Interactions between Three Fungi Associated with Esca of Grapevine, and Their Secondary Metabolites

The effect of the culture filtrates, crude organic extracts of culture filtrates, metabolites such as scytalone, pullulan and oligosaccharides produced by three fungi associated with esca, Phaeomoniella chlamydospora (Pch), Phaeoacremonium aleophilum (Pal) and Fomitiporia punctata (Fop), on the growth of the fungi themselves, was studied in vitro. At 1:1 dilution, the culture filtrates of Pal and Pch inhibited Fop completely, whereas at 1:2 dilution they only increased Fop growth latency. Fop was not inhibited by crude organic extracts of Pal or Pch. Growth of Pal was slightly stimulated at the lowest concentrations of Pch crude extracts, whereas it was inhibited at the highest concentration. Scytalone (at 1 mg ml-1), pullulan (at 0.2 mg ml-1) and oligosaccharides up to 2.5 kDa (2 mg ml-1) did not affect the radial growth of Fop

Two Naphthalenone Pentakides from Liquid Cultures of «Phaeoacremonium aleophilum», a Fungus Associated with Esca of Grapevine

Several phytotoxic metabolites were extracted from culture filtrates of Phaeoacremonium aleophilum, a fungus associated with the esca of grapevine and related diseases. Two of these metabolites were identified by chemical and spectroscopic methods as scytalone (23.9 mg l-1) and isosclerone (2.4 mg l-1), two naphthalenone pentaketides already known as fungal metabolites. Assayed on detached leaves of grapevine cv. Italia, scytalone at 0.05 mg ml-1 caused light green to chlorotic, rounded to irregular, interveinal or marginal spots, and isosclerone at 0.1 mg ml-1 caused large, coalescent chlorotic and necrotic spots followed by distortion of the lamina and withering. This is the first report on the production in vitro of scytalone and isosclerone by P. aleophilum, and on the phytotoxic activity of these compounds

Infection of Grapevines by Some Fungi Associated with Esca. I. «Fomitiporia punctata» as a Wood-Rot Inducer

Inoculation experiments with three strains of Fomitiporia punctata on grapevine cv. Sangiovese and on grafted ‘Italia’ rootstocks were carried out in southern Italy in 1992-1993. Inoculations were performed on fresh wounds made on the spurs, branches and trunks of vines showing no symptoms of esca. The fungus developed in the discoloured wood around the inoculation site, and caused white rot within two years. No symptoms were induced on foliage or fruit of the infected vines, nor was there any significant difference in virulence of the strains of F. punctata. After 2 years, re-isolation of F. punctata from the diseased woody tissues was successful, whereas no other species of fungi suspected to act as a “precursor” of wood decay were isolated. In 1999, further experiments were carried out with one strain of F. punctata on standing vines cv. Italia and Matilde free of any sign of wood deterioration. The development of internal symptoms was recorded monthly. The results indicated that the cv. Matilde was less susceptible than the cv. Italia. The first signs of spongy wood decay appeared 6 months after inoculation on both cultivars. F. punctata was re-isolated from the infected vines, whereas no species of Phaeoacremonium or other wood-decaying fungi were isolated from either inoculated or non-inoculated vines. These findings suggest that F. punctata behaves as a primary pathogen, being able to cause wood deterioration and spongy decay both on adult and young grapevines in a relatively short time, without the prior or concurrent action of other fungi

Interaction between Esca-Associated Fungi, Grapevine Calli and Micropropagated Shoot Cultures of Grapevine

Callus of the grapevine cv. Italia, micropropagated shoot cultures of the cv. Italia, Matilde and Sangiovese and callus of the tobacco cv. White Burley were cultivated on modified LS and MS-media. Phaeomoniella chlamydospora (Pch), Phaeoacremonium aleophilum (Pal) and Fomitiporia punctata (Fop) grew well in the presence of calli and vitroplants of all the grapevine cultivars which stimulated the growth of the fungi, but growth of the calli and vitroplants was reduced. All three fungi inhibited callus growth more strongly in grapevine than in tobacco. Fop had the greatest inhibitory effect on grapevine callus. Symptom severity also differed between cultivars. All three fungi invaded callus tissue after entirely overgrowing its surface. Hyphae were observed within the callus mass, where several cellular aggregates showed melanin-like deposits, particularly when invaded by Pal. On the foliar lamina of vitroplants symptoms were produced before and after the invasion of the plantlets by each fungus. Symptoms consisted in light green or chlorotic, rounded or irregular spots between the veins or along the leaf margin. The combination Pal vs. vitroplant gave the fastest host-pathogen response. The susceptibility or resistance of calli or vitroplants to escaassociated fungi could be a means to select grapevine for resistance to these fungi

Effects on Plants of Metabolites Produced in Culture by «Phaeoacremonium chlamydosporum», «P. Aleophilum» and «Fomitiporia punctata»

Several phytotoxic metabolites were extracted and purified from culture filtrates of Phaeoacremonium chlamydosporum, P. aleophilum and Fomitiporia punctata, three fungi associated with esca of grapevine. Those identified and characterised so far were a-glucans of various molecular weight (pullulans) produced by both species of Phaeoacremonium, and two naphthalenone pentaketides (scytalone and isosclerone) produced by P. aleophilum. Absorbed at very low doses by detached leaves of grapevine or injected into the woody tissue of shoots and branches of standing grapevines, these metabolites produced foliar symptoms similar to those shown by the esca-affected vines. The same results were obtained with preparations of pullulan extracted from the discoloured woody tissue of a grapevine infected with P. chlamydosporum, and with samples of commercial pullulan

Infection of Grapevines by Some Fungi Associated with Esca. II. Interaction among «Phaeoacremonium chlamydosporum, P. aleophilum» and «Fomitiporia punctata»

Cross-inoculation experiments were designed to study the interaction among the three fungi, Phaeoacremonium chlamydosporum (Pch), P. aleophilum (Pal) and Fomitiporia punctata (Fop) most commonly associated with esca of grapevine. The experiments were carried out in southern Italy on grapevines cv. Italia and Matilde, and the inoculations were performed in January 1999 by infecting trunks (cv. Italia) and branches (cv. Matilde) through wounds. Fop, Pch and Pal were inoculated singly or in groups of two or three in all possible combinations. Pch, Pal or both were inoculated above or below the inoculation site of Fop. From the results obtained in the first eight months of experiments planned for three years, the following conclusions can be drawn. All fungi inoculated singly spread along the woody tissue and caused brown streaking downward and upward along the trunk and branches of inoculated vines. On the basis of internal symptoms, vines cv. Italia were more susceptible than those cv. Matilde; in particular, the brown wood-streaking induced by P. aleophilum was much more severe on cv. Italia. Co-infection with the two tracheiphilous species of Phaeoacremonium caused wood gummosis and discoloration, whereas the lignicolous basidiomycete F. punctata alone caused wood deterioration and decay (white rot). Severity of internal symptoms, assessed as extent and colour of the discoloured wood, varied with the growth and interaction of the inoculated fungi. The wood discoloration caused by F. punctata was not hampered by P. chlamydosporum, but it was always limited by P. aleophilum. A similar interaction was observed in vitro with cultures of the three fungi together, which showed a marked antagonistic effect of P. aleophilum against F. punctata. Although not consistent, foliar symptoms (interveinal and marginal chlorotic areas) developed within six months on ‘Matilde’ vines co-inoculated with either species of Phaeoacremonium, or within three months after a syringe containing a liquid culture of P. chlamydosporum was inserted into current-season ‘Italia’ shoots