First Report of Neofusicoccum australe Associated with Botryosphaeria Canker of Grapevine in Chile.

Abstract

A survey of trunk diseases was conducted in 2010 in vineyards (n = 14) in central Chile (latitude 33°51' to 36°30'), specifically of Vitis vinifera 'Cabernet Sauvignon,' which is the main wine-grape cultivar (38,806 ha) in Chile. The following symptoms of trunk disease were observed in 5- to 19-year-old grapevines: short internodes, dead spurs, dead cordons (arms), and shoot dieback. Upon cutting into cordons and trunks of symptomatic vines, brown, V-shaped cankers of hard consistency were observed. A total of 56 wood cankers were collected, and small pieces of symptomatic wood (approximately 4 mm in diameter) taken from the canker margin were surface disinfected (75% ethanol, 30 s) and placed on acidified PDA (0.5 ml of 96% lactic acid per liter; APDA), which was incubated for 4 to 7 days at 24°C. Colonies, tentatively identified as a species within the Botryosphaeriaceae based on the presence of whitish-to-gray aerial mycelium and exhibiting rapid growth (4 to 5 cm colony diameter in 48 h), were hyphal-tip purified to APDA for identification. Colonies produced globose, black pycnidia with unicellular, hyaline, ellipsoidal, densely granulate, externally smooth, and thin-walled conidia of 17.0 ± 0.7 ± 6.7 ± 0.4 μm (n = 20). A yellow pigmentation was observed at the center of 48-h colonies on APDA. Morphologically, these isolates were identified as Neofusicoccum australe (Slippers, Crous & M.J. Wingfield) Crous, Slippers & A.J.L. Phillips (2,3). BLASTn searches of the ITS rDNA region, amplified with PCR primers ITS4/ITS5 (532 bp), and a 400-bp section of the beta-tubulin subunit 2 gene amplified with primers Bt2a and Bt2b of N. australe (GenBank Accession No. JX290091 and JX679868, respectively) revealed 99% similarity with the ITS and beta-tubulin sequences of N. australe reference strains EF638778 and HQ392761, respectively. Pathogenicity tests were conducted using N. australe isolate Vid1559 on 2-year-old Cabernet Sauvignon plants (n = 4), which were inoculated by wounding the woody stem with a scalpel approximately 1 cm below the most basal bud, placing an 8-mm mycelial plug taken from a 7-day culture into the wound, and then sealing the wound with Parafilm. Non-inoculated controls (n = 4) were 'mock' inoculated with sterile agar plugs. After 3 months under field conditions, during spring and summer, the woody stems were examined for vascular discoloration (VD), characteristic of a wood canker. Inoculated plants had stems with light-brown, necrotic VD with a mean length of 15.2 cm, measured from the inoculation point. No VD was observed on the controls. N. australe was reisolated from 100% of the inoculated plants, completing Koch's postulates. Of 14 vineyards surveyed, 8% were infected with N. australe. N. australe is known as a trunk pathogen of grape (4), and other species of Botryosphaeriaceae have been associated with grapevine trunk disease in Chile (1). To our knowledge, this is the first report of N. australe causing Botryosphaeria canker of grape in Chile, where the pathogen is previously reported on blueberry (2).