Anne Grandmougin-Ferjani

Sterol distribution in arbuscular mycorrhizal fungi

Abstract The sterol composition of spores from 16 species of arbuscular mycorrhizal fungi belonging to the order Glomales were examined by GC–MS. The major compound was found to be 24-ethylcholesterol (up to 85%) followed by cholesterol (up to 15%). Several other sterols such as 24-methylcholesterol, Δ5-avenasterol and 24-ethylcholesta-5,22-dien-3 β -ol were also detected. Significant amounts of α -amyrin, a common vascular plant triterpene, were present in the spores of all the fungal species analyzed. The absence of ergosterol, a classical fungal sterol, is discussed in relation to fungal evolution.

Glomus proliferum sp. nov.: a description based on morphological, biochemical, molecular and monoxenic cultivation data

A new arbuscular mycorrhizal fungus, Glomus proliferum (Glomales, Zygomycetes) is described. The description, based on a monoxenic culture established in association with a Ri T-DNA transformed carrot root, combines sequencing of the small subunit (SSU) rDNA, spore sterols and fatty acid profiles with more classical taxonomic tools such as optical and electron microscopy. The fungus forms clusters containing hundreds of small, hyaline, four-layered spores. The necessity to use different tools for identification of arbuscular mycorrhizal fungi is discussed.

Sterol biosynthesis by the arbuscular mycorrhizal fungus Glomus intraradices

Ri-T-DNA-transformed carrot roots were used for investigating sterol metabolism by the arbuscular mycorrhizal (AM) fungus Glomus intraradices under three distinct experimental conditions: (i) a symbiotic stage (fungus still attached to the host roots); (ii) a detached stage (fungus physically separated from the roots); and (iii) a germinating stage (germinating spores). In all three stages, G. intraradices was found to contain a mixture of 24-alkylated sterols, with 24-methyl and 24-ethyl cholesterol as the main compounds, but no ergosterol, the predominant sterol in most fungi. Feeding experiments with [1-14C]sodium acetate were performed to check the ability of the fungus to synthesize sterols. Whatever the experimental conditions, G. intraradices was able to actively take up exogenous acetate and to incorporate it into sterols and their precursors. Our data provide first evidence for de novo sterol synthesis by an AM fungus.

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.

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.

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.

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 oilin- 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.

Métabolisme lipidique du champignon endomycorhizien: Glomus intraradices

Abstract The use of monoxenic cultures of the obligately biotrophic vesicular arbuscular fungus Glomus intraradices now permits investigation of the lipid metabolism of this organism. In bicompartmental culture plates, sporulating extraradical hyphae can be obtained, totally free of roots, and then provided with 14 C-acetate as lipid precursor. Three experimental stages were studied: i) stage A, symbiotic stage corresponding to the fungus still attached to the host plant roots, ii) stage B, consisting of the fungus detached from the host roots, iii) stage C, germinating spores. In each case, the fungus proved to be able to synthesise its own lipids: 1,2- and 1,3-diacylglycerols, triacylglycerols, phospholipids, sterols and free fatty acids, de novo. Lipid metabolism varied with the experimental conditions. Phospholipid synthesis was intensive in germinating spores. Thus the obligately biotrophic status of this fungus cannot be explained by a deficiency in synthesis of these various lipid classes.

Propiconazole toxicity on the non-target organism, the arbuscular mycorrhizal fungus, Glomus irregulare.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts that colonize the roots of most terrestrial plants. Indeed, 80% of vegetal species realize this symbiosis (Bonfante and Perotto, 1995). Plants generally benefit from this AMF association through increased plant nutrient uptake, plant growth and survival rates (Smith and Read, 2008). The symbiotic association may also increase host plant resistance/tolerance against biotic (Hol and Cook, 2005; Akhtar and Siddiqui, 2008) and abiotic stresses, including salinity, drought and pollution (Gerdemann, 1968; Franco-Ramirez et al., 2007; Giri et al., 2007; Sudova et al., 2007; Cartmill et al., 2008; Debiane et al., 2008, 2009; Campagnac et al., 2010). The functioning of AMF may be impaired by cultural practices such as fungicides application (Sukarno et al., 2006). Unfortunately, the use of fungicides is generalized in modern agriculture for the control of fungal diseases. Most of fungicides act directly on essential fungal functions such as respiration, lipid synthesis or cell division (Leroux, 2003). Consequently, they can exhibit undesirable effects on non-target organisms. Among the fungicides, the Sterol Biosynthesis Inhibitor (SBI) family is one of the most used in agriculture (Hewitt, 1998). Four main classes can be distinguished according to their action target site: (i) squalene epoxidation (e.g. naftifine, terbinafine, tolnaftate), (ii) Δ14 demethylation or DMIs (e.g. imazalil, prochloraz, triadimenol, propiconazole), (iii) Δ14reduction and/or Δ8→ Δ7 isomerisation (e.g. fenpropidine, fenpropimorph, tridemorph), (iiii) C4 demethylation (e.g. fenhexamid) (Leroux, 2003). Several studies carried out on SBI fungicide impact on mycorrhizal plants showed contradictory results on the plant growth, on AM fungal development and on the symbiosis functioning (Dodd and Jeffries, 1989; Von Alten et al., 1993; Schweiger and Jacobsen, 1998; Kjoller and Rosendahl, 2000; Schweiger et al., 2001). The use of different experimental procedure in the reported studies (plant species, culture conditions, fungicide formulation, application methods...) did not allow easy comparison with the results obtained and led to some difficulties to give clear conclusion concerning the SBI fungicides effect on AMF (Sancholle et al., 2001).