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We investigated the possible transgenerational transfer of bacterial seed endophytes across three consecutive seed generations of Crotalaria pumila growing on a metal mining site in Mexico. Seeds were collected during three successive years in the semi-arid region of Zimapan, Mexico. Total communities of seed endophytes were investigated using DNA extraction from surface sterilized seeds and 454 pyrosequencing of the V5-V7 hypervariable regions of the 16S rRNA gene. The communities consisted of an average of 75 operational taxonomic units (OTUs); richness and diversity did not change across years. Methylobacterium, Staphylococcus, Corynebacterium, Propionibacterium and eight other OTUs constituted >60% of the community in each generation. The microbiome was dominated by Methylobacterium (present in >80% of samples). Functions associated with the microbiome were C and N fixation, oxidative phosphorylation and photosynthesis activity. The bacterial endophytic communities were similar across three consecutive seed generations. Among the core microbiome Methylobacterium strains were the most abundant and they can contribute to nutrient acquisition, plant growth promotion and stress resilience to their host in metal contaminated mine residues. Identification of the seed microbiome of C. pumila may lead to novel and more efficient inoculants for microbe-assisted phytoremediation.

The Citadel, part of the pre-Hispanic city of Teotihuacan and listed as a World Heritage Site, harbors irreplaceable archaeological walls and murals. This city was abandoned by the 7th century and its potential deterioration represents a noteworthy loss of the world's cultural heritage. This research consisted of isolation and identification of bacteria and fungi contributing to this deterioration from walls of a pre-Hispanic city. In addition, silver nanoparticles (AgNP) produced, using a green synthesis method, were tested as potential inhibitors of microbes. AgNP of different sizes and concentrations were tested using in situ assays. Leaf aqueous extracts from two plants species (Foeniculum vulgare and Tecoma stans) and two extraction procedures were used in the NP synthesis. The potential of AgNP as preventive/corrective treatments to protect stucco materials from biodeterioration, as well as the microbial inhibition on three stone materials (stucco, basalt and calcite) was analyzed. Twenty-three bacterial species belonging to eight genera and fourteen fungal species belonging to seven genera were isolated from colored stains, patinas and biofilms produced on the surfaces of archaeological walls from the pre-Hispanic city, Teotihuacan. AgNP from F. vulgare were more effective for in vitro microbial growth inhibition than those from T. stans. Bacteria were less sensitive to AgNP than fungi; however, sensitivity mainly depended on the microbial strain and the plant extract used to prepare AgNP. The use of AgNP as a preventive or corrective treatment to decrease microbial colonization in three kinds of stone used in historical walls was successful. Calcite was more colonized by Alternaria alternata, but less by Pectobacterium carotovorum. This is the first study at different scales (in vitro and tests on different stone types) of inhibition of biodeterioration-causing microorganisms isolated from an archaeological site by green synthesized AgNP.

Plant surfaces are known as an important sink for various air pollutants, including particulate matter and its associated potentially toxic elements (PTE). Moreover, leaves surface or phylloplane is a habitat that harbors diverse bacterial communities (epiphytic). However, little is known about their possible functions during phytoremediation of air pollutants like PTE. The study of leaf epiphytic bacteria of plants colonizing mine residues (MR) containing PTE is thus a key to understand and exploit plant–epiphytic bacteria interactions for air phytoremediation purposes. In this research, we aimed (i) to characterize the functions of epiphytic bacteria isolated from the phylloplane of Brickellia veronicifolia, Flaveria trinervia, Gnaphalium sp., and Allionia choisyi growing spontaneously on multi-PTE contaminated MR and (ii) to compare these against the same plant species in a non-polluted control site (NC). Concentrations (mg kg-1) of PTE on MR leaf surfaces of A. choisyi reached up to 232 for Pb, 13 for Cd, 2,728 for As, 52 for Sb, 123 for Cu in F. trinervia, and 269 for Zn in Gnaphalium sp. In the four plant species, the amount of colony-forming units per cm2 was superior in MR leaves than in NC ones, being A. choisyi the plant species with the highest value. Moreover, the proportion of isolates tolerant to PTE (Zn, Cu, Cd, and Sb), UV light, and drought was higher in MR leaves than in those in NC. Strain BA15, isolated from MR B. veronicifolia, tolerated 150 mg Zn L-1, 30 mg Sb L-1, 25 mg Cu L-1; 80 mg Pb L-1, and was able to grow after 12 h of continuous exposition to UV light and 8 weeks of drought. Plant growth promotion related traits [N fixation, indole acetic acid (IAA) production, and phosphate solubilization] of bacterial isolates varied among plant species isolates and between MR and NC sampling condition. The studied epiphytic isolates possess functions interesting for phytoremediation of air pollutants. The results of this research may contribute to the development of novel and more efficient inoculants for microbe-assisted phytoremediation applied to improve air quality in areas exposed to the dispersion of metal mine tailings.

Vanilla is an orchid of economic importance widely cultivated in tropical regions and native to Mexico. We sampled three species of Vanilla (V. planifolia, V. pompona, and V. insignis) in different crop systems. We studied the effect of crop system on the abundance, type of fungi, and quality of pelotons found in the roots using light and electron microscopy and direct sequencing of mycorrhizal structures. Fungi were identified directly from pelotons obtained from terrestrial roots of vanilla plants in the flowering stage. Root samples were collected from plants in crop systems located in the Totonacapan area in Mexico (states of Puebla and Veracruz). DNA was extracted directly from 40 pelotons and amplified using ITS rRNA sequencing. Peloton-like structures were observed, presenting a combination of active pelotons characterized by abundant hyphal coils and pelotons in various stages of degradation. The most active pelotons were observed in crop systems throughout living tutors (host tree) in comparison with roots collected from dead or artificial tutors. Fungi identified directly from pelotons included Scleroderma areolatum, a common ectomycorrhizal fungus that has not been reported as a mycorrhizal symbiont in orchids. Direct amplification of pelotons also yielded common plant pathogens, including Fusarium and Pyrenophora seminiperda, especially in those sites with low colonization rates, and where large numbers of degraded pelotons were observed. This research reports for the first time the potential colonization of Vanilla by Scleroderma, as a putative orchid mycorrhizal symbiont in four sites in Mexico and the influence of crop system on mycorrhizal colonization on this orchid.

We utilized the two-compartment system to study the effect of arsenic (As) on the expression of the Glomus intraradices high-affinity phosphate transporter GiPT, and the GiArsA gene, a novel protein with a possible putative role as part of an arsenite efflux pump and similar to ArsA ATPase. Our results show that induction of GiPT expression correlates with As(V) uptake in the extra-radical mycelium of G. intraradices. We showed a time-concerted induction of transcript levels first of GiPT, followed by GiArsA, as well as the location of gene expression using laser microdissection of these two genes not only in the extra-radical mycelium but also in arbuscules. This work represents the first report showing the dissection of the molecular players involved in arbuscular mycorrhizal fungus (AMF)-mediated As tolerance in plants, and suggests that tolerance mediated by AMF may be caused by an As exclusion mechanism, where fungal structures such as the extra-radical mycelium and arbuscules may be playing an important role. Our results extend knowledge of the mechanisms underlying As efflux in arbuscular mycorrhizal fungi and mechanisms related to As tolerance.

In this study, the capacity of an ornamental species (Coleus blumei) to extract and accumulate aluminum was evaluated. The analyzed parameters were amount of soluble aluminum, radical growth, tolerance rate, bioaccumulation factor, and tissues aluminum concentration. The main limiting factor for aluminum accumulation is the availability of the metal. However, Coleus blumei can grow and accumulate up to 1445.7 mg kg(-1) of aluminum dry base. This plant can play an important role in the treatment of polluted water with metals, since it can grow in conditions with a pH of around 4.8. The aluminum tolerance rate showed for this plant ranged between 18.8% and 25%. Therefore, this species behaves as a non-accumulator, even though the bioaccumulation factor was 3098.5 L kg(-1).

A field study of the natural attenuation occurring in a slag heap contaminated with high available cadmium was carried out. The aims of this research were: to determine plants colonizing this slag heap; to analyze colonization and morphological biodiversity of spores of arbuscular mycorrhizal fungi (AMF); to determine spore distribution in undisturbed samples; to know mycelium and glomalin abundance in the rhizosphere of these plants, and to investigate glomalin participation in Cd-stabilization. Forming vegetal islands, 22 different pioneering plant species from 11 families were colonizing the slag heap. The most common plants were species of Fabaceae, Asteraceae and Poaceae. Almost all plants were hosting AMF in their roots, and spores belonging to Gigaspora, Glomus, Scutellospora and Acaulospora species were observed. Micromorphological analysis showed that spores were related to decomposing vegetal residues and excrements, which means that mesofauna is contributing to their dispersion in the groundmass. Mycelium mass ranged from 0.11 to 26.3 mg/g, which contained between 13 and 75 mg of glomalin/g. Slag-extracted total glomalin was between 0.36 and 4.74 mg/g. Cadmium sequestered by glomalin extracted from either slag or mycelium was 0.028 mg/g. The ecological implication of these results is that organisms occupying vegetal patches are modifying mine residues, which contribute to soil formation.

Phytoremediation is a technology for extracting or inactivating pollutants. Echinochloa polystachya [(H.B.K.) Hitchcock] (Poaceae) is a fast-growing perennial grass that is common in tropical areas and is often found in oil-polluted soils that contain high concentrations of heavy metals. However, its tolerance to heavy metals, and its ability to accumulate them, has yet to be investigated. Here we test the hypothesis that E. polystachya is able to accumulate high concentrations of cadmium (Cd). Plants were grown hydroponically with different levels of Cd(2+) (0, 0.25, 1, 2, 10, 50, and 100mgL(-1)), and were found to be tolerant to Cd(2+) at all levels. No metal-toxicity symptoms were observed at any Cd(2+) level. Root and leaves Cd concentrations were 299+/-13.93 and 233+/-8.77mgkg(-1) (on a dry weight basis), respectively. Scanning electron microscopy showed the inclusion of Cd within the xylem; this result was confirmed by energy dispersive X-ray spectrometry. Leaf tissues also accumulated Cd, especially within the bulliform cells of the epidermis. We conclude that E. polystachya is a hyperaccumulator of Cd. While data for other metals are not yet available, E. polystachya shows promise in the phytoextraction of Cd from polluted tropical sites.

Metal contaminated soils are increasing worldwide. Metal-tolerant plants growing on metalliferous soils are fascinating genetic and microbial resources. Seeds can vertically transmit endophytic microorganisms that can assist next generations to cope with environmental stresses, through yet poorly understood mechanisms. The aims of this study were to identify the core seed endophyte microbiome of the pioneer metallophyte Crotalaria pumila throughout three generations, and to better understand the plant colonisation of the seed endophyte Methylobacterium sp. Cp3. Strain Cp3 was detected in C. pumila seeds across three successive generations and showed the most dominant community member. When inoculated in the soil at the time of flowering, strain Cp3 migrated from soil to seeds. Using confocal microscopy, Cp3-mCherry was demonstrated to colonise the root cortex cells and xylem vessels of the stem under metal stress. Moreover, strain Cp3 showed genetic and in planta potential to promote seed germination and seedling development. We revealed, for the first time, that the seed microbiome of a pioneer plant growing in its natural environment, and the colonisation behaviour of an important plant growth promoting systemic seed endophyte. Future characterization of seed microbiota will lead to a better understanding of their functional contribution and the potential use for seed-fortification applications. This research was supported by a BOF-BILA grant from Hasselt University, the UHasselt Methusalem project 08M03VGRJ

The protective mechanisms employed by arbuscular mycorrhizal fungi (AMF) to reduce the toxic effects of arsenic on host plants remain partially unknown. The goal of this research was identifying the in situ localization and speciation of arsenic (As) in the AM fungus Rhizophagus intraradices [formerly named Glomus intraradices] exposed to arsenate [As(V)]. By using a two-compartment in vitro fungal cultures of R. intraradices-transformed carrot roots, microspectroscopic X-ray fluorescence (μ-XRF), and microspectroscopic X-ray absorption near edge structure (μ-XANES), we observed that As(V) is absorbed after 1 h in the hyphae of AMF. Three hours after exposure a decrease in the concentration of As was noticed and after 24 and 72 h no detectable As concentrations were perceived suggesting that As taken up was pumped out from the hyphae. No As was detected within the roots or hyphae in the root compartment zone three or 45 h after exposure. This suggests a dual protective mechanism to the plant by rapidly excluding As from the fungus and preventing As translocation to the plant root. μ-XANES data showed that gradual As(V) reduction occurred in the AM hyphae between 1 and 3 h after arsenic exposure and was completed after 6 h. Principal component analysis (PCA) and linear combination fitting (LCF) of μ-XANES data showed that the dominant species after reduction of As(V) by R. intraradices extra-radical hyphal was As(III) complexed with a reduced iron(II) carbonate compound. The second most abundant As species present was As(V)–iron hydroxides. The remaining As(III) compounds identified by the LCF analyses suggested these molecules were made of reduced As and S. These results increase our knowledge on the mechanism of As transport in AMF and validate our hypotheses that R. intraradices directly participates in arsenic detoxification. These fungal mechanisms may help AMF colonized plants to increase their tolerance to As at contaminated sites.

To find if ornamental plants are applicable to the remediation of metal-polluted areas, the tolerance of chrysanthemum plants (Chysanthemum maximum) var. Shasta to different metals under hydroponic conditions was studied. Their responses as influenced by the mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe BEG25 on substrates containing mine residues were also investigated. Our results showed that chrysanthemum is a metal-tolerant plant under hydroponic conditions, plants behaving as Pb-excluders, whereas Cd, Cu and Ni were accumulated in roots. Low accumulation in flowers was observed for Cd and Cu but it was concentration-dependent. Ni and Pb were not translocated to flowers. Shoot biomass was not significantly affected by the different rates of mine residue addition for both mycorrhizal and non-mycorrhizal plants. Mycorrhizal plants accumulated less Pb and Cu in both shoots and roots than non-mycorrhizal plants. Chysanthemum could be a prospective plant for revegetation of tailings and the use of inoculation may decrease plant metal accumulation in polluted soils.

Plant establishment, presence of arbuscular mycorrhizal fungi (AMF) and other rhizospheric fungi were studied in mine wastes from Zimapan, Hidalgo state, Mexico, using a holistic approach. Two long-term afforested and three non-afforested mine tailings were included in this research. Fifty-six plant species belonging to 29 families were successfully established on the afforested sites, while unmanaged tailings had only a few native plant species colonizing the surrounding soils. Almost all plant roots collected were associated to AMF in these sites. The genus Glomus was the most abundant AMF species found in their rhizosphere; however, the Acaulospora genus was also observed. Other rhizospheric fungi were identified by 18S rDNA sequencing analysis. Their role in these substrates, i.e. biocontrol, pollutant- and organic matter-degradation, and aides that increase plant metal tolerance is discussed. Our results advance the understanding of fungal diversity in sites polluted with metals and present alternative plants for remediation use.

Naturally occurring soil organic compounds stabilize potentially toxic elements (PTEs) such as Cu, Cd, Pb, and Mn. The hypothesis of this work was that an insoluble glycoprotein, glomalin, produced in copious amounts on hyphae of arbuscular mycorrhizal fungi (AMF) sequesters PTEs. Glomalin can be extracted from laboratory cultures of AMF and from soils. Three different experiments were conducted. Experiment 1 showed that glomalin extracted from two polluted soils contained 1.6–4.3 mg Cu, 0.02–0.08 mg Cd, and 0.62–1.12 mg Pb/g glomalin. Experiment 2 showed that glomalin from hyphae of an isolate of Gigaspora rosea sequestered up to 28 mg Cu/g in vitro. Experiment 3 tested in vivo differences in Cu sequestration by Cu-tolerant and non-tolerant isolates of Glomus mosseae colonizing sorghum. Plants were fed with nutrient solution containing 0.5, 10 or 20 μM of Cu. Although no differences between isolates were detected, mean values for the 20 μM Cu level were 1.6, 0.4, and 0.3 mg Cu/g for glomalin extracted from hyphae, from sand after removal of hyphae and from hyphae attached to roots, respectively. Glomalin should be considered for biostabilization leading to remediation of polluted soils.