Isolation of esca-associated fungi, chemical composition of xylem exudate from bleeding spurs of infected grapevines and annual trend of sap flux

Phaeomoniella chlamydospora (Pch), Phaeoacremonium aleophilum (Pal) and Fomitiporia mediterranea (Fme) caused at least a part of the external symptoms of esca by phytotoxic metabolites produced in the discoloured or decayed woody tissue. Both Pch and Pal produced in vitro several phytotoxic compounds: exopolysaccharides (pullulans) and two pentaketides (scytalone and isosclerone). The aim of this study was to: 1. isolate fungal pathogens; 2. detect phytotoxic pullulans and pentaketides; 3. detect proteins, glycoproteins and polyphenols of host response; 4. assess the annual trend of sap flux and its chemical composition. During vine bleeding (early spring: 2000-2004), the xylem sap was collected from 'Sangiovese' vines showing severe symptoms of “brown wood-streaking" or in combination with "white rot". Aliquots of sap were poured on malt-agar dishes or filtered and assayed on leaves of grapevine cvs. Italia and Sangiovese. Numerous phialidic conidia were found in the xylem sap from esca-affected grapevines; isolated in pure culture, these conidia gave origin to colonies of Pch and Pal. In addition, strains of Sphaeropsis malorum, Alternaria sp., Diplodia sp., Fusarium sp. and, bacteria were also isolated. Symptoms appeared on the leaves after absorption of the xylem sap solutions were: irregular, pale green areas located among the main veins or at the leaf margins, yellow-brown or red-brown necrotic spots. Significant differences between the data referring to each year in the trend of the parameters (flux, pH, and organic compounds) examined were recorded. The main components of the EPS were pullulans, galactomannans, glucogalactomannans, arabinogalactans. Proteins and glycoproteins were detected ranging from 14 to 60 kDa. Benzoic acid derivatives, stilbenes and flavonol-O-glycosides were the dominant polyphenols of the xylem sap. The results of this study provide new information on the production of toxic metabolites in planta by Pch and Pal associated with esca disease. Conidia of Pch and Pal, their metabolites and host response compounds were shown to occur in the xylem sap of naturally infected vines, indicating that the pathogens, their by-products and defense substances are translocated from the infected woody tissue of the trunk to the aerial part of the affected vines.