First report of Neoscytalidium dimidiatum causing black canker and root rot of walnut in Turkey

Abstract

Walnut (Juglans regia L.) is commercially important with about 87,670 ha grown in Turkey, the fourth largest producer worldwide. During summer 2018, unfamiliar and serious decline symptoms associated with stem and branch cankers were observed in walnut trees in two orchards in S¸ anlıurfa Province, Southeastern Anatolia Region. In a 3-year-old orchard of 450 trees (cv. Chandler) in Kayseri, Siverek District, about 62% of trees had deep, large, and black cankers in the trunks and on main branches, drastically reduced tree vigor, and yellowing, wilting, and browning of the leaves, followed by leaf drop. In a 12-year-old orchard with 30 trees (cv. Kaman), in Yaylak, Bozova District, 40% of trees had similar symptoms. Symptoms were first noticed in trunks. Bark cracks, cankers in trunks, and dark discoloration or a black sooty, dusty mass of fungal spores under peeling bark on the trunk and main branches were the most striking symptoms. Deep cankers expanded longitudinally, extending along trunks and into the phloem of the trees, which consequently died, and occasionally laterally extending into the sapwood. Declining trees had dry root rot symptoms, with black necrosis on the surface, and under the cracked and coarse bark extending from the taproot and some large roots to the crown, and black rot and loss of fine roots. Samples were taken from under the bark of trunk cankers, crown tissues, and root tissues of five trees per Q:1 orchard. The sections of samples were surface sterilized in 2% NaOCl for 2 min and rinsed twice in sterile distilled water, blotted dry with sterilized filter paper, placed on PDA, and incubated at 28°C. A fungus with identical cultural characteristics was consistently isolated. On PDA, the mycelium growth was white and then turned black with age. Rapidly growing colonies (average, 40 mm/day) produced mycelia that disarticulated into 0- to 1-septate, cylindrical-truncate or rod-shaped, thick-walled, hyaline to brown arthroconidia occurring singly or in arthric chains and averaged 9.6 ± 3.7 × 5.5 ± 1.6 μm with a length/width ratio of 1.79. These characteristics were consistent with the description of the arthric synanamorph of Q:3 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 (one from under bark tissues; one from root tissues) were amplified with ITS6/ITS4 and NL1/NL4 primer pairs, respectively, and the amplicons sequenced. The ITS and LSU sequences deposited in GenBank (ITS, MK430050 and MK430051; LSU, MK430056 and MK430057) had 100% identity with many sequences of N. dimidiatum, including recently available ones (T¨urk¨olmez et al. 2019; ITS, MH114591; LSU, MH114592), in line with the morphological features. Pathogenicity tests were performed on 20 replicates of 1-year-old container-grown J. regia ‘Chandler’ seedlings with a N. dimidiatum isolate by removing a 5-mm-diameter bark plug from the stem with a cork borer and placing an equivalent-sized 7-day-old culture disc of N. dimidiatum onto the exposed vascular cambium. The wound was covered with wet, autoclaved cotton wool and sealed with Parafilm. Treated plants were maintained in a growth chamber with a 16/8-h light/dark photoperiod at 26°C and 55% RH and watered on demand. Within 3 weeks, average lesion lengths under the bark of all inoculated stems were >150 mm, and the lesions were covered with a black sooty layer. Leaf blight was also observed, similar to naturally affected trees. Control plants remained healthy. N. dimidiatum was reisolated only from lesions of inoculated stems, fulfilling Koch’s postulates. In Turkey, N. dimidiatum has been reported as a destructive new blight of tomato (T¨urk¨olmez et al. 2019) and canker, shoot blight, and root rot of pistachio (Dervis¸ et al. 2019). This is the first report of N. dimidiatum infection of walnuts in Turkey. By the end of 2018, all symptomatic trees had died. Recent occurrence of this severe disease suggests it could have a significant potential for limiting walnut production.