R. Dargent

Purification, characterisation and analysis of melanin extracted from Tuber melanosporum Vitt.

Abstract A new method for the purification of melanin extracted from Tuber melanosporum Vitt. with KOH under nitrogen was developed. The physical and chemical properties of the resulting dark pigment were determined, including its elemental composition and ultraviolet (UV) and infrared (IR) spectra. The results of this study were calibrated using data obtained with synthetic dihydroxyphenylalanine (DOPA) melanin and indicate that (1) purification with chloroform gave the best results, as shown by elemental and infrared analysis; and (2) the synthetic and natural melanins have the same physical and chemical properties, with the same percentage of nitrogen, the same functional groups and the same degree of polymerisation. However, they showed large differences in: (1) the time necessary to become decolorised by oxidizing agents (NaOCl and H2O2); (2) the slope of log absorbance vs wavelength. These results and data found in the literature lead to a proposal that melanin of T. melanosporum is derived from a new nitrogen precursor.

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.

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

Addition of the essential oil ofHyssopus officinalis to the culture medium ofAspergillus 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.

Cytochemical characterization of Golgi apparatus in some filamentous fungi

Abstract The Golgi apparatus is an important part of the endomembranous system of eukaryotic cells consisting of regions of stacked cisternae (dictyosomes) that are not commonly found in fungi. The present report illustrates the morphological difference of the Golgi apparatus in two species. A typical dictyosome with cisternae and vesicles has been observed in thin sections of Achlya bisexualis . Modified dictyosomes consisting of smooth-membrane cisternae have been found in Hypomyces chlorinus . The localization of carbohydrates, inosine diphosphatase and thiamine pyrophosphatase, in typical dictyosome and single Golgi cisternae has been demonstrated. The results obtained with A. bisexualis and H. chlorinus support the idea that the smooth-membrane cisternae have a function similar to those of the typical dictyosome.

Effects of triazoles on fungi. III. Composition of a plasma membrane-enriched fraction of Taphrina deformans *

Comparative chemical analyses were conducted with plasma membrane-enriched fractions of Taphrina deformans cells grown in a medium with or without the C-14 demethylation inhibitor propiconazole at a concentration that gives 50% growth inhibition. The membrane fractions were prepared using differential and discontinuous sucrose density gradient centrifugation, and characterized by cytochemical, enzymatic and chemical analyses. Membranes of nontreated cells were similar to those from other fungi with a protein/lipid ratio of 1.2, 13% phospholipid content in the membrane lipid (122 μg/mg protein), and a relatively high sterol/phospholipid molar ratio of 0.69. The corresponding membrane fraction from propiconazole-treated cells had 24% less lipid, 27% less phospholipid, 5-times more triacylglycerol relative to other neutral acyl lipids, and over a 2-fold higher sterol/phospholipid ratio. The greater sterol/phospholipid ratio was due to a higher C-14 methyl sterol content rather than less functional sterol (brassicasterol). Membranes from treated cells contained slightly less protein than those from nontreated cells, but there was little difference in the electrophoretic separation patterns of solubilized membrane polypeptides.

The biological cycle of Sporisorium reilianum f.sp. zeae: an overview using microscopy

Sporisorium reilianum f.sp. zeae is the causal agent of maize head smut. Using microscopy, we describe the development of the fungus during its saprophytic and parasitic phase. When compatible, the yeast forms fused to produce dicaryotic hyphae. These hyphae were infectious and penetrated the maize in the root. Surprisingly, the formation of conjugation tubes was rarely observed in vitro. In contrast, extensive development of long hyphae was observed from the haploid form of the yeast, these hyphae being able to fuse when arising from compatible strains. In planta, the fungus acted as a biotrophic endophyte until sporogenesis, which occurred in the floral meristem of the maize. The symptoms of the infection were reduced. Penetration in the root was never accompanied by drastic damage of the host cell and we did not observe thickening or apposition of plant material to reinforce the wall structure. Moreover, the fungus was embedded in an amorphous matrix and thus appeared isolated from the host cell. In the floral meristem, radical changes were observed, the host cell was totally invaded by the fungus in the course of sporogenesis. The deposits observed on the fungal wall are likely related to the echinulation of the teliospores.

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.

Early infection of maize roots bySporisorium reilianum f. sp.zeae

SummaryA cytological study was carried out to describe the initial steps of infection of maize roots by the soil fungusSporisorium reilianum f. sp.zeae. Morphogenetic changes of the fungal cells were induced in the presence of maize roots. Extensive hyphal growth led to the formation of a thick fungal layer colonising the maize root surface. This structure is original in interactions of members of the family Ustilaginaceae with plants. In the thick fungal layer, we observed fimbriae inserted into the host cell wall, suggesting a direct role of these fibrillar structures in cell adhesion and infection processes. During infection, no reaction of host cells was observed. In this way, the fungus acts as a biotrophic endophyte during the initial steps of infection.

Effects of a fraction from maize root exudates on haploid strains of Sporisorium reilianum f. sp. zeae

Sporisorium reilianum f. sp. zeae is the causal agent of head smut of maize. Although the main symptom of this disease is the formation of a black fungal sorus on the reproductive parts of the maize, the infection always occurs via the roots. Early infection stages are characterised by a hyphal proliferation of the fungus around the roots. In this paper, we describe effects of a fraction extracted from maize root exudates on growth of S. reilianum f. sp. zeae. The fungus grew as a yeast form on artificial medium, but in presence of these fractions, some yeasts switched to a hyphal form. In addition, an increased proliferation of the yeast form was also observed with exudates from a variety of maize susceptible to head smut. In the presence of exudates obtained from a tolerant variety of maize, proliferation of the yeast form was inhibited, whereas the induction of yeast-hypha transition was always observed. These results indicated that some molecules in root exudates could play a role in the pre-infectious stage between maize and S. reilianum f. sp. zeae.

Supporting matrix influences protoplast-derived colony formation: Structural analysis

SummaryFlax (Linum usitatissimum) protoplasts were immobilized in agarose and in Ca-alginate, medium (MV) and high viscosity (HV) grades. Protoplast viability was markedly decreased, probably as a result of the entrapment procedure itself. On the other hand, mitotic activity of surviving protoplasts was not influenced by the type of matrix agarose or alginate HV grade. Light and electron microscopical observations revealed compact heterogeneous cell colonies in agarose surrounded by cells in a more or less advanced state of lysis. In Ca-alginate (MV and HV) cell colonies were compact and spherical with poorly vacuolated cells. In this matrix, cell walls were intensely stained and sinuous, separated from the plasma membrane by a large periplasmic space.