First report of Greeneria uvicola as cause of grapevine dead-arm dieback in Uruguay

In Uruguay, there are 8000 ha of several cultivars of Vitis vinifera planted in different regions. Symptoms corresponding to trunk diseases have been ascribed to Petri, Esca, dead-arm dieback and black foot (Abreo et al. 2008). Dead-arm dieback occurs on plants older than 6 year, affecting spurs and eventually sections of the cordons and trunks in which Eutypa lata causes brown and wedge-shaped necrosis (Rudelle et al. 2005). Fungi other than E. lata, such as Eutypella vitis, E. leptoplaca, Cryptovalsa ampelina (Mostert et al. 2004; Trouillas and Gubler 2004; Jordan and Schilder 2005) and Botryosphaeria spp. (Amponsah et al. 2009; Úrbez-Torres and Gubler 2009) can cause the same symptoms. In Uruguay, several fungi have been isolated from typical wedge-shaped necrotic tissues in dead arms of grapevines. Among them, Eutypella vitis, Botryosphaeria spp. and Greeneria uvicola have been misidentified as Pilidiella sp. (Abreo et al. 2008). Greeneria uvicola is an Ascomycete (Diaporthales) that causes bitter rot of grape bunches in different countries. This is an anamorphic fungus with unknown teleomorph. The fungus overwinters on stem lesions and in mummified berries (Farr et al. 2001). The objective of this work was to characterise and test the pathogenicity of isolates of G. uvicola on canes. Isolates were obtained from wedge-shaped necrotic tissues, and as an endophyte from healthy canes.

Fungi were isolated from healthy canes of cv. Paulsen rootstock used for propagation in nurseries, and cvv. Ugni Blanc and Cabernet Sauvignon plants showing symptoms of dead-arm dieback, onto potato dextrose agar (PDA) at 25°C. Emerging colonies with morphological characteristics of G. uvicola, such as pycnidia, conidiogenous cell shape, and conidial shape and size (Fig. 1), were grown as pure cultures for DNA isolation and molecular characterisation. Partial DNA amplification of LSU (large subunit) was performed with LR0R and LR7 primers (Vilgalys and Hester 1990) and sequenced by Macrogen Korea (Seoul, South Korea). Four sequences were submitted to GeneBank with accession numbers GQ870619 (FI12007), GQ870620 (FI12008), GQ870622 and GQ870621. Corrected sequences were aligned with G. uvicola (AF362570) and other Diaporthales from GenBank using Clustal W. The phylogenetical analysis was carried out with MEGA 4.0 using the method of Maximum Parsimony with a Boostrap test of 1000 pseudosamples (Fig. 2). Pathogenicity of two isolates of G. uvicola (FI12007 from wedge-shaped tissues and FI12008 from healthy canes) was evaluated on seven canes of cvv. Tannat and Cabernet Sauvignon. In total, 28 canes were inoculated. Discs of agar (5 mm diam.) with mycelia of G. uvicola were obtained from actively growing colonies on PDA and inoculated directly on wounds made with a 5-mm cork borer, and covered with Parafilm (Pechiney Plastic Packaging, Chicago, IL, USA). Control canes were inoculated with agar discs without mycelium. Canes were incubated in the dark under moist conditions in the laboratory at 25°C. Size of lesions was measured 30 days after inoculation, and eight segments (5×3 mm) from each lesion were transferred to fresh PDA medium, incubated at 25°C and 24-h illumination to recover the inoculated fungus. Percentage of canes with discolouration and percentage of recovered fungi was calculated based on total number of inoculated canes.

Phylogenetic analysis confirmed that all isolates could be assigned to G. uvicola as their sequences formed a well-supported clade (99%) with G. uvicola AF362570 from GenBank. (Fig. 2). Isolates FI12007 and FI12008 of G. uvicola were able to produce wood discolouration in xylem tissue of canes of cvv. Tannat and Cabernet Sauvignon (Fig. 3). The size of lesions was variable, and the inoculated fungus was recovered in 86% of the canes inoculated with the strain isolated from diseased tissues (FI12007) and from 71% of the canes inoculated with the strain isolated from non-symptomatic tissues (FI12008) considering both cultivars (Table 1).

Table 1.  Pathogenicity of two Greeneria uvicola isolates on canes of Cabernet Sauvignon and Tannat cultivars

The t-test analysis of length of discolouration showed that FI12008 produced more extended wood discolouration than FI12007 for both varieties (P = 0.07). When Cabernet Sauvignon was analysed separately, the statistical difference between fungal strains was evident (P = 0.0005). When cultivar sensitivity was assessed, there was no difference in length of discolouration between cvv. Tannat and Cabernet Sauvignon (P = 0.88). Finally, the fungus could be recovered in some of the canes that did not show discolouration. (Table 1).

This is the first report that G. uvicola isolated from healthy and dead-arm affected grapevines, able to produce wood discolouration similar to that caused by E. lata and Botryosphaeria spp.