Fungal wood-degrading enzymes in esca-diseased grapevine and effects of carbohydrate environment on fungal development.

In esca disease affecting grapevines, Phaeomoniella chlamydospora and Phaeoacremonium minimum colonize the woody parts of the trunks and arms, where they obtain nutrition from xylem sap and, potentially, from residues resulting from the enzymatic breakdown of lignified cell walls, particularly osidic residues. We quantified the secretion of lignin peroxidase, manganese peroxidase and laccase by these fungi in woody tissues of selectively infected cuttings using immunolabeling and transmission electron microscopy. Our results indicated that the detection of these enzymes was generally higher in tissues infected with Phaeoacremonium minimum. These data were confirmed through immunodetection of enzymes secreted by hyphae of fungi grown in vitro. Additionally, we observed that the supply of various carbohydrates (mono, di, tri and tetrasaccharides and polymers) differentially influenced fungal growth and polypeptide secretion. Since some secreted polypeptides display detrimental effects on grapevine cells, these results raise the question of whether the carbohydrate environment could be a factor affecting the aggressiveness of these pathogens.

Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells.

The typical fungal membrane component ergosterol was previously shown to trigger defence responses and protect plants against pathogens. Most of the elicitors mobilize the second messenger calcium, to trigger plant defences. We checked the involvement of calcium in response to ergosterol using Nicotiana plumbaginifolia and Nicotiana tabacum cv Xanthi cells expressing apoaequorin in the cytosol. First, it was verified if ergosterol was efficient in these cells inducing modifications of proton fluxes and increased expression of defence-related genes. Then, it was shown that ergosterol induced a rapid and transient biphasic increase of free [Ca²âº](cyt) which intensity depends on ergosterol concentration in the range 0.002-10 µM. Among sterols, this calcium mobilization was specific for ergosterol and, ergosterol-induced pH and [Ca²âº](cyt) changes were specifically desensitized after two subsequent applications of ergosterol. Specific modulators allowed elucidating some events in the signalling pathway triggered by ergosterol. The action of BAPTA, LaCl3, nifedipine, verapamil, neomycin, U73122 and ruthenium red suggested that the first phase was linked to calcium influx from external medium which subsequently triggered the second phase linked to calcium release from internal stores. The calcium influx and the [Ca²âº](cyt) increase depended on upstream protein phosphorylation. The extracellular alkalinization and ROS production depended on calcium influx but, the ergosterol-induced MAPK activation was calcium-independent. ROS were not involved in cytosolic calcium rise as described in other models, indicating that ROS do not systematically participate in the amplification of calcium signalling. Interestingly, ergosterol-induced ROS production is not linked to cell death and ergosterol does not induce any calcium elevation in the nucleus.

Early changes in membrane permeability, production of oxidative burst and modification of PAL activity induced by ergosterol in cotyledons of Mimosa pudica.

Ergosterol (a fungal membrane component) was shown to induce transient influx of protons and membrane hyperpolarization in cotyledonary cells of Mimosa pudica L. By contrast, chitosan (a fungal wall component with known elicitor properties) triggered membrane depolarization. In the processes induced by ergosterol, a specific desensitization was observed, since cells did not react to a second ergosterol application but did respond to a chitosan treatment. This comparative study correspondingly shows that ergosterol and chitosan were perceived in a distinct manner by plant cells. Generation of O2*-, visualized by infiltration with nitroblue tetrazolium, was displayed in organs treated with ergosterol and chitosan. This AOS production was preceded by an increase in activity of NADPH oxidase measured in protein extracts of treated cotyledons. In all the previously described processes, cholesterol had no effect, thereby indicating that ergosterol specifically induced these physiological changes known to participate in the reaction chain activated by characteristic elicitors. Contrary to chitosan, ergosterol did not greatly activate secondary metabolism as shown by the small change in content of free phenolics and by the low modification in activity of PAL, the key enzyme of this metabolic pathway. Therefore, future studies have to clarify the signalling cascade triggered by ergosterol recognition.

Antifungal effects of cysteine towards Eutypa lata, a pathogen of vineyards.

Cysteine inhibited mycelial growth of the pathogenic fungus affecting grapevines Eutypa lata Pers. Fr. Tul. and C. Tul. in a concentration-dependent manner. The threshold value (defined by the concentration inducing a growth inhibition higher than 5%) was 0.5 mM. A 10 mM concentration induced a complete inhibition of growth and triggered necrotic processes as evidenced by an increasing number of nuclei stained by propidium iodide. In conditions mimicking the plant environment (in particular, a pH near the apoplastic value, i.e. 5.5), 6 mM cysteine induced dramatic modifications in the structural organization of the mycelium (wall, mitochondria, vacuoles and nucleus) leading to death of the hyphae. The antifungal effect of the molecule increased at the acidic experimental pH (pH 4.1). The effect was highly specific to cysteine since modifying the molecular arrangement or masking the SH-function hindered the antifungal efficiency. Cysteine spectrum of action was broad among the various strains of E. lata tested. However, a lower efficiency was observed against fungal species intervening in other grapevine diseases (esca, black dead arm). Besides its direct antifungal effect, the role of cysteine presents particular interest in the fight against fungal pathogens since it triggered an excretion of ergosterol, a compound with elicitor properties. Therefore, cysteine may indirectly increase plant defense reactions.