To identify fungal species associated with grapevine trunk diseases (GTDs) in Hungary, 230 wood samples were collected from vines exhibiting typical GTD symptoms. Samples were washed and bark tissues removed before being transversally sectioned to produce 0.5 to 1 cm thick disks from parts exhibiting both healthy and necrotic tissues. Discs were surface sterilized in 1% chloramine B solution for 5 min, rinsed in sterile distilled water, and dried in a sterile box. Small woody pieces were removed with a sterile needle from the margins of necrotic and healthy plant tissues, placed on potato dextrose agar (PDA), and incubated at room temperature. The ITS region was amplified and sequenced from pure cultures using the primer pair ITS1F-ITS4. The ITS sequences of four strains, isolated in April 2013 in Maklár, Hungary, from trunks of 30-year-old grapevines cv. Pinot Gris, were identical to those of Seimatosporium vitis (GenBank accession no. KR920363) isolated from dead stems of Vitis vinifera in Italy (Senanayake et al. 2015), a Seimatosporium sp. (KU162941) isolated from a dead branch of Vitis sp. in Iran, and 99% similar to the ITS sequences of a number of closely related fungi. The ITS sequence of one strain was deposited in GenBank (KX555652). Therefore, the four strains isolated in Hungary were tentatively identified as S. vitis. To induce sporulation, these strains were transferred to agar containing autoclaved pine needles, but no conidia were produced. The pathogenicity of two strains was verified using ∼5-month-old shoots of 1.5-year-old potted vines. Shoots were surface sterilized with 70% alcohol and wounded to the pith with a transfer needle. Each plant had one wounded shoot. Four millimter diameter mycelial discs cut from 2-week-old cultures were sealed with Parafilm on the wounded surface of the shoots and plants were kept in a greenhouse. Control shoots received the same treatment with sterile PDA discs. Three plants were inoculated with each strain and two others served as controls. The pathogenicity test was carried out twice. Three weeks after inoculations, each shoot inoculated with S. vitis exhibited a 2 to 3.5 cm long lesion that extended both upward and downward from the point of inoculation. No symptoms were observed in the controls. The pathogen was reisolated from the symptomatic tissue and the ITS sequences, determined in three isolates, were identical to those previously obtained. To our knowledge, this is the first record of pathogenic strains of S. vitis isolated from grapevine with GTD symptoms in Hungary and worldwide. S. vitis was recently described by Senanayake et al. (2015), but no pathogenicity tests were conducted. Similarly, a Seimatosporium sp. was isolated from grapevine in the United States, but its pathogenicity was not demonstrated (Morales et al. 2010). Seimatosporium spp. are not considered as important pathogens associated with these diseases (Bertsch et al. 2013; Úrbez-Torres et al. 2014). Recently, pathogenic S. botan strains were reported as being associated with GTDs in Chile (Díaz et al. 2012) but these cannot be confused with S. vitis based on ITS sequences. Our report suggests that S. vitis can be associated with GTDs.
This work was carried out in the framework of COST Action FA1303 and EU H2020 project no. 652601.