First Report of Branch Canker of Pomegranate Caused by Neoscytalidium dimidiatum in China

Abstract

Pomegranate (Punica granatum L.) is an important specialty fruit crop in southern Xinjiang, China. In July 2025, branch canker and dieback symptoms were observed in pomegranate orchards in Kashi Prefecture, Xinjiang Uygur Autonomous Region, China, with a representative sampling site located at 39°29′23.9″N, 76°06′56.0″E. A survey of 3,200 pomegranate trees in 25 orchards showed an average disease incidence of approximately 7.5%. Symptoms appeared as longitudinally expanding, brown to dark brown, slightly sunken necrotic lesions on branches, sometimes accompanied by localized bark cracking. After the outer bark was removed, the phloem and cambial tissues beneath the lesions were brown and necrotic. Severely affected branches became blighted, resulting in partial canopy dieback. To identify the causal agent, small pieces approximately 5 × 5 mm were excised from the margins between diseased and healthy tissues of symptomatic branches, surface-disinfested in 75% ethanol for 30 s and 1% sodium hypochlorite for 60 s, rinsed three times with sterile distilled water, blotted dry, and placed on potato dextrose agar (PDA). Plates were incubated at 25°C in the dark. Twenty-two fungal isolates with similar colony morphology were obtained. Three representative isolates, PGSL01, PGSL15, and PGSL19, were purified by hyphal-tip isolation and used for morphological characterization, multigene identification, and pathogenicity tests. Colonies of the representative isolates grew rapidly on PDA, were initially white to grayish white, and gradually became gray to dark gray. Arthroconidia were produced on PDA, occurring singly or in short chains. They were initially hyaline to pale brown and became brown to dark brown at maturity. Arthroconidia were short-cylindrical, ellipsoid to short-rod-shaped, or barrel-shaped, with rounded to nearly truncate ends; most were aseptate, and a few had one septum. They measured 3.2 to 8.6 × 1.8 to 3.8 μm, with a mean ± standard deviation of 5.0 ± 1.1 × 2.6 ± 0.4 μm (n = 50). The colony and arthroconidial characteristics were consistent with those reported for Neoscytalidium dimidiatum (Nouri et al. 2018). Genomic DNA was extracted from the three representative isolates, and the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (tef1-α), and β-tubulin gene (tub2) were amplified using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, and BT2a/BT2b, respectively (White et al. 1990; Carbone and Kohn 1999; Glass and Donaldson 1995). The resulting sequences were deposited in GenBank under accession numbers PZ628252, PZ628253, and PZ628254 for ITS; PZ687086, PZ687087 and PZ687088 for tef1-α; and PZ687092, PZ687093, and PZ687094 for tub2. A phylogenetic tree inferred from the concatenated ITS, tef1-α, and tub2 sequences placed PGSL01, PGSL15, and PGSL19 in the same clade as reference isolates of N. dimidiatum. Based on morphology and multigene phylogenetic analyses, the isolates were identified as N. dimidiatum (Crous et al. 2006). To confirm pathogenicity, 5-mm-diameter mycelial plugs from each representative isolate were inoculated onto wounded 1- to 2-year-old branches of healthy pomegranate trees under field conditions. Sterile PDA plugs of the same size were used as controls. For each isolate, three plants were inoculated, with one inoculation site per plant; three plants were used as controls. Inoculation sites were covered with moist sterile cotton and sealed with Parafilm to maintain humidity. Four days after inoculation, all three isolates produced brown to dark brown branch canker lesions similar to those observed in the field, whereas control branches remained asymptomatic. Fungi with morphology identical to the original isolates were consistently reisolated from the margins of inoculated lesions and confirmed as N. dimidiatum by ITS sequencing; the fungus was not isolated from control tissues. The pathogenicity test was conducted twice with consistent results, fulfilling Koch’s postulates. N. dimidiatum causes canker, dieback, shoot blight, and fruit rot on a wide range of woody hosts (Nouri et al. 2018), and it has recently been reported on Euphrates poplar in Xinjiang and on fig in China (Xie et al. 2024, 2025). Although N. dimidiatum was previously detected from necrotic wood tissues of pomegranate in Iran (Kadkhoda-Hematabadi et al. 2023), to our knowledge, this is the first report of N. dimidiatum causing branch canker of pomegranate in China.