bacterial and fungal community structures of 14 woody plant families planted in three contrasting sites contaminated by metals (Pb, Cd, Zn, Mn, Fe, S). The rhizospheric bacterial communities in the given sites showed no significant difference between the various woody species but did differ significantly between sites. The Proteobacteria phylum was dominant, accounting for over 25 % of the overall relative abundance, followed by Actinobacteria, Bacteroidetes and Gemmatimonadetes. Site was also the main driver of fungal community composition, yet unlike bacteria, tree species identity significantly affected fungal communities. The Betulaceae, Salicaceae and Fagaceae families had a high proportion of Basidiomycota, particularly ectomycorrhizal fungi, and the lowest diversity and richness. The other tree families and the unplanted soil harboured a greater abundance of Ascomycota and Mucoromycota. Consequently, for both bacteria and fungi, the site effect significantly impacted their community richness and composition, while the influence of plants on the richness and composition of rhizospheric microbial communities stayed consistent across sites and was dependent on the microbial kingdom. Finally, we highlighted the importance of considering this contrasting response of plant rhizospheric microbial communities in relation to their host identity, particularly to improve assisted revegetation efforts at contaminated sites.

Global deletome profile of Saccharomyces cerevisiae exposed to lithium.

The increasing use of lithium (Li) in new technologies raises the question of its impact on living microorganisms. In the present study, we aimed to identify putative Li targets and resistance mechanisms in the yeast model Saccharomyces cerevisiae using a deletomic approach based on the screening of a collection of 4733 knockout mutants under Li exposure. This screening highlighted 60 mutants resistant to Li and 124 mutants sensitive to Li. Through functional enrichment analyses, transport systems were identified as playing a central role in cell resistance to toxic concentrations of Li. In contrast, the AKT/protein kinase B family, signal transduction or cell communication were identified as potential toxic targets of Li. The majority of the mutants with a Li-sensitive phenotype were also sensitive to other alkali and alkaline earth metals, whereas the Li-resistance phenotype was mostly resistant to Na but poorly resistant to other metals. A comparison with the results of deletomics studies carried out in the presence of other metals highlighted Li-specific phenotypes. Three genes (NAM7, NMD2, UPF3) of the nonsense-mediated decay pathway were specifically involved in resistance to Li. In contrast, mutants with the NCA2, SPT20, GCN5, YOR376W, YPK3, and DCW1 genes deleted were specifically resistant to Li. These genes encode various functions from putative mannosidase to constitution of the Spt-Ada-Gcn5 acetyltransferase complex. This work provides a better understanding of potential specific resistance mechanisms and cellular targets of Li in yeast.

Yeast Deletomics to Uncover Gadolinium Toxicity Targets and Resistance Mechanisms.

Among the rare earth elements (REEs), a crucial group of metals for high-technologies. Gadolinium (Gd) is the only REE intentionally injected to human patients. The use of Gd-based contrasting agents for magnetic resonance imaging (MRI) is the primary route for Gd direct exposure and accumulation in humans. Consequently, aquatic environments are increasingly exposed to Gd due to its excretion through the urinary tract of patients following an MRI examination. The increasing number of reports mentioning Gd toxicity, notably originating from medical applications of Gd, necessitates an improved risk-benefit assessment of Gd utilizations. To go beyond toxicological studies, unravelling the mechanistic impact of Gd on humans and the ecosystem requires the use of genome-wide approaches. We used functional deletomics, a robust method relying on the screening of a knock-out mutant library of Saccharomyces cerevisiae exposed to toxic concentrations of Gd. The analysis of Gd-resistant and -sensitive mutants highlighted the cell wall, endosomes and the vacuolar compartment as cellular hotspots involved in the Gd response. Furthermore, we identified endocytosis and vesicular trafficking pathways (ESCRT) as well as sphingolipids homeostasis as playing pivotal roles mediating Gd toxicity. Finally, tens of yeast genes with human orthologs linked to renal dysfunction were identified as Gd-responsive. Therefore, the molecular and cellular pathways involved in Gd toxicity and detoxification uncovered in this study underline the pleotropic consequences of the increasing exposure to this strategic metal.

High-energy interference-free K-lines synchrotron X-ray fluorescence microscopy of rare earth elements in hyperaccumulator plants.

Synchrotron-based micro-X-ray fluorescence analysis (µXRF) is a nondestructive and highly sensitive technique. However, element mapping of rare earth elements (REEs) under standard conditions requires care, since energy-dispersive detectors are not able to differentiate accurately between REEs L-shell X-ray emission lines overlapping with K-shell X-ray emission lines of common transition elements of high concentrations. We aim to test REE element mapping with high-energy interference-free excitation of the REE K-lines on hyperaccumulator plant tissues and compare with measurements with REE L-shell excitation at the microprobe experiment of beamline P06 (PETRA III, DESY). A combination of compound refractive lens optics (CRLs) was used to obtain a micrometer-sized focused incident beam with an energy of 44 keV and an extra-thick silicon drift detector optimized for high-energy X-ray detection to detect the K-lines of yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (Pr), and neodymium (Nd) without any interferences due to line overlaps. High-energy excitation from La to Nd in the hyperaccumulator organs was successful but compared to L-line excitation less efficient and therefore slow (∼10-fold slower than similar maps at lower incident energy) due to lower flux and detection efficiency. However, REE K-lines do not suffer significantly from self-absorption, which makes XRF tomography of millimeter-sized frozen-hydrated plant samples possible. The K-line excitation of REEs at the P06 CRL setup has scope for application in samples that are particularly prone to REE interfering elements, such as soil samples with high concomitant Ti, Cr, Fe, Mn, and Ni concentrations.

Impact of dark septate endophytes on salt stress alleviation of tomato plants.

Fungal endophytes can improve plant tolerance to abiotic stress conditions. Dark septate endophytes (DSEs) belong to phylogenetically non-related groups of root colonizing fungi among the Ascomycota with high melanin-producing activities. They can be isolated from roots of more than 600 plant species in diverse ecosystems. Still the knowledge about their interaction with host plants and their contribution to stress alleviation is limited. The current work aimed to test the abilities of three DSEs (Periconia macrospinosa, Cadophora sp., Leptodontidium sp.) to alleviate moderate and high salt stress in tomato plants. By including an albino mutant, the role of melanin for the interaction with plants and salt stress alleviation could also be tested. P. macrospinosa and Cadophora sp. improved shoot and root growth 6 weeks after inoculation under moderate and high salt stress conditions. No matter how much salt stress was applied, macroelement (P, N, and C) contents were unaffected by DSE inoculation. The four tested DSE strains successfully colonized the roots of tomato, but the colonization level was clearly reduced in the albino mutant of Leptodontidium sp. Any difference in the effects on plant growth between the Leptodontidium sp. wild type strain and the albino mutant could, however, not be observed. These results show that particular DSEs are able to increase salt tolerance as they promote plant growth specifically under stress condition. Increased plant biomasses combined with stable nutrient contents resulted in higher P uptake in shoots of inoculated plants at moderate and high salt conditions and higher N uptake in the absence of salt stress in all inoculated plants, in P. macrospinosa-inoculated plants at moderate salt condition and in all inoculated plants except the albino mutants at high salt condition. In summary, melanin in DSEs seems to be important for the colonization process, but does not influence growth, nutrient uptake or salt tolerance of plants.

A reduced but stable core microbiome found in seeds of hyperaccumulators.

The seed microbiota is currently of great interest in the scientific community since seed germination is a critical stage in plant life cycle. Some seed endophytic bacteria could be commonly found in seeds of hyperaccumulating plants and may confer them an evolutionary advantage over non-hyperaccumulating plants when confronted to biotic or abiotic stress. This study focuses on the endophytic bacterial diversity of a wide diversity of metal hyperaccumulating and non-hyperaccumulating plants (93 seed samples from Mediterranean regions, Oceania, South-East Asia) to reveal the core endophyte communities specific of hyperaccumulating plants. The rather low richness of the seed bacterial communities found in all seeds suggest that a sub-population of specialized endophytic strains is able to colonize seeds and survive. The factor that shapes the diversity of those bacterial communities was first the botanical family and secondly the hyperaccumulation trait of the host plants. Based on the taxonomic affiliation, we revealed that the Brassicales had 1349 OTUs that were specific to them and the Asterales 204 OTUs, independently of their metal accumulation strategy. Nonetheless, a set of 12 OTUs were shared by the seeds of all the hyperaccumulators independently of the taxonomic order of the plants (among Asterales and Brassicales) and could be considered as a 'stable' core microbiome. Those OTUs identified as Luteibacter, Alphaproteobacteria unclassified, Sphingopyxis, Alishewanella, bacteria unclassified, Heliimonas, Aeromicrobium, Proteobacteria unclassified, Xanthomonadales unclassified and Micromonosporaceae unclassified may constitute an endophytic bacterial core with PGP traits. Further studies are needed to extend our knowledge of the possible role played by those bacteria.

Taxonomic and functional responses of soil and root bacterial communities associated with poplar exposed to a contamination gradient of phenanthrene.

Polycyclic aromatic hydrocarbon (PAH) contamination of industrial wasteland soils affects microbial diversity, but little is known about the dose-response effects of such contaminants on taxonomic and functional diversities of rhizospheric and plant endophytic bacteria. This study focused on the response of soil and root bacterial communities associated to poplar grown in a contamination gradient of phenanthrene (PHE). It was hypothesized that the increase in contamination would modify gradually the bacterial diversity and functions. The effects of the PHE contamination were limited to soil communities and did not affect the poplar root endophytome where Streptomyces and Cutibacterium were the most abundant genera. Along the PHE gradient, alpha-diversity indices decreased and the community structure of soil bacteria at the taxonomic level shifted. The abundance of genes involved in PAH-degradation pathways and the relative proportion of certain microbial taxa such as Polaromonas, Sphingopyxis, Peredibacter, Phenylobacterium, Ramlibacter, Sphingomonas, and Pseudomonas, often described as potential PAH biodegraders, increased with the PHE concentration in the soil community. Conversely, the contamination negatively impacted other taxa like Nocardioides, Streptomyces, Gaiella, Solirubrobacter, Bradyrhizobium, and Nitrospira. Functional inference and enzymatic activity measurements revealed that some bacterial functions related to carbon, nitrogen and phosphorus cycles were modified in soil throughout the PHE gradient. This study allowed a deeper understanding of the complex plant-bacteria interactions in the case of soil PAH contamination and the potential impact on soil functioning.

Locked up Inside the Vessels: Rare Earth Elements Are Transferred and Stored in the Conductive Tissues of the Accumulating Fern Dryopteris erythrosora.

Rare earth elements (REEs) are strategic metals strongly involved in low-carbon energy conversion. However, these emerging contaminants are increasingly disseminated into ecosystems, raising concern regarding their toxicity. REE-accumulating plants are crucial subjects to better understand REE transfer to the trophic chain but are also promising phytoremediation tools. In this analysis, we deciphered REE accumulation sites in the REE-accumulating fern Dryopteris erythrosora by synchrotron X-ray µfluorescence (µXRF). This technique allows a high-resolution and in situ analysis of fresh samples or frozen-hydrated cross sections of different organs of the plant. In the sporophyte, REEs were translocated from the roots to the fronds by the xylem sap and were stored within the xylem conductive system. The comparison of REE distribution and accumulation levels in the healthy and necrotic parts of the frond shed light on the differential mobility between light and heavy REEs. Furthermore, the comparison emphasized that necrotized areas were not the main REE-accumulating sites. Finally, the absence of cell-to-cell mobility of REEs in the gametophyte suggested the absence of REE-compatible transporters in photosynthetic tissues. These results provide valuable knowledge on the physiology of REE-accumulating ferns to understand the REE cycle in biological systems and the expansion of phytotechnologies for REE-enriched or REE-contaminated soils.

Mycorrhizal inoculation effects on growth and the mycobiome of poplar on two phytomanaged sites after 7-year-short rotation coppicing.

Aims: Afforestation of trace-element contaminated soils, notably with fast growing trees, has been demonstrated to be an attractive option for bioremediation due to the lower costs and dispersion of contaminants than conventional cleanup methods. Mycorrhizal fungi form symbiotic associations with plants, contributing to their tolerance towards toxic elements and actively participating to the biorestoration processes. The aim of this study was to deepen our understanding on the effects of mycorrhizal inoculation on plant development and fungal community at two trace-element contaminated sites (Pierrelaye and Fresnes-sur-Escaut, France) planted with poplar (Populus trichocarpa x Populus maximowiczii). Methods: The 2 sites were divided into 4 replicated field blocks with a final plant density of 2200 tree h-1. Half of the trees were inoculated with a commercial inoculum made of a mix of mycorrhizal species. The sites presented different physico-chemical characteristics (e.g., texture: sandy soil versus silty-loam soil and organic matter: 5.7% versus 3.4% for Pierrelaye and Fresnes-sur-Escaut, respectively) and various trace element contamination levels. Results: After 7 years of plantation, inoculation showed a significant positive effect on poplar biomass production at the two sites. Fungal composition study demonstrated a predominance of the phylum Ascomycota at both sites, with a dominance of Geopora Arenicola and Mortierella elongata, and a higher proportion of ectomycorrhizal and endophytic fungi (with the highest values observed in Fresnes-sur-Escaut: 45% and 28% for ECM and endophytic fungi, respectively), well known for their capacity to have positive effects on plant development in stressful conditions. Furthermore, Pierrelaye site showed higher frequency (%) of mycorrhizal tips for ectomycorrhizal fungi (ECM) and higher intensity (%) of mycorrhizal root cortex colonization for arbuscular mycorrhizal fungi (AMF) than Fresnes-sur-Escaut site, which translates in a higher level of diversity. Conclusions: Finally, this study demonstrated that this biofertilization approach could be recommended as an appropriate phytomanagement strategy, due to its capacity to significantly improve poplar productivity without any perturbations in soil mycobiomes.

Response of Poplar and Associated Fungal Endophytic Communities to a PAH Contamination Gradient.

Microbial populations associated to poplar are well described in non-contaminated and metal-contaminated environments but more poorly in the context of polycyclic aromatic hydrocarbon (PAH) contamination. This study aimed to understand how a gradient of phenanthrene (PHE) contamination affects poplar growth and the fungal microbiome in both soil and plant endosphere (roots, stems and leaves). Plant growth and fitness parameters indicated that the growth of Populus canadensis was impaired when PHE concentration increased above 400 mg kg-1. Values of alpha-diversity indicators of fungal diversity and richness were not affected by the PHE gradient. The PHE contamination had a stronger impact on the fungal community composition in the soil and root compartments compared to that of the aboveground organs. Most of the indicator species whose relative abundance was correlated with PHE contamination decreased along the gradient indicating a toxic effect of PHE on these fungal OTUs (Operational Taxonomic Units). However, the relative abundance of some OTUs such as Cadophora, Alternaria and Aspergillus, potentially linked to PHE degradation or being plant-beneficial taxa, increased along the gradient. Finally, this study allowed a deeper understanding of the dual response of plant and fungal communities in the case of a soil PAH contamination gradient leading to new perspectives on fungal assisted phytoremediation.

Nettle, a Long-Known Fiber Plant with New Perspectives.

The stinging nettle Urticadioica L. is a perennial crop with low fertilizer and pesticide requirements, well adapted to a wide range of environmental conditions. It has been successfully grown in most European climatic zones while also promoting local flora and fauna diversity. The cultivation of nettle could help meet the strong increase in demand for raw materials based on plant fibers as a substitute for artificial fibers in sectors as diverse as the textile and automotive industries. In the present review, we present a historical perspective of selection, harvest, and fiber processing features where the state of the art of nettle varietal selection is detailed. A synthesis of the general knowledge about its biology, adaptability, and genetics constituents, highlighting gaps in our current knowledge on interactions with other organisms, is provided. We further addressed cultivation and processing features, putting a special emphasis on harvesting systems and fiber extraction processes to improve fiber yield and quality. Various uses in industrial processes and notably for the restoration of marginal lands and avenues of future research on this high-value multi-use plant for the global fiber market are described.

Genome-Wide Mutant Screening in Yeast Reveals that the Cell Wall is a First Shield to Discriminate Light From Heavy Lanthanides.

The rapidly expanding utilization of lanthanides (Ln) for the development of new technologies, green energies, and agriculture has raised concerns regarding their impacts on the environment and human health. The absence of characterization of the underlying cellular and molecular mechanisms regarding their toxicity is a caveat in the apprehension of their environmental impacts. We performed genomic phenotyping and molecular physiology analyses of Saccharomyces cerevisiae mutants exposed to La and Yb to uncover genes and pathways affecting Ln resistance and toxicity. Ln responses strongly differed from well-known transition metal and from common responses mediated by oxidative compounds. Shared response pathways to La and Yb exposure were associated to lipid metabolism, ion homeostasis, vesicular trafficking, and endocytosis, which represents a putative way of entry for Ln. Cell wall organization and related signaling pathways allowed for the discrimination of light and heavy Ln. Mutants in cell wall integrity-related proteins (e.g., Kre1p, Kre6p) or in the activation of secretory pathway and cell wall proteins (e.g., Kex2p, Kex1p) were resistant to Yb but sensitive to La. Exposure of WT yeast to the serine protease inhibitor tosyl phenylalanyl chloromethyl ketone mimicked the phenotype of kex2∆ under Ln, strengthening these results. Our data also suggest that the relative proportions of chitin and phosphomannan could modulate the proportion of functional groups (phosphates and carboxylates) to which La and Yb could differentially bind. Moreover, we showed that kex2∆, kex1∆, kre1∆, and kre6∆ strains were all sensitive to light Ln (La to Eu), while being increasingly resistant to heavier Ln. Finally, shotgun proteomic analyses identified modulated proteins in kex2∆ exposed to Ln, among which several plasmalemma ion transporters that were less abundant and that could play a role in Yb uptake. By combining these different approaches, we unraveled that cell wall components not only act in Ln adsorption but are also active signal effectors allowing cells to differentiate light and heavy Ln. This work paves the way for future investigations to the better understanding of Ln toxicity in higher eukaryotes.

Combined omics approaches reveal distinct responses between light and heavy rare earth elements in Saccharomyces cerevisiae.

The rapid development of green energy sources and new medical technologies contributes to the increased exploitation of rare earth elements (REEs). They can be subdivided into light (LREEs) and heavy (HREEs) REEs. Mining, industrial processing, and end-use practices of REEs has led to elevated environmental concentrations and raises concerns about their toxicity to organisms and their impact on ecosystems. REE toxicity has been reported, but its precise underlying molecular effects have not been well described. Here, transcriptomic and proteomic approaches were combined to decipher the molecular responses of the model organism Saccharomyces cerevisiae to La (LREE) and Yb (HREE). Differences were observed between the early and late responses to La and Yb. Several crucial pathways were modulated in response to both REEs, such as oxidative-reduction processes, DNA replication, and carbohydrate metabolism. REE-specific responses involving the cell wall and pheromone signalling pathways were identified, and these responses have not been reported for other metals. REE exposure also modified the expression and abundance of several ion transport systems, with strong discrepancies between La and Yb. These findings are valuable for prioritizing key genes and proteins involved in La and Yb detoxification mechanisms that deserve further characterization to better understand REE environmental and human health toxicity.

Natural Fungal Endophytes From Noccaea caerulescens Mediate Neutral to Positive Effects on Plant Biomass, Mineral Nutrition and Zn Phytoextraction.

Phytoextraction using hyperaccumulating plants is a method for the remediation of soils contaminated with trace elements (TEs). As a strategy for improvement, the concept of fungal-assisted phytoextraction has emerged in the last decade. However, the role played by fungal endophytes of hyperaccumulating plants in phytoextraction is poorly studied. Here, fungal endophytes isolated from calamine or non-metalliferous populations of the Cd/Zn hyperaccumulator Noccaea caerulescens were tested for their growth promotion abilities affecting the host plant. Plants were inoculated with seven different isolates and grown for 2 months in trace element (TE)-contaminated soil. The outcomes of the interactions between N. caerulescens and its native strains ranged from neutral to beneficial. Among the strains, Alternaria thlaspis and Metapochonia rubescens, respectively, isolated from the roots of a non-metallicolous and a calamine population of N. caerulescens, respectively, exhibited the most promising abilities to enhance the Zn phytoextraction potential of N. caerulescens related to a significant increase of the plant biomass. These strains significantly increased the root elemental composition, particularly in the case of K, P, and S, suggesting an improvement of the plant nutrition. Results obtained in this study provide new insights into the relevance of microbial-assisted phytoextraction approaches in the case of hyperaccumulating plants.

Dark septate endophytes isolated from non-hyperaccumulator plants can increase phytoextraction of Cd and Zn by the hyperaccumulator Noccaea caerulescens.

Dark septate endophytes (DSEs) can improve plant stress tolerance by promoting growth and affecting element accumulation. Due to its ability to accumulate high Cd, Zn, and Ni concentrations in its shoots, Noccaea caerulescens is considered a promising candidate for phytoextraction in the field. However, the ability of DSEs to improve trace element (TE) phytoextraction with N. caerulescens has not yet been studied. The aim of this study was therefore to determine the ability of five DSE strains, previously isolated from poplar roots collected at different TE-contaminated sites, to improve plant development, mineral nutrient status, and metal accumulation by N. caerulescens during a pot experiment using two soils differing in their level of TE contamination. Microscopic observations revealed that the tested DSE strains effectively colonised the roots of N. caerulescens. In the highly contaminated (HC) soil, a threefold increase in root biomass was found in plants inoculated with the Leptodontidium sp. Pr30 strain compared to that in the non-inoculated condition; however, the plant nutrient status was not affected. In contrast, the two strains Phialophora mustea Pr27 and Leptodontidium sp. Me07 had positive effects on the mineral nutrient status of plants without significantly modifying their biomass. Compared to non-inoculated plants cultivated on HC soil, Pr27- and Pr30-inoculated plants extracted more Zn (+ 30%) and Cd (+ 90%), respectively. In conclusion, we demonstrated that the responses of N. caerulescens to DSE inoculation ranged from neutral to beneficial and we identified two strains (i.e. Leptodontidium sp. (Pr30) and Phialophora mustea (Pr27)) isolated from poplar that appeared promising as they increased the amounts of Zn and Cd extracted by improving plant growth and/or TE accumulation by N. caerulescens. These results generate interest in further characterising the DSEs that naturally colonise N. caerulescens and testing their ability to improve phytoextraction.

Salt Stress Tolerance of Dark Septate Endophytes Is Independent of Melanin Accumulation.

Dark septate endophytes (DSEs) represent a diverse group of root-endophytic fungi that have been isolated from plant roots in many different natural and anthropogenic ecosystems. Melanin is widespread in eukaryotic organisms and possesses various functions such as protecting human skin from UV radiation, affecting the virulence of pathogens, and playing a role in development and physiology of insects. Melanin is a distinctive feature of the cell walls of DSEs and has been thought to protect these fungi from abiotic stress. Melanin in DSEs is assumed to be synthesized via the 1,8-dihydroxynaphthalene (DHN) pathway. Its function in alleviation of salt stress is not yet known. The aims of this study were: (i) investigating the growth responses of three DSEs (Periconia macrospinosa, Cadophora sp., and Leptodontidium sp.) to salt stress, (ii) analyzing melanin production under salt stress and, (iii) testing the role of melanin in salt stress tolerance of DSEs. The study shows that the three DSE species can tolerate high salt concentrations. Melanin content increased in the hyphae of all DSEs at 100 mM salt, but decreased at 500 mM. This was not reflected in the RNA accumulation of the gene encoding scytalone dehydratase which is involved in melanin biosynthesis. The application of tricyclazole, a DHN-melanin biosynthesis inhibitor, did not affect either salt stress tolerance or the accumulation of sodium in the hyphae. In addition, melanin biosynthesis mutants of Leptodontidium sp. did not show decreased growth performance compared to the wild-type, especially not at high salt concentrations. This indicates that DSEs can live under salt stress and withstand these conditions regardless of melanin accumulation.

Unravelling the Role of Melanin in Cd and Zn Tolerance and Accumulation of Three Dark Septate Endophytic Species.

Dark septate endophytes (DSEs) are often trace element (TE)-tolerant fungi and are abundant in TE-polluted environments. The production of melanin, a black polymer found in cell walls, was hypothesized by several authors to play a role in the TE tolerance of DSEs. To test this hypothesis, we established a series of experiments using albino strains and melanin inhibitors and examined the responses to Cd and Zn. Six DSEs belonging to genera Cadophora sp., Leptodontidium sp. and Phialophora mustea, were evaluated. The strains mainly produced 1,8-dihydroxynaphthalene (DHN) melanin whereas 3,4-dihydroxyphenylalanin melanin was also synthetized. Cd and Zn decreased melanin synthesis in most of the strains. A reduction in melanin concentration in hyphae through the use of tricyclazole, an inhibitor of DHN-melanin synthesis, did not reduce the tolerance of the strains to Cd and Zn. Similarly, albino mutants of Leptodontidium sp. were not more sensitive to Cd and Zn than the WT strain. Moreover, tricyclazole-treated colonies accumulated less Cd but more Zn compared to untreated colonies. The Cd and Zn contents of Leptodontidium albino strains were variable and similar to that of the WT. The results suggest that melanin production is not an important functional trait that contributes to Cd and Zn tolerance, but might contribute to Cd accumulation.