First Report of Neofusicoccum parvum Associated with Grapevine Trunk Diseases in Croatia.

In September 2010, during survey of diseased grapevines (Vitis vinifera L.) in vineyards at localities Zmajevac (BZ), Orahovica (SO), Cilipi (KC), and Novalja (PN), symptoms characteristic of grapevine trunk diseases (GTD) (3) were observed, showing on cross-sectioned cordons and trunks as brown, wedge-shaped perennial cankers and/or dark streaking of the wood. In Croatia, these symptoms were traditionally associated with Eutypa Tul. & C.Tul. and with fungi from Diaporthaceae (2). From affected grapevines (cvs. Grasevina, Pinot bijeli, Malvazija dubrovacka, and Gegic), samples of symptomatic cordons and trunks were collected (n ≥ 35). To isolate the causal agents from the samples, woodchips of symptomatic tissue, surface-sterilized in 2% sodium hypochlorite for 2 min, were placed on potato dextrose agar amended with streptomycin sulphate (50 μg/ml) and incubated for 7 days at 25°C in darkness. A percentage of samples (72, 15, 27, and 54% from BZ, SO, KC, and PN, respectively) yielded fungal colonies with abundant aerial mycelium, initially white, but turning olivaceous grey after 5 days. From these colonies, monohyphal isolates were obtained and pycnidial formation stimulated by cultivation on 2% water agar with stems of plant species Foeniculum vulgare Mill. at 25°C under diffuse light for 3 weeks. Pycnidia contained conidia that were hyaline, unicellular, ellipsoid with round apices and truncated bases, and thin walled with smooth surface. Dimensions of conidia (n ≥ 50) were (12.8) 15.3 ± 1.4 (17.6) × (5.4) 6.3 ± 0.8 (7.6) μm, with length/width ratio (2.0) 2.5 ± 0.5 (3.2). Based on morphological data, species Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips was suspected (1). For molecular identification, isolates BZ330, SO334, KC342, and PN121 were used for PCR to amplify internal transcribed spacer region and partial translation elongation factor 1-alpha gene, using primers ITS5/ITS4 and EF1-728F/EF1-986R, respectively. Obtained sequences were shown to be identical between the four isolates (GenBank: KF296318, KF296319) and when compared with sequences for reference N. parvum isolate CMW9080 (AY236942, AY236887) they showed >99% homology, confirming the isolates as species N. parvum. Pathogenicity tests were done by inoculation of detached green shoots (GS) and lignified canes (LC) (n = 5) of grapevine cv. Skrlet by either mycelial plugs of the same four isolates, or sterile agar plugs for the controls. Inoculated GS were kept in flasks with sterile water in a glasshouse for 10 days, and LC in humid dark chambers for 30 days, at 25°C. Resulting vascular necrosis measured 62 to 81 mm (GS) and 215 to 246 mm (LC), but was absent on controls. Koch's postulates were satisfied by successful reisolation of N. parvum only from plants inoculated with mycelial plugs. N. parvum has been recognized as a serious grapevine pathogen, causing similar symptoms worldwide (3). To our knowledge, this is the first report of N. parvum associated with GTD in Croatia, and due to its relatively high incidence at surveyed localities, it could present considerable threat, particularly for neighboring vine growing regions. Diplodia seriata De Not., a weak pathogen (3), was also identified from a percentage of samples in this survey. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) J. Kaliterna et al. Arh. Hig. Rada Toksikol. 63:471, 2012. (3) J. R. Urbez-Torres. Phytopathol. Mediterr. 50(Suppl.):S5, 2011.