First report of Neoscytalidium dimidiatum causing dieback, shoot blight and branch canker of willow trees in Turkey

Abstract

Salix alba L. (Salicaceae), a deciduous tree with ascending branches, naturally occurs throughout Turkey and is extensively used as a shade tree in city parks and botanical gardens of urban ecosystems and mostly for its timber in forestry. In June 2018, declining willow trees were observed at Haliliye and central districts of Şanlıurfa in the southeastern Anatolia region. The affected stands were located in public parks and boulevards (37°10′08.3″N, 38°49′16.9″E; 37°10′53.5″N, 38°49′07.1″E) with a high incidence in 3- to 8-year-old S. alba trees. The trees exhibited lack of vigor, foliar chlorosis, dieback of the crown with sudden wilting of shoots and branches followed by light to dark brown discoloration of leaves, reduced foliage, cankers, and dead branches. In most cases the bark had cracked and peeled off, thereby exposing a black sooty mass of fungal conidia. Longitudinal cracks also extended in the wood tissue beneath the broken bark. In some cases, bark cracks, cankers, and the black sooty spore masses also occurred on trunks. Symptomatic trees were scattered in the whole survey area. Samples of shoots and cankered branches of 20 affected trees from various points in these localities were cut into discs and surface disinfected by immersing in 2% NaOCl for 30 s, rinsed in sterile distilled water (SDW), blotted dry with sterile filter paper, placed onto potato dextrose agar (PDA), and incubated at 28°C. The sooty layer of conidia under the peeled barks of branches was also plated onto PDA using a sterile needle. A fungal species with dark, gray to black, and fast-growing colonies was consistently isolated from shoots, branches, and the sooty layer. Mycelia were branched and septate and formed arthric chains of zero- to one-septate arthroconidia, cylindrical-truncate or rod-shaped, 5.8 to 11.1 × 2.6 to 5.7 μm, initially hyaline that became dark brown and with a thick wall with age. These traits concurred with those of Neoscytalidium dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). The rDNA internal transcribed spacer (ITS) and large subunit (LSU) genes of three single-conidial isolates were amplified with ITS6/ITS4 and NL1/NL4 primer pairs, respectively, and the amplicons were sequenced (GenBank accession nos. MK430052 to MK430054 for ITS and MK430058 to MK430060 for LSU). The ITS sequences were 99.54 to 99.82% identical to N. dimidiatum accession number KR867696 from willow (Hashemi and Mohammadi 2016), and the LSU region sequences showed 100% identity to N. dimidiatum accession number MH114592 from tomato (Türkölmez et al. 2019). Pathogenicity tests were performed on 60-cm-long detached branch segments of 5-year-old S. alba, which were surface disinfected with 75% alcohol for 3 to 5 s and sealed with wax at one end. Branch segments were then wounded (5-mm diameter, 2-mm deep) between two nodes using a sterilized scalpel. Five-millimeter-diameter mycelial plugs from the margin of actively growing colonies (PDA) of an isolate were placed in the wounds and wrapped with Parafilm. Noncolonized PDA plugs were used as a control. The wax-free ends of the inoculated and control branches (15 each) were dipped individually in 100 to 150 ml of SDW in 250-ml Erlenmeyer flasks. After 6 weeks, all inoculated branches showed necrotic lesions, with an average length of 14.2 ±1.8 cm. No symptoms were observed in the control branches. The pathogen was reisolated from all inoculated shoots and identified by conidial morphology to fulfill Koch’s postulates. N. dimidiatum is known to cause gummosis, dieback, branch wilt, decline, leaf spot, tip rot, and canker of many woody plants in various families worldwide (Farr and Rossman 2019). On Salix spp., it was previously only isolated from internal wood lesions of S. alba trees in Iran (Hashemi and Mohammadi 2016). To our knowledge, this is the first report of N. dimidiatum causing willow dieback, shoot blight, and branch canker in Turkey. One of the biggest concerns should be paid to the provenance of this fungus, which is significantly involved in human pathology (Machouart et al. 2013).