Botryosphaeriaceae species affecting table grape vineyards in Chile and cultivar susceptibility

Cien. Inv. Agr. 39(3):445-458. 2012 www.rcia.uc.cl crop protection research paper Botryosphaeriaceae species affecting table grape vineyards in Chile and cultivar susceptibility Andrea Morales1, Bernardo A. Latorre2, Eduardo Piontelli3, and Ximena Besoain1 Facultad de Agronomía, Pontificia Universidad Católica de Valparaíso, Casilla 4-D, Quillota, Chile. Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Casilla 306-22, Santiago, Chile. 3 Facultad de Medicina, Universidad de Valparaíso, Casilla 92-V, Valparaíso, Chile. 1 2 Abstract A. Morales, B.A. Latorre, E. Piontelli, and X. Besoain. 2012. Botryosphaeriaceae species affecting table grape vineyards in Chile and cultivar susceptibility. Cien. Inv. Agr. 39(3): 445-458. Several Botryosphaeriaceae species have been identified as the causes of cankers and dieback of Vitis vinifera in several grape-growing regions around the world. This research was conducted to further study the species of Botryosphaeriaceae associated with table grapes in Chile, to estimate the prevalence and severity of the disease as a function of vineyard age, to study the susceptibility of table grape cultivars to infection by Botryosphaeriaceae species, and to evaluate the effect of tissue age on the infection caused by Botryosphaeriaceae species. Symptoms were characterized by the presence of the partial or total death of the grapevine cordons and distorted leaves. Brown V-shaped or U-shaped cankers and black spots were observed in cross-sections, while brown vascular streaks were observed in longitudinal sections of the cordons and trunks. Pathogenic isolates of Diplodia seriata, D. mutila and Spencermartinsia viticola were consistently obtained from wood cankers and/or vascular streaking; D. seriata was the most common (83.3%) Botryosphaeriaceae species. In 11- to 20-year-old vineyards, the disease incidence varied between 22.0 and 69.0%, and the severity varied between 6.0 and 21.3%. The table grape cultivars ‘Thompson Seedless’, ‘Redglobe’ and ‘Flame Seedless’ were equally susceptible to infection by D. mutila, D. seriata and S. viticola. The age of the inoculated tissue had no significant effect on the development of the vascular necrosis. This is the first report of D. mutila and S. viticola infections of grapevines in Chile. Key words: Diplodia, grapevine diseases, Spencermartinsia viticola, trunk diseases, Vitis vinifera. Introduction Table grapes (Vitis vinifera L.) are high-value export crops cultivated on 55,119 ha across a range of diverse climate zones in Chile. ‘ThompReceived: March 5, 2012. Accepted July 17, 2012. Corresponding author: son Seedless’, ‘Flame Seedless’ and ‘Redglobe’ are the most prevalent cultivars planted in Chile (ODEPA, 2009). Several grapevine trunk diseases have been described worldwide including Botryosphaeria cankers, black dead arm (BDA), esca, excoriose, and Eutypa dieback (Bulit et al., 1972; Lehoczky, 446 ciencia e investigación agraria 1974; Moller and Kasimatis, 1978; Latorre et al., 1986; Mugnai et al., 1999; Larignon et al., 2001; Phillips et al., 2005; Úrbez-Torres et al., 2006; Úrbez-Torres and Gubler, 2009; Úrbez-Torres, 2011). Many studies have associated canker diseases with the presence of Botryosphaeriaceae species, Diatrypaceae species, and Phomopsis viticola (Sacc.) Sacc. (Moller and Kasimatis, 1978; Larignon and Dubos, 1997; Armengol et al., 2001; Phillips, 2002; Auger et al., 2004; Trouillas et al., 2010). BDA was identified for the first time in Hungary by Lehoczky (1974), who associated this disease with Diplodia mutila Fr. (as Botryosphaeria stevensii Shoemaker). Later it was reported in Italy (Cristinzio, 1978), where the disease was associated with D. seriata De Not. (as B. obtusa Schwein. Shoemaker), and later in France (Larignon et al., 2001), where it was associated with D. seriata and B. dothidea (Moug.:Fr.) Ces & De Not. However, Chamberlain et al. (1964) were the first to suggest the role of D. seriata (as Sphaeropsis malorum Peck.) in the etiology of wood cankers of grapes. The aims of the present study were 1. to further study the Botryosphaeriaceae species associated with the dieback in table grape vineyards in Chile, 2. to estimate the prevalence and severity of the disease as a function of vineyard age, 3. to evaluate the susceptibility of table grape cultivars to infection by Botryosphaeriaceae species, and 4. to study the effect of tissue age on the infection caused by Botryosphaeriaceae species. Materials and methods Sampling locations and fungal isolations Fungal isolates were obtained from grapevines showing decline, small and distorted leaves and chlorosis. The isolations were performed between July 2008 and April 2009 from a total of 79 samples taken either from wood necrosis or vascular streaking from the following table grape cultivars: ‘Thompson Seedless’, ‘Redg- lobe’, ‘Crimson Seedless’, ‘Flame Seedless’ and ‘Superior’. The samples were collected from 22 vineyards located between Ovalle (lat. 30°, 37” S) and Paine (lat. 33°, 55” S). Small pieces of wood (<1 cm2) were selected from the margins both of V-shaped cankers and black spots observed in the cross-sections of affected arms. The samples were disinfected for 30 s in 95% ethanol, rinsed with sterile distilled water (SDW), dried and plated onto potato dextrose agar (PDA) plates acidified with 0.5 mL per liter of 96% lactic acid (APDA). The plates were incubated at 24 °C for at least 5 days or until fungi were observed growing from the symptomatic wood. The hyphal tips were sub-cultured in water agar (WA) and then transferred to APDA. The isolates were maintained in SDW at 5ºC. Morphological characterization To stimulate sporulation, the isolates were cultivated on autoclaved lignified 1-year-old apple twigs (2 cm in length) placed on WA. The cultures were incubated for 5 days at 24°C in the dark and for an additional 45 days at room temperature (18–22ºC) under ultraviolet (UV) light (λ=320 nm). For each isolate, 30 conidia were characterized according to their shape, length, width, color, and septum development. The presence or absence of pycnidium development for each isolate in APDA at 24°C was also recorded. For each isolate, ten conidiogenous cells were measured, and colony characterization was also performed. For species identification, the taxonomic key proposed by Phillips (2007) was used. Molecular characterization Genomic DNA was extracted from 3- to 4-day-old cultures using a DNeasy Plant Mini Kit (Qiagen GmbH, Duesseldorf, Germany). The internal transcribed spacer (ITS) was amplified, including the 5.8S gene of the nuclear ribosomal DNA, using VOLUME 39 Nº3 SEPTEMBER – DECEMBER 2012 primers ITS1 and ITS4 (White et al., 1990). The polymerase chain reaction (PCR) was performed in a thermal cycler (BIOER, TC-96/T/H(a), Bioer Technology Co. LTD, Tokyo, Japan), as described by Úrbez-Torres et al. (2008). The PCR products were separated by agarose gel (...truncated)