Polyaromatic hydrocarbons impair phosphorus transport by the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Phosphate uptake by plant roots is mainly mediated by arbuscular mycorrhizal fungi (AMF). However, the impact on phosphorus (P) transport of polycyclic aromatic hydrocarbons (PAH), persistent organic pollutants widely found in altered soils, is not known up today. Here, we monitored the Rhizophagus irregularis fungal growth and the fungal P transport ability from the extraradical mycelium to the host transformed chicory roots in the presence of anthracene and benzo[a]pyrene (B[a]P) and the combination of both PAH, under in vitro conditions. Firstly, our findings showed that PAH have detrimental effect on the fungal growth. The combination of both PAH was more toxic than each of the PAH individually due to synergistic effects. Secondly, PAH affected the P transport by the fungus from the medium to the roots. This was evidenced by either the decrease in (33)P quantity transported in the roots as well as the decrease in acid phosphatase activity in the mycorrhizal roots. Moreover, the fungal alkaline phosphatase activities remained constant in the extraradical mycelium as well as in the roots in the absence and in the presence of PAH. The GintPT and GiALP (encoding a P transporter and an alkaline phosphatase respectively) gene expressions were also found to be similar in the extraradical mycelium treated with PAH or not (control). These findings suggested that the P uptake by R. irregularis was not affected by PAH but probably the transport from the extraradical mycelium to the intraradical mycelium.

The arbuscular mycorrhizal Rhizophagus irregularis activates storage lipid biosynthesis to cope with the benzo[a]pyrene oxidative stress.

The phytoremediation assisted by arbuscular mycorrhizal fungi (AMF) could constitute an ecological and economic method to restore polycyclic aromatic hydrocarbon (PAH) polluted soils. Unfortunately, little is known about the PAH impact on the beneficial symbiotic AMF. Using radiolabelling experiments, our work aims to understand how benzo[a]pyrene (B[a]P), a representative of high molecular weight PAH, acts on the AMF lipid metabolism. Our results showed decreases in the sterol precursors as well as in total phospholipid quantities, in link with the [1-(14)C]acetate incorporation decreases in these lipids. Interestingly, a concomitant increase of [1-(14)C]acetate incorporation by 29.5% into phosphatidylcholine with its content decrease in Rhizophagus irregularis extraradical mycelium was observed, suggesting a membrane regeneration. A second concomitant increase (estimated to 69%) of [1-(14)C]acetate incorporation into triacylglycerols (TAG) with the content decrease was also observed. This suggests a fungal TAG biosynthesis activation probably to offset the decrease in storage lipid content when the fungus was grown under B[a]P pollution. In addition, our findings showed that lipase activity was induced by more than 3 fold in the presence of B[a]P in comparison to the control indicating that the drop in TAG content could be a consequence of their active degradation. Taken together, our data suggest the involvement of the fungal TAG metabolism to cope B[a]P toxicity through two means: (i) by providing carbon skeletons and energy necessary for membrane regeneration and/or for B[a]P translocation and degradation as well as (ii) by activating the phosphatidic acid and hexose metabolisms which may be involved in cellular stress defence.

Benzo[a]pyrene induced lipid changes in the monoxenic arbuscular mycorrhizal chicory roots.

Arbuscular mycorrhizal (AM) colonization may be one of the means that protects plants and allows them to thrive on polycyclic aromatic hydrocarbon-polluted soils including the carcinogenic benzo(a)pyrene (B[a]P). To understand the mechanisms involved in the AM symbiosis tolerance to B[a]P toxicity, the purpose of this study was to compare the lipid compositions as well as the contents between mycorrhizal and non-mycorrhizal chicory root cultures grown in vitro under B[a]P pollution. Firstly, B[a]P induced significant decreases of the Glomalean lipid markers: C16:1ω5 and 24-methyl/methylene sterol amounts in AM roots indicating a reduced AM fungal development inside the roots. Secondly, whereas increases in fatty acid amounts after B[a]P application were measured in non-mycorrhizal roots, no changes were shown in mycorrhizal roots. On the other hand, while, after treatment with B[a]P, the total phospholipid contents were unmodified in non-mycorrhizal roots in comparison with the control, drastic reductions were observed in mycorrhizal roots, mainly owing to decreases in phosphatidylethanolamine and phosphatidylcholine. Moreover, B[a]P affected AM root sterols by reducing stigmasterol. In conclusion, the findings presented in this paper have highlighted, for the first time, significant changes in the AM root lipid metabolism under B[a]P pollution and have culminated on their role in the defense/protection mechanisms.

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.

Role of arbuscular mycorrhizal symbiosis in root mineral uptake under CaCO3 stress.

This study investigated the effects of increasing CaCO(3) concentrations (0, 5, 10, 20 mM) on arbuscular mycorrhizal (AM) symbiosis establishment as well as on chicory root growth and mineral nutrient uptake in a monoxenic system. Although CaCO(3) treatments significantly decreased root growth and altered the symbiosis-related development steps of the AM fungus Rhizophagus irregularis (germination, germination hypha elongation, root colonization rate, extraradical hyphal development, sporulation), the fungus was able to completely fulfill its life cycle. Even when root growth decreased more drastically in mycorrhizal roots than in non-mycorrhizal ones in the presence of high CaCO(3) levels, the AM symbiosis was found to be beneficial for root mineral uptake. Significant increases in P, N, Fe, Zn and Cu concentrations were recorded in the mycorrhizal roots. Whereas acid and alkaline phosphatase enzymatic activities remained constant in mycorrhizal roots, they were affected in non-mycorrhizal roots grown in the presence of CaCO(3) when compared with the control.

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.

Lipid content disturbance in the arbuscular mycorrhizal, Glomus irregulare grown in monoxenic conditions under PAHs pollution.

Most polycyclic aromatic hydrocarbons (PAHs) are ubiquitous natural and/or anthropogenic pollutants that have adverse effects on the human health and the environment. Little is known about their potential effects on arbuscular mycorrhizal fungi (AMF). Thus, using monoxenic cultures, this work aims to study the impact of increasing concentrations (140 and 280 µM) of two PAHs [anthracene and benzo[a]pyrene (B[a]P)] on Glomus irregulare lipid content in relation with its development. Changes in the total lipids [fatty acids (FA), sterols, phospholipids (PL) and their associated FA (PLFA)] compositions and contents as well as [malondialdehyde (MDA)] production, of the AMF G. irregulare were examined. Direct toxic effects of both PAHs on the AMF were shown as compared to the control culture. The extraradical hyphae length and spore production were drastically restricted in the presence of PAHs. Significant decreases of the main membrane constituents, phosphatidylcholine (PC) and sterols (in particular 24-methycholesterol) were shown in G. irregulare grown under PAHs treatment. Moreover, PAHs exposure caused an oxidative stress in the AMF extraradical structures pointed out by an increase of the lipid peroxidation biomarker production (MDA). All the observed changes were less marked in presence of anthracene, which was found to be less toxic than B[a]P. Taken together, our results suggested that the drastic decrease of the AMF growth under PAHs pollution could partially be explained by depletions in sterols, PC and MDA accumulation.

Endobacteria affect the metabolic profile of their host Gigaspora margarita, an arbuscular mycorrhizal fungus.

The aim of this paper was to understand whether the endobacterium identified as Candidatus Glomeribacter gigasporarum has an effect on the biology of its host, the arbuscular mycorrhizal fungus Gigaspora margarita, through the study of the modifications induced on the fungal proteome and lipid profile. The availability of G. margarita cured spores (i.e. spores that do not contain bacteria), represented a crucial tool to enable the comparison between two fungal homogeneous populations in the presence and the absence of the bacterial components. Our results demonstrate that the endobacterial presence leads to a modulation of fungal protein expression in all the different conditions we tested (quiescent, germinating and strigolactone-elicited germinating spores), and in particular after treatment with a strigolactone analogue. The fungal fatty acid profile resulted to be modified both quantitatively and qualitatively in the absence of endobacteria, being fatty acids less abundant in the cured spores. The results offer one of the first comparative metabolic studies of an AM fungus investigated under different physiological conditions, reveal that endobacteria have an important impact on the host fungal activity, influencing both protein expression and lipid profile, and suggest that the bacterial absence is perceived by G. margarita as a stimulus which activates stress-responsive proteins.

Arbuscular mycorrhiza partially protect chicory roots against oxidative stress induced by two fungicides, fenpropimorph and fenhexamid.

The present work examined the oxidative stress induced by different concentrations (0.02 and 0.2 mg l-1) of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) in non-target chicory root colonised or not by Glomus intraradices in a monoxenic system. The fungicides were found to cause oxidative damage by increasing lipid peroxidation measured by malondialdehyde production in non-colonised roots. Detoxification of the H(2)O(2) product was measured at 0.2 mg l-1 of fenpropimorph by an increase in peroxidase activities suggesting an antioxidant capacity in these roots. Moreover, this study pointed out the ability of arbuscular mycorrhiza to alleviate partially the oxidative stress in chicory roots, probably by lowering reactive oxygen species concentrations, resulting from increases in antioxidant defences. Our results suggest that the enhanced fungicide tolerance in the AM symbiosis could be related to less cell membrane damage.

Mycorrhization alleviates benzo[a]pyrene-induced oxidative stress in an in vitro chicory root model.

Among chemicals that are widely spread both in terrestrial and aquatic ecosystems, benzo[a]pyrene is a major source of concern. However, little is known about its adverse effects on plants, as well as about the role of mycorrhization in protection of plant grown in benzo[a]pyrene-polluted conditions. Hence, to contribute to a better understanding of the adverse effects of polycyclic aromatic hydrocarbons on the partners of mycorrhizal symbiotic association, benzo[a]pyrene-induced oxidative stress was studied in transformed Cichorium intybus roots grown in vitro and colonized or not by Glomus intraradices. The arbuscular mycorrhizal fungus development (colonization, extraradical hyphae length, and spore formation) was significantly reduced in response to increasing concentrations of benzo[a]pyrene (35-280 microM). The higher length of arbuscular mycorrhizal roots, compared to non-arbuscular mycorrhizal roots following benzo[a]pyrene exposure, pointed out a lower toxicity of benzo[a]pyrene in arbuscular mycorrhizal roots, thereby suggesting protection of the roots by mycorrhization. Accordingly, in benzo[a]pyrene-exposed arbuscular mycorrhizal roots, statistically significant decreases were observed in malondialdehyde concentration and 8-hydroxy-2'-desoxyguanosine formation. The higher superoxide dismutase activity detected in mycorrhizal chicory roots could explain the benzo[a]pyrene tolerance of the colonized roots. Taken together, these results support an essential role of mycorrhizal fungi in protecting plants submitted to polycyclic aromatic hydrocarbon, notably by reducing polycyclic aromatic hydrocarbon-induced oxidative stress damage.

Functional characterization of a C-4 sterol methyl oxidase from the endomycorrhizal fungus Glomus intraradices.

Sterols are crucial components of eukaryotic membranes that control membrane fluidity and permeability. They play an important role in cell signaling, polarity and sorting. Since many steps in the pathway are essential, sterol biosynthesis inhibitors (SBI) are widely used as antifungal agents. This work reports the identification and the characterization of a C-4 sterol methyl oxidase (SMO), the first gene involved in the sterol biosynthetic pathway, so far described from an arbuscular mycorrhizal fungus. The sequence, called GintSMO, shows a primary structure, a hydrophobicity profile and a pattern of histidine-rich motifs which are typical of C-4 methyl sterol oxidases. The complementation assay in a Saccharomyces cerevisiae mutant strain demonstrates that GintSMO encodes a functional SMO. Changes in GintSMO transcript levels and in the amount of the sterol precursor squalene were observed in in vitro grown extraradical structures exposed to the fenpropimorph SBI fungicide.

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.

Effects of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) on the development of an arbuscular mycorrhizal fungus.

The effects of different concentrations (0.2, 2, 20, 200mgl(-1)) of two sterol biosynthesis inhibitor (SBI) fungicides, i.e. fenpropimorph and fenhexamid, were evaluated on the spore germination, germ tube elongation, sporulation, and root colonization of Glomus intraradices grown monoxenically in association with transformed carrot roots. The percentage of germinated spores incubated on the SBI fungicides and the length of the germ tubes decreased with increasing concentrations of both fungicides. However, for spore germination this impact was fungistatic rather than fungicidal. Extraradical mycelium architecture and spore production in contact with the SBI fungicides were also strongly impacted at high concentration (20mgl(-1)). Conversely, the colonization of roots developing in the fungicide-free compartment, but interconnected with the extraradical mycelium developing on the SBI fungicides, appeared unaffected. Our results demonstrated that the monoxenic culture system could be used as a standardized, reproducible technique to compare the impacts of different molecules on arbuscular mycorrhizal fungi, and for the initial screening of new candidate molecules before registration.

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.

Effect of the high polycyclic aromatic hydrocarbon, benzo[a]pyrene, on the lipid content of Fusarium solani.

The purpose of the present paper was to study the effect of the high polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene, on the lipid [fatty acid (FA) and sterol] composition and content of the fungi Fusarium solani and F. oxysporum, respectively recognized as good and poor PAH degraders. The major FAs and the major sterol that characterized the tested Fusarium strains were C16:0, C18:1, C18:2, and ergosterol. Lipid profiles of F. solani remained unchanged with the addition of benzo[a]pyrene in the culture media at all concentrations and duration of treatment. However, in the presence of benzo[a]pyrene, significant decreases in FA content, which reached 18% in young cultures and 28% in mature colonies, were registered. Similarly, the sterol content of F. solani was reduced by 27% in the presence of benzo[a]pyrene. In contrast, no modification in lipid profile and lipid content were observed with F. oxysporum, a strain recognized as a low benzo[a]pyrene degrader.

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.

The oil of Adenanthera pavonina L. seeds and its emulsions.

The oil of Adenanthera pavonina L. seeds was analysed by chromatographic and instrumental means. The oil was found to be rich in neutral lipids (86.2%), and low in polar lipids (13.8%). The neutral lipids consisted mainly of triacylglycerols (64.2%). Unsaturated fatty acids were found as high as 71%, while the percentage of saturated fatty acids was only 29%. GC and GC/MS analyses revealed linoleic, oleic and lignocerotic acid to be predominant among all fatty acids in the A. pavonina oil, whereas stigmasterol was the major steroid identified within this study. Subsequently, the oil was used for preparation of submicron oil-in-water (o/w) lipid emulsions. Lipid emulsions were formulated by using soybean lecithin (SL) to investigate their particle size, Zeta potential and stability at the different oil and SL ratios. The results obtained indicate possible applications of the tested oil in pharmaceutical and medical fields as drug and cosmetic active ingredient carriers.