Structural and biochemical characterization of SmoPG1, an exo-polygalacturonase from Selaginella moellendorffii.

Polygalacturonases (PGs) can modulate chemistry and mechanical properties of the plant cell wall through the degradation of pectins, one of its major constituents. PGs are largely used in food, beverage, textile, and paper industries to increase processes' performances. To improve the use of PGs, knowledge of their biochemical, structural and functional features is of prime importance. Our study aims at characterizing SmoPG1, a polygalacturonase from Selaginella moellendorffii, that belongs to the lycophytes. Transcription data showed that SmoPG1 was mainly expressed in S. moellendorffii shoots while phylogenetic analyses suggested that SmoPG1 is an exo-PG, which was confirmed by the biochemical characterization following its expression in heterologous system. Indeed, LC-MS/MS oligoprofiling using various pectic substrates identified galacturonic acid (GalA) as the main hydrolysis product. We found that SmoPG1 was most active on polygalacturonic acid (PGA) at pH 5, and that its activity could be modulated by different cations (Ca2+, Cu2+, Fe2+, Mg2+, Mn2+, Na2+, Zn2+). In addition, SmoPG1 was inhibited by green tea catechins, including (-)-epigallocatechin-3-gallate (EGCG). Docking analyses and MD simulations showed in detail amino acids responsible for the SmoPG1-EGCG interaction. Considering its expression yield and activity, SmoPG1 appears as a prime candidate for the industrial production of GalA.

Plant testing with hemp and miscanthus to assess phytomanagement options including biostimulants and mycorrhizae on a metal-contaminated soil to provide biomass for sustainable biofuel production.

The need of biofuels from biomass, including sustainable aviation fuel, without using agricultural land dedicated to food crops, is in constant demand. Strategies to intensify biomass production using mycorrhizal fungi, biostimulants and their combinations could be solutions for improving the cultivation of lignocellulosic plants but still lack well-established validation on metal-contaminated soils. This study aimed to assess the yield of Miscanthus x giganteus J.M. Greef & Deuter and Cannabis sativa L. grown on a metal-contaminated agricultural soil (11 mg Cd, 536 mg Pb and 955 mg Zn kg-1) amended with biostimulants and/or arbuscular mycorrhizal fungi, and the shoot Cd, Pb and Zn uptake. A pot trial was carried out with soil collected from a field near a former Pb/Zn smelter in France and six treatments: control (C), protein hydrolysate (a mixture of peptides and amino acids, PH), humic/fulvic acids (HFA), arbuscular mycorrhizae fungi (AMF), PH combined with AMF (PHxAMF), and HFA combined with AMF (HFAxAMF). Metal concentrations in the soil pore water (SPW), pH and electrical conductivity were measured over time. Miscanthus and hemp shoots were harvested on day 90. Both PH and PHxAMF treatments increased SPW Cd, Pb, and Zn concentrations (e.g. by 26, 1.9, and 22.9 times for miscanthus and 9.7, 4.7, and 19.3 times for hemp in the PH and PHxAMF treatments as compared to the control one, respectively). This led to phytotoxicity and reduced shoot yield for miscanthus. Conversely, HFA and HFAxAMF treatments decreased SPW Cd and Zn concentrations, increasing shoot yields for hemp and miscanthus. Shoot Cd, Pb, and Zn uptakes peaked for PH and PHxAMF hemp plants (in µg plant-1, Cd: 310-334, Pb: 34-38, and Zn: 232-309 for PHxAMF and PH, respectively), while lowest values occurred for PH miscanthus plants mainly due to low shoot yield. Overall, this study suggested that humic/fulvic acids can be an effective biostimulant for increasing shoot biomass production in a metal-contaminated soil. These results warrant further investigations of the HFAxAMF in field trials.

Phytoextraction of Zn and Cd with Arabidopsis halleri: a focus on fertilization and biological amendment as a means of increasing biomass and Cd and Zn concentrations.

The current work aims to investigate the influence of fertilization (fertilizer) and fungal inoculation (Funneliformis mosseae and Serendipita indica (formerly Piriformospora indica), respectively arbuscular mycorrhizal (AMF) and endophytic fungi) on the phytoextraction potential of Arabidopsis halleri (L.) O'Kane & Al-Shehbaz (biomass yield and/or aboveground part Zn and Cd concentrations) over one life plant cycle. The mycorrhizal rates of A. halleri were measured in situ while the fungal inoculation experiments were carried out under controlled conditions. For the first time, it is demonstrated that the fertilizer used on A. halleri increased its biomass not only at the rosette stage but also at the flowering and fruiting stages. Fertilizer reduced the Zn concentration variability between developmental stages and increased the Cd concentration at fruiting stage. A. halleri roots did not show AMF colonization at any stage in our field conditions, neither in the absence nor in the presence of fertilizer, thus suggesting that A. halleri is not naturally mycorrhizal. Induced mycorrhization agreed with this result. However, S. indica has been shown to successfully colonize A. halleri roots under controlled conditions. This study confirms the benefit of using fertilizer to increase the phytoextraction potential of A. halleri. Overall, these results contribute to the future applicability of A. halleri in a phytomanagement strategy by giving information on its cultural itinerary.

Phytomanagement of a metal(loid)-contaminated agricultural site using aromatic and medicinal plants to produce essential oils: analysis of the metal(loid) fate in the value chain.

Phytomanagement uses plants and soil conditioners to create value on contaminated land while minimizing environmental risk. This work was carried out on a metal(loid)-contaminated site and aimed at assessing the suitability of Salvia sclarea L. (sage) and Coriandrum sativum L. (coriander) combined with an arbuscular mycorrhizal fungus (AMF) inoculant to immobilize metal(loid)s and produce essential oils (EO). The effect of the inoculant on the transfer of metal(loid)s (ML, i.e., Cd, Cu, Pb, Zn, As, Ni, and Sb) to plants and the ML soil mobility were investigated. The ML concentrations in EO from both plant species and the valorization options for the distillation residues (soil conditioner, animal fodder, and anaerobic digestion) were studied. Sage was a suitable candidate for this value chain because it presents an excluder phenotype and the residues of oil extraction could be used as a soil conditioner. The metal concentrations in the sage EO were similar to those obtained from plants cultivated on an uncontaminated soil. These results indicate the suitability of sage harvested on the contaminated soil according to the ML fate in the whole value chain. Like the EO of sage, ML concentrations in the coriander EO did not differ from those in the commercial EO that were obtained from plants grown on uncontaminated soil. However, the use of distillation residues of coriander was limited by their relatively elevated Cd concentrations. The use of a mycorrhizal inoculum did not decrease the Cd mobility in soil for the coriander.

Aided Phytoremediation to Clean Up Dioxins/Furans-Aged Contaminated Soil: correlation between microbial communities and pollutant dissipation.

To restore and clean up polluted soils, aided phytoremediation was found to be an effective, eco-friendly, and feasible approach in the case of many organic pollutants. However, little is known about its potential efficiency regarding polychlorinated dibenzo-p-dioxins and furans-contaminated soils. Thus, phytoremediation of aged dioxins/furans-contaminated soil was carried out through microcosm experiments vegetated with alfalfa combined with different amendments: an arbuscular mycorrhizal fungal inoculum (Funneliformis mosseae), a biosurfactant (rhamnolipids), a dioxins/furans degrading-bacterium (Sphingomonas wittichii RW1), and native microbiota. The total dioxins/furans dissipation was estimated to 23%, which corresponds to 48 ng.kg-1 of soil, after six months of culture in the vegetated soil combined with the four amendments compared to the non-vegetated soil. Our findings showed that the dioxins/furans dissipation resulted from the stimulation of soil microbial enzyme activities (fluorescein diacetate hydrolase and dehydrogenase) and the increase of bacterial abundance, richness, and diversity, as well as fungal diversity. Amplicon sequencing using Illumina MiSeq analysis led to identification of several bacterial (Bacillaceae, Sphingomonadaceae) and fungal (Chaetomium) groups known to be involved in dioxins/furans degradation. Furthermore, concomitant cytotoxicity and dioxins/furans concentration decreases were pointed out in the phytoremediated soil. The current study demonstrated the usefulness of combining different types of amendments to improve phytoremediation efficacy of aged dioxins/furans-contaminated soils.

Ecotoxicity evaluation and human risk assessment of an agricultural polluted soil.

The present study aims to evaluate the nature and level of chemical pollution as well as the potential toxicity and ecotoxicity of an agricultural soil irrigated by the water of Litani River. Our findings showed that the soil was mainly contaminated by alkanes (hentriacontane, octadecane, hexadecane) and metal trace elements (nickel, vanadium, chromium, and manganese). Soil organic extracts showed high cytotoxicity against human hepatic (HepG2) and bronchial epithelial cells (Beas-2B). Soil ecotoxicity was revealed by seed germination inhibition of several plant species (wheat, clover, alfalfa, tall fescue, and ryegrass) ranging from 7 to 30% on the polluted soil compared to non-polluted one. In addition, significant decreases in telluric microbial biomasses (bacterial and fungal biomasses), quantified by phospholipid fatty acids (PLFA) analysis were observed in polluted soil compared to non-contaminated soils. The density of the arbuscular mycorrhizal fungal (AMF) spores isolated from the polluted soil was about 316 spores/100 g. Three main AMF species were identified as Funelliformis mosseae, Septoglomus constrictum, and Claroideoglomus lamellosum. Moreover, 16 indigenous plant species were inventoried with Silybum marianum L. as the dominant one. Plant biodiversity indices (Shannon, Simpson, Menhinick, and Margaleff) were lower than those found in other contaminated soils. Finally, it was found that all the present plant species on this polluted site were mycorrhized, suggesting a possible protection of these plants against encountered pollutants, and the possibility to use AMF-assisted phytoremediation to clean-up such a site.

Defence mechanisms associated with mycorrhiza-induced resistance in wheat against powdery mildew.

To develop a more sustainable agriculture using alternative control strategies, mechanisms involved in the biocontrol ability of the arbuscular mycorrhizal fungus Funneliformis mosseae to protect wheat against the foliar biotrophic pathogen Blumeria graminis f. sp. tritici were investigated under controlled conditions. B. graminis infection on wheat leaves was reduced by 78% in mycorrhizal plants compared with non-mycorrhizal ones (control). Wheat roots inoculated with F. mosseae revealed a systemic resistance in leaves to B. graminis, after a 6-week co-culture. Accordingly, this resistance was associated with a significant reduction of B. graminis haustorium formation in epidermal leaf cells of mycorrhizal wheat and an accumulation of phenolic compounds and H2O2 at B. graminis penetration sites. Moreover, gene expression analysis demonstrated upregulation of genes encoding for several defence markers, such as peroxidase, phenylalanine ammonia lyase, chitinase 1 and nonexpressor of pathogenesis-related proteins 1 in mycorrhizal wheat only in the absence of the pathogen. This study showed that protection of wheat obtained against B. graminis in response to mycorrhizal inoculation by F. mosseae could be interpreted as a mycorrhiza-induced resistance (MIR). Our findings also suggest that MIR-associated mechanisms impaired the B. graminis development process and corresponded to a systemic elicitation of plant defences rather than a primed state in wheat leaves.

Impact of soil salinity on arbuscular mycorrhizal fungi biodiversity and microflora biomass associated with Tamarix articulata Vahll rhizosphere in arid and semi-arid Algerian areas.

Soil salinization is an increasingly important problem in many parts of the world, particularly under arid and semi-arid areas. Unfortunately, the knowledge about restoration of salt affected ecosystems using mycorrhizae is limited. The current study aims to investigate the impact of salinity on the microbial richness of the halophytic plant Tamarix articulata rhizosphere. Soil samples were collected from natural sites with increasing salinity (1.82-4.95 ds.m(-1)). Six arbuscular mycorrhizal fungi (AMF) species were isolated from the different saline soils and identified as Septoglomus constrictum, Funneliformis mosseae, Funneliformis geosporum, Funneliformis coronatum, Rhizophagus fasciculatus, and Gigaspora gigantea. The number of AMF spores increased with soil salinity. Total root colonization rate decreased from 65 to 16% but remained possible with soil salinity. Microbial biomass in T. articulata rhizosphere was affected by salinity. The phospholipid fatty acids (PLFA) C16:1ω5 as well as i15:0, a15:0, i16:0, i17:0, a17:0, cy17:0, C18:1ω7 and cy19:0 increased in high saline soils suggesting that AMF and bacterial biomasses increased with salinity. In contrast, ergosterol amount was negatively correlated with soil salinity indicating that ectomycorrhizal and saprotrophic fungal biomasses were reduced with salinity. Our findings highlight the adaptation of arbuscular and bacterial communities to natural soil salinity and thus the potential use of mycorrhizal T. articulata trees as an approach to restore moderately saline disturbed arid lands.

Propiconazole inhibits the sterol 14α-demethylase in Glomus irregulare like in phytopathogenic fungi.

The increasing concentrations impact (0.02, 0.2 and 2 mg L(-1)) of a Sterol Biosynthesis Inhibitor (SBI) fungicide, propiconazole, was evaluated on development and sterol metabolism of two non-target organisms: mycorrhizal or non-mycorrhizal transformed chicory roots and the arbuscular mycorrhizal fungus (AMF) Glomus irregulare using monoxenic cultures. In this work, we provide the first evidence of a direct impact of propiconazole on the AMF by disturbing its sterol metabolism. A significant decrease in end-products sterols contents (24-methylcholesterol and in 24-ethylcholesterol) was observed concomitantly to a 24-methylenedihydrolanosterol accumulation indicating the inhibition of a key enzyme in sterol biosynthesis pathway, the sterol 14α-demethylase like in phytopathogenic fungi. A decrease in end-product sterol contents in propiconazole-treated roots was also observed suggesting a slowing down of the sterol metabolism in plant. Taken together, our findings suggest that the inhibition of the both AM symbiotic partners development by propiconazole results from their sterol metabolism alterations.

Calcareous impact on arbuscular mycorrhizal fungus development and on lipid peroxidation in monoxenic roots.

The present work underlined the negative effects of increasing CaCO(3) concentrations (5, 10 and 20 mM) both on the chicory root growth and the arbuscular mycorrhizal fungus (AMF) Glomus irregulare development in monoxenic system. CaCO(3) was found to reduce drastically the main stages of G. irregulare life cycle (spore germination, germinative hyphae elongation, root colonization, extraradical hyphae development and sporulation) but not to inhibit it completely. The root colonization drop was confirmed by the decrease in the arbuscular mycorrhizal fungal marker C16:1ω5 amounts in the mycorrhizal chicory roots grown in the presence of CaCO(3). Oxidative damage evaluated by lipid peroxidation increase measured by (i) malondialdehyde (MDA) production and (ii) the antioxidant enzyme peroxidase (POD) activities, was highlighted in chicory roots grown in the presence of CaCO(3). However, MDA formation was significantly higher in non-mycorrhizal roots as compared to mycorrhizal ones. This study pointed out the ability of arbuscular mycorrhizal symbiosis to enhance plant tolerance to high levels of CaCO(3) by preventing lipid peroxidation and so less cell membrane damage.

Differential effects of fenpropimorph and fenhexamid, two sterol biosynthesis inhibitor fungicides, on arbuscular mycorrhizal development and sterol metabolism in carrot roots.

Sterols composition of transformed carrot roots incubated in presence of increasing concentrations of fenpropimorph (0.02; 0.2; 2mgl(-1)) and fenhexamid (0.02; 0.2; 2; 20mgl(-1)), colonized or not by Glomus intraradices was determined. In mycorrhizal roots treated with fenpropimorph, normal Delta(5)-sterols were replaced by unusual compounds such as 9beta,19-cyclopropylsterols (24-methylpollinastanol), Delta(8,14)-sterols (ergosta-8,14-dienol, stigmasta-8,14-dienol), Delta(8)-sterols (Delta(8) sitosterol) and Delta(7)-sterols (ergosta-7,22-dienol). After application of fenpropimorph, a drastic reduction of the mycorrhizal root growth, root colonization and extraradical fungal development was observed. Application of fenhexamid did not modify sterol profiles and the total colonization of roots. But the arbuscule frequency of the fungal partner was significantly affected. Comparison of the effects caused by the tested fungicides indicates that the usual phytosterols may be involved in symbiosis development. Indeed, observed modifications of root sterols composition could explain the high fenpropimorph toxicity to the AM symbiosis. However, the absence of sterolic modifications in the roots treated with fenhexamid could account for its more limited impact on mycorrhization.

Changes in lipid composition of Blumeria graminis f.sp. tritici conidia produced on wheat leaves treated with heptanoyl salicylic acid.

Treatment of wheat leaves with heptanoyl salicylic acid (HS) and trehalose at concentrations of 0.1 and 15 g l(-1), prior to fungal inoculation, resulted in 40% and 60% protection, respectively, against powdery mildew. The total lipid composition of Blumeria graminis f.sp. tritici (Bgt) conidia, the causal agent of wheat powdery mildew, was compared when produced on wheat leaves, respectively, untreated and treated with the two elicitors, HS and trehalose. An obvious effect was observed on lipid composition (sterol and fatty acid (FA)) of Bgt conidia produced on wheat leaves treated with HS. A total of 16 FA (C12-C24 saturated and unsaturated) as well as unusual methoxylated Fatty Acids (mFA) (3-methoxydocosanoic and 3-methoxytetracosanoic acids) were detected in the conidia. Medium chain FA were predominant in HS treated conidia (64.65%) while long chain fatty acids constituted the major compounds in untreated conidia (62%). The long chain/medium chain FA ratio decreased from 1.8 in the conidia produced on untreated leaves to 0.5 in the conidia obtained from HS treated leaves. When comparing the sterol composition of Bgt conidia produced on leaves treated with HS versus conidia obtained from untreated ones, very important changes within the two major classes can be seen. In particular, 24-methylsterols, e.g., 24-methylenecholesterol and 24-methylcholesta-7,24-dien were reduced by about 82% whereas 24-ethylsterols, e.g., 24-ethylcholesterol and 24-ethylcholesta-5,22-dienol were increased by about 85%. The 24-methylsterols/24-ethylsterols ratio was reduced by ninefold in the conidia produced from HS treated leaves.

Methoxylated fatty acids in Blumeria graminis conidia.

The total fatty acids (FA) composition of Blumeria graminis f.sp. tritici conidia, the causal agent of wheat powdery mildew, was analyzed as a function of their age. A total of 19 FA (C12-C24 saturated and unsaturated) and unusual methoxylated fatty acids (mFA) were detected in young, intermediate and old conidia. Two very long chain methoxylated FA were identified by GC-MS as 3-methoxydocosanoic and 3-methoxytetracosanoic acids. Medium chain FA were predominant in young conidia (75%, including 13% of mFA) while very long chain fatty acids constituted the major compounds in old conidia (74%, including 30% of mFA). We have shown for the first time that the total FA composition is strongly correlated with the age of B. graminis f.sp. tritici (Bgt) conidia.