First report of Neoscytalidium dimidiatum causing shoot and needle blight of pines (Pinus spp.) in Turkey

Abstract

Several Pinus spp. (Pinaceae) cover ∼12 million ha in Turkey (15% of the national territory). In Şanlıurfa Province of Southeastern Anatolia Region, small areas have been occupied by several pine species, and their use in afforestation and landscaping is common. A new needle blight disease causing needle browning or drop was observed on 3- to 20-year-old P. nigra Arn. (European black pine), P. sylvestris L. (Scots pine), and P. eldarica Medw. (Afghan pine) trees in parks, streets, and boulevards at Tülmen, Karaköprü, Hilvan, and central districts of Şanlıurfa during spring and summer of 2018. At first, needles turned yellow starting from the tip to the bottom of the needle and dried all the way down. Later, affected needles and shoots turned light or dark brown in color, and black fungal masses of conidia appeared on some branches, shoots, and at the base of needles. The branches covered with black fungal mass were partially or totally defoliated. Disease incidence and severity varied significantly among species; P. eldarica (∼70% of stands and 80% of needles) and P. sylvestris (∼40% of stands and 30% of needles) were affected most and least severely, respectively. The blight was most pronounced in the lower part of the crown, and the most damaging effect was observed on small trees, in which defoliation, slow growth, and eventual death were inevitable. Other Pinus species, such as P. brutia Ten. (Calabrian or Turkish red pine) and P. pinea L. (stone or Mediterranean pine), remained healthy. Shoots and needle samples exhibiting blight symptoms were collected from all Pinus spp. across all locations. These samples were cut into small pieces (5 to 7 mm), surface sterilized (2% NaOCl for 30 s), rinsed three times in sterile distilled water (SDW), transferred to sterile filter paper to dry, plated onto potato dextrose agar, and grown at 28°C. The resulting colonies of a consistently isolated fungus from all samples were initially dark gray and turned black 4 to 7 days later. The mycelia formed arthric chains of dark, thick-walled, cylindrical-truncate to rod-shaped, zero- to one-septate arthroconidia (4.9 to 11.5 × 2.5 to 8.3 μm). These traits were typical of Neoscytalidium dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). The rDNA internal transcribed spacer (ITS) and large subunit (LSU) genes of two single-conidial isolates (ND183 and ND184) were amplified with ITS6/ITS4 and NL1/NL4 primer pairs, respectively, and the amplicons were sequenced (GenBank MK430055 for ITS and MK430061 and MK430062 for LSU). The LSU region sequences were 99.43% identical to N. dimidiatum DQ377925 from mango (Crous et al. 2006) and 99.81 to 100% identical to MH114592 from tomato (Türkölmez et al. 2019). The ITS sequences exhibited 99.81% identity to N. dimidiatum KJ513460 from pitahaya (Retana-Sánchez et al. 2019). Pathogenicity tests were carried out on ten 6-month-old seedlings from each of P. eldarica, P. nigra, and P. sylvestris grown in plastic pots (10-cm diameter, 3-liter volume) by spraying needles and shoots with 50 ml of conidial suspension (1 × 105 conidia/ml) of ND183. For the control, five healthy seedlings of each Pinus species were sprayed with SDW in a similar manner. All plants were maintained in a growth chamber with a 16 h light/8 h dark photoperiod at 28°C with 55% RH and watered as needed. Spray-inoculated needles initially showed yellow, small, round or oval spots within 2 weeks. Diameter of the lesions gradually increased, coalesced, and turned the entire needle dark brown. Six weeks after inoculation, infected shoots also turned brown with the needles on, whereas all control seedlings remained healthy and symptomless. The pathogen was reisolated from the inoculated needles and shoots, fulfilling Koch’s postulates. This is the first report of this pathogen on Pinus spp. in Turkey as well as worldwide. P. eldarica, P. nigra, and P. sylvestris were, therefore, added to the world host list of N. dimidiatum. The outbreak of this disease is a threat to Pinus forest lands in Turkey.