During 2009 and 2010, a survey (n = 520) of diseased grapevines (Vitis vinifera L.) was done in vineyards located in Maipo and Colchagua valleys (33°43′ to 34°36′S) in Chile. Symptoms of trunk diseases (TD) were observed on >10-year-old grapevines and consisted of short internodes, dead spurs and arms, and dieback. In cross sections, diseased arms and trunks exhibited brown, V-shaped cankers of hard consistency. Collected canker samples from cvs. Cabernet Sauvignon, Carménère, Red Globe, Syrah, and Thompson Seedless were surface sterilized in 75% ethanol for 45 s and plated onto potato dextrose agar modified with 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (MPDA; Sigma-Aldrich, St. Louis, MO) for 7 days at 20°C. White-to-gray colonies with aerial mycelium growth turned dark gray after 3 to 5 days and tentatively identified as Botryosphaeriaceae. Hyphal tips of these colonies were transferred to MPDA and kept at 20°C with continuous light. After 30 days, colonies developed black, globose pycnidia with unicellular, hyaline, ellipsoidal, densely granulate, externally smooth, and thin-walled conidia that measured (16.3) 19.3 ± 2.3 (25.9) × (5.8) 7.4 ± 0.8 (9.2) μm (n = 20). Morphologically, these isolates were identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (2). Nucleotide BLAST analysis of the region ITS1-5.8S-ITS2 of rDNA of N. parvum isolates HMUC-104 and HMUC-105 (GenBank Accession Nos. JF273631 and JF273632) were amplified with ITS4 and ITS5 primers and revealed >99% similarity with the sequence of reference isolate (EU833984). Pathogenicity tests were conducted using isolates HMUC-104 and HMUC-105 on 30-day-old Carménère grapevines (n = 8) rooted in vitro by placing a 3- to 5-mm mycelial plug on the surface of the propagation medium. Additionally, detached green shoots (GS) (n = 5) and dormant canes (DC) (n = 6) 15-cm long were inoculated by placing a 3- to 5-mm mycelial plug underneath a cut aseptically made in the cortex. The GS and DC were placed in humidity chambers at 20 and 25°C, respectively. For controls, an equal number of rooted vines, in vitro vines, GS, and DC were treated with sterile agar plugs. Leaf number (LN), shoot length (SL), and root length (RL) were assessed on rooted plants in vitro after 30 days at 20°C. The extent of vascular discoloration (VD) of GS and DC were determined 15 and 45 days, respectively. N. parvum significantly (P < 0.05) reduced the LN, SL, and RL relative to the control plants. The length of VD varied from 54.86 to 55.39 mm and 14.8 to 15.48 mm in inoculated GS and DC, respectively. No VD symptoms were observed on the controls. N. parvum was reisolated from 100% of the inoculated in vitro plants, GS, and DC, completing Koch's postulates. N. parvum has been documented as a canker pathogen on V. vinifera and is known to contribute to the decline of grapevines. To our knowledge, this is the first report of N. parvum causing bot canker on grapevines in Chile, but has previously been reported in Australia, Spain, and the United States. Of 520 diseased plants in this study, 10 to 15% prevalence was estimated for TD and almost 2% prevalence was associated to N. parvum. Other Botryosphaeriaceae spp. were isolated with N. parvum from grapevine TD in Chilean vineyards (1,3,4).
References: (1) J. Auger et al. Plant Dis. 88:1286, 2004. (2) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (3) B. A. Latorre et al. Phytopathology 76:1112, 1986. (4) A. Morales et al. Phytopathol. Mediterr. 49:112, 2010.