Effects of Cymbopogon nardus (L.) W. Watson essential oil on the growth and morphogenesis of Aspergillus niger.

The growth inhibitory effect of Cymbopogon nardus (L.) W. Watson var. nurdus essential oil on Aspergillus niger (Van Tieghem) mycelium was determined on agar medium. The mycelium growth was completely inhibited at 800 mg/L. This concentration was found to be lethal under the test conditions. Essential oil at 400 mg/L caused growth inhibition of 80% after 4 days of incubation, and a delay in conidiation of 4 days compared with the control. Microscopic observations were carried out to determine the ultrastructural modifications of A. niger hyphae after treatment with C. nardus essential oil. The main change observed by transmission electron microscopy concerned the hyphal diameter and the hyphal wall, which appeared markedly thinner. These modifications in cytological structure might be caused by the interference of the essential oil with the enzymes responsible for wall synthesis which disturb normal growth. Moreover, the essential oil caused plasma membrane disruption and mitochondrial structure disorganization. The findings thus indicate the possibility of exploiting Cymbopogon nardus essential oil as an effective inhibitor of biodegrading and storage-contaminating fungi.

Molecular tools for the identification of Tuber melanosporum in agroindustry.

Tuber melanosporum Vitt., Tuber magnatum Pico, and Tuber uncinatum Chat. can be differentiated by their morphological characters. Fraud problems have arisen recently with the importation to Europe of truffles from China. T. melanosporum is morphologically very close, but distinct from the Chinese species [Tuber indicum (Cooke and Massee) and T. himalayense BC (Zhang and Winter)]. We have optimized molecular tools to unequivocally identify T. melanosporum. DNA extraction from ascocarps of black truffles is not straightforward. Problems to obtain pure DNA are due to high contents of phenolic compounds, melanine, and various polymers (proteins, polysaccharides, etc). These compounds coprecipitate with the DNA during extraction and strongly inhibit the PCR reaction. We have developed an efficient and reliable protocol for DNA extraction from truffle ascocarps. It was used successfully for DNA extraction from mycorrhizal root tips as well as from canned preparations of T. melanosporum. Several approaches to identify T. melanosporum by PCR were developed. Two specific primers for T. melanosporum were designed after comparison of the ITS region of this species with those of three Chinese fungi. They proved to be efficient to specifically detect the presence of T. melanosporum by PCR. The mycorrhizal status of trees inoculated with T. melanosporum but unable to produce truffles was confirmed in a single-step PCR reaction. A multiplex PCR approach was also developed with three sets of primers (including a specific one for Chinese truffles) to detect, in one PCR reaction, the presence of any other Tuber species mixed with T. melanosporum ascocarps. This optimized protocol, in association with the specific primers we designed, is applicable to quality control in the truffle industry from the production stages to final commercial products.

Ultrastructural Studies of the Mode of Penetration by Phoma macdonaldii in Sunflower Seedlings.

ABSTRACT An ultrastructural investigation of the artificial inoculation of sunflower with Phoma macdonaldii conidia was undertaken using light, scanning, and transmission electron microscopy to elucidate the host-parasite relationship. The behavior of the conidia deposited on the cotyledon petiole was investigated at various time intervals after inoculation. Conidia adhesion and germination were observed first. The cotyledon petiole was invaded by the fungus directly through the cuticle and via stomata. Externally, the spore and germ tube were covered with a mucilaginous polysaccharide sheath of a cotton-like appearance and of variable thickness. At the time of penetration, the host cuticle was perforated mechanically. The cuticle was slightly depressed and no enzymatic alteration could be observed. The fungus did not form appressoria on the surface of the host tissues but developed an infection peg. As soon as the cuticle barrier was crossed, the fungus rapidly colonized the host parietal layer. In a first step, the plasmalemma of the host cell appeared to be stuck against the cell wall. As soon as the fungus passed through the epidermal cell wall to reach the host cytoplasm, the plasmalemma was disrupted, and the subsequent rapid breakdown of cell integrity favored the colonization of the tissues by the pathogen.

Phylogenetic relationships between European and Chinese truffles based on parsimony and distance analysis of ITS sequences.

Phylogenetic relationships among truffle species from Europe and China were investigated through parsimony analysis of the ITS sequences. Three major clades were obtained among the species analysed. The so-called white truffles appeared polyphyletic since Tuber magnatum was grouped with brown truffles and not with the other white species (T. maculatum, T. borchii, T. dryophilum, T. puberulum). The black truffles investigated in this study, T. brumale, T. melanosporum, T. indicum and T. himalayense, were grouped in an independent clade. The Périgord black truffle T. melanosporum and the Chinese black truffles T. indicum and T. himalayense, were very closely related. The delimitation of these species was estimated by a distance analysis on several isolates collected from different geographic areas. In spite of intraspecific variations of the internal transcribed spacers (ITS) sequences, T. melanosporum and the Chinese black truffles can be unambiguously attributed to distinct taxa.

Biotrophic Development of Sporisorium reilianum f. sp. zeae in Vegetative Shoot Apex of Maize.

ABSTRACT Sporisorium reilianum f. sp. zeae is the causal agent of maize head smut, a disease present in several regions of France. A cytological study was carried out to describe a key step of the fungal etiology, in which the mycelium invades the vegetative shoot apex. Light and transmission electron microscopy observations show that the fungus is mostly intracellular and suggest that it passes through the host cell wall by lysis and mechanical pressure. The hyphae are surrounded by an amorphous vesicle-rich layer limited by a membrane related to the host plasmalemma. The encasement can be considered as an exchange zone between the plant and the fungus. The infected host cells appear normal; therefore, the fungus seems to act like a biotrophic endophyte.

Influence of essential oil of Hyssopus officinalis on the chemical composition of the walls of Aspergillus fumigatus (Fresenius).

The cell walls of the growing hyphae of Aspergillus fumigatus (Fresenius) cultured in the presence or absence of the essential oil of Hyssopus officinalis were isolated and their chemical composition analysed. The presence of the essential oil led to a reduction in levels of neutral sugars, uronic acid and proteins, whereas amino sugars, lipids and phosphorus levels were increased. HPLC analysis of the neutral sugars showed that they consisted mainly of glucose, mannose and galactose, while the amino sugars consisted of glucosamine and galactosamine. The presence of the essential oil in the culture medium induced marked changes in the content of galactose and galactosamine. Cell walls were fractionated by treatment with alkali and acid. The essential oil induced similar alterations in the various fractions with a more marked effect on the major constituents. The alterations were related to changes in the structure of the cells.

Purification, Elicitor Activity, and Cell Wall Localization of a Glycoprotein from Phytophthora parasitica var. nicotianae, a Fungal Pathogen of Tobacco.

ABSTRACT A glycoprotein of 34 kDa (GP 34) was solubilized at acidic pH from the mycelium of Phytophthora parasitica var. nicotianae and was purified by ion exchange and gel permeation chromatography. Whole tobacco plants treated with GP 34 through their roots showed an enhanced lipoxygenase activity as well as hydroxyproline-rich glycoprotein accumulation, indicating that this molecule had elicitor properties. An antiserum raised against the pure glycoprotein allowed localization of GP 34 by immunogold-labeling on the cell surface of the mycelium when the fungus was grown in vitro. In the wall-less zoospores, GP 34 was limited to the flagellum surface. It was then abundantly synthesized at the onset of encystment. During infection of tobacco plants, labeling was very faint at early stages of colonization, particularly in the susceptible host cultivar. It appeared earlier in the resistant host cultivar and was restricted to the living fungus, declining with mycelium cell death.

Identification and quantification of Tuber melanosporum Vitt. sterols.

The sterol composition of Tuber melanosporum was examined by medium-pressure liquid and high-performance liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. Ergosterol (ergosta-5,7,22-trienol) and brassicasterol (ergosta-5,22-dienol) were identified as the major components (90%). Their quantities and their relative concentrations, probably good indicators for differentiating mature truffles from immature colored ones, were determined during the maturation of the fungus. The results show that the total quantity of sterols decreased with the age of the fungus, but that the ergosterol/brassicasterol ratio remained relatively constant.

[Effect of monensin on the growth and secretion of exopolysaccharides in Botrytis cinerea Pers. and Sclerotium rolfsii Sacc]. / Influence de la monensine sur la croissance et la sécrétion des exopolysaccharides chez le Botrytis cinerea Pers. et le Sclerotium rolfsii Sacc.

The addition of various concentrations of monensin (1,5, and 10 micrograms/mL) to the culture medium inhibits the fungal growth and perturbs exopolysaccharides secretion, provoking a decrease of production in Botrytis cinerea and an increase in Sclerotium rolfsii. The ionophore induces also modifications in both polymer composition and structure. New monomers were observed in the two species and a decreased branching rate for Sclerotium rolfsii. These modifications show that monensin affects the enzymes responsible for normal wall synthesis and therefore vesicular traffic.

Effect of essential oil of Hyssopus officinalis on the lipid composition of Aspergillus fumigatus.

Addition of the essential oil of Hyssopus officinalis to the culture medium of Aspergillus fumigatus induced alterations in both growth and lipid composition of this mould. Total lipids and sterols were reduced, whereas total phospholipids were increased. There were alterations in the proportions of fatty acids, neutral lipid and phospholipid fractions.

Effect of keratin on the lipid composition of Scopulariopsis brevicaulis (Bainier).

Lipid content and composition of Scopulariopsis brevicaulis cultured with or without its natural substrate keratin was investigated. Lipid content was found to be lower in the presence of keratin (maximum 12% against 19% in the absence of keratin), with differences in levels of linoleic acid (C18 = 2), palmitic (C16:0) and to a lesser extent palmitoleic (C16 = 1) and alpha-linolenic (C18 = 3) acids. The degree of unsaturation was correspondingly lower in the organisms cultured with keratin as substrate.

Evidence for a geranyl-diphosphate synthase located within the plastids of Vitis vinifera L. cultivated in vitro.

Intact plastids from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan, free of detectable contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy, exhibit geranyl-diphosphate synthase activity (EC 2.5.1.1). This synthase activity remains stable after tryptic digestion of unlysed organelles and is enhanced by plastid disruption. We conclude that the enzyme is located within the organelle. The possibility of an isopentenyl diphosphate/dimethylallyl diphosphate translocating system which would play a major role in the regulation of monoterpene metabolism is discussed.

Aspartate aminotransferase isoenzymes in Leptosphaeria michotii. Properties and intracellular location.

Two forms of aspartate aminotransferase were obtained from the fungus Leptosphaeria michotii and purified to a state of apparent homogeneity by a five-step purification procedure ending with blue Ultrogel chromatography. Holoenzyme specific activities were 13430 and 9110 nkat oxalacetate/mg protein-1 and isoelectric points were 7.1 and 7.0 for forms A and B, respectively. Both isoenzymes were isologous dimers of Mr 92,000. They differed mainly in their Km for 2-oxoglutarate and aspartate, their ability to use cysteine sulfinate as a substrate and their ability in vitro to be specifically tightly associated as follows: form A with a malate dehydrogenase monomer of Mr 25,000; form B with an unidentified protein of Mr 40,000-44,000. Rabbit antiserum raised against the form A holoenzyme was not reactive against the form B holoenzyme and vice versa. Association of the holoenzyme with the complex essentially provoked a shift of the isoelectric point to 5.8 for form B [corrected] and to 5.2 for form B, without affecting kinetic parameters. In order to localize in situ the two transaminase forms, ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl-K4M-embedded colonies. Antiserum against form A essentially labelled cytoplasm and cell wall and, to some extent, mitochondria, while antiserum against form B heavily labelled mitochondria and cell wall and to a lesser extent cytoplasm. Moreover, mitochondria were isolated and purified by Percoll-density-gradient centrifugation. Only form A was identified in this subcellular fraction using ELISA.

[Effect of nitrogenous sources on the growth and morphogenesis of Scopulariopsis brevicaulis Bainier]. / Effet de la source azotée sur la croissance et la morphogénèse de Scopulariopsis brevicaulis Bainier.

Growth and morphology of hyphae grown on a synthetic amino-acid supplemented medium were compared with those grown on a control medium. Dry weight was lower in the amino-acids-containing medium and some ultrastructural changes were observed. Cell wall thickness increase, and the cultures were seen to colonize the cellophane support. These observations may be related to a physiological requirement of this organism when grown on amino-acids of keratin.

[The effect of cytochalasin A on the composition of subcellular fractions of hyphae in the growth of Mucor mucedo. II. Composition of the cell wall]. / Influence de la cytochalasine A sur la composition de fractions subcellulaires d'hyphes en croissance du Mucor mucedo L. II. Composition des parois.

Walls of young hyphae of Mucor mucedo L. growing in the presence or absence of cytochalasin A were isolated and their chemical content determined. Cytochalasin A induced modified proportions of various monomers resulting in a reduction of the (neutral sugars + glucuronic acid)/glucosamine ratio. The walls contained less proteins but more chitin-chitosan and phosphate. These modifications are discussed in relation to ultrastructural changes described previously.

[The effect of cytochalasin A on the composition of subcellular fractions of hyphae in the growth of Mucor mucedo L. I. Composition of the plasmalemma]. / Influence de la cytochalasine A sur la composition de fractions subcellulaires d'hyphes en croissance du Mucor mucedo L. I. Composition du plasmalemme.

The plasma membrane of young hyphae of Mucor mucedo L. growing in presence or absence of cytochalasine A was isolated by continuous density gradient centrifugation using Percoll at 10% or on discontinuous sucrose density gradient. Isolated membranes were characterized by enzymatic markers and cytochemical reactions, using electron microscopy. Lipid composition and protein content were determined. From the enzymatic point of view, the cytochalasine A induced a decrease (60%) in ATPase activity and with regard to the chemical composition of the membrane, a decrease in sterol content and in the sterol-phospholipid ratio as well as a decrease in protein content and an increase in the proportion of cysteine relative to other amino acids.

[Preliminary study of the chemical composition of crude membranes of Scopulariopsis brevicaulis (Bainier)]. / Etude préliminaire de la composition chimique des membranes 'brutes' de Scopulariopsis brevicaulis (Bainier).

Whole membrane constituents isolated from Scopulariopsis brevicaulis in culture in vitro have been studied with respect to ultracytological and biochemical composition. The ratio of proteins to lipids is near 1. Neutral lipids and phospholipids have been studied separately. The neutral lipids are triglycerids, diglycerids, free sterols and sterol esters, phospholipids are phosphatidylethanolamine and phosphatidylcholine.