First report of leaf spot disease caused by Neopestalotiopsis chiangmaiensis on Phoebe zhennan in China

Abstract

Phoebe zhennan is one of the main cultivated tree species on Longquan Mountain. P. zhennan is a precious timber species endemic to China with significant economic importance. Its fine-grained, durable, and fragrant wood is highly prized for high-end furniture, traditional architecture, and handicrafts, commanding premium market prices. The species also supports rural economic development through timber production, seedling cultivation, and understory forestry industries. Additionally, it has ornamental value for landscape gardening. May 8, 2025, leaf lesions were found on 20% of P. zhennan trees within the approximately 45.5-ha survey area (Location: 30°35′50.25″N, 104°22′58.76″E, altitude 680 m). Black fruiting bodies, semi-immersed, solitary, and scattered, were produced on the diseased leaf parts. A total of 80 diseased leaves were collected from 20 P. zhennan plants showing typical symptoms. Five fungal isolates were obtained by single-conidium isolation and cultured on PDA. After 7 days of growth on potato dextrose agar (PDA) at 25 °C, the colony diameter reached 50 mm. After 30 days, fruiting bodies were produced, which were black, solitary or clustered, spherical, and released black conidial masses. Conidiophores were inconspicuous and reduced to conidiogenous cells. Conidiogenous cells were cylindrical, hyaline to pale olive. Conidia were fusiform to elliptical, mostly straight, occasionally slightly curved, with 4 septa, measuring 19–27 × 6–8 μm (x̄ = 23 ± 2.1 × 7 ± 0.4 μm, n = 50); the basal cell was conical to obconical, hyaline, 3–5 μm long (x̄ = 4 ± 0.7 μm, n = 50); the three middle cells were polygonal to cylindrical, 13–19 μm long (x̄ = 15 ± 1.5 μm, n = 50), slightly constricted at the septa, pale olive to dark brown, with the central cell darker and the flanking cells slightly lighter, thick-walled, wrinkled, and dark brown to black; the apical cell was hyaline, conical to trapezoidal, thin-walled, and smooth; it possessed 2–4 tubular apical appendages, unbranched, filamentous, hyaline, 18–29 μm long (x̄ = 23 ± 2.9 μm, n = 50); the basal appendage was solitary, tubular, straight, occasionally slightly curved, hyaline, 2–4 μm long (x̄ = 3 ± 0.5 μm, n = 50). The detailed symptoms observed after inoculation on detached healthy P. zhennan leaves with mycelial plugs of N. chiangmaiensis were recorded. The morphological characteristics were similar to those of species in the genus Neopestalotiopsis (Maharachchikumbura et al. 2014). Two representative isolates (TXC202505-1 and TXC202505-2) were selected for DNA extraction, and the ITS, tef1-α, and tub2 gene regions were amplified and sequenced (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999; Maharachchikumbura et al. 2014). All sequences have been deposited in GenBank (ITS: PX930896–PX930897; tub2: PX938982–PX938983; tef1-α: PX938978–PX938979). BLAST analysis showed that these sequences shared 93.55%–99.32% similarity with the species N. chiangmaiensis (type strain MFLUCC 18-0113): ITS: 174/186 bp (93.55% and 93.55%); tef1-α: 925/927 bp (99.78% and 99.78%); tub2: 438/441 bp (99.32% and 99.32%). Based on multi-gene phylogenetic analysis, the isolates clustered together with N. chiangmaiensis with a support rate of 72%. Morphologically, the isolated strains were consistent with N. chiangmaiensis, and their nucleotide sequences exhibited high similarity. Integrating morphological and molecular phylogenetic evidence, the isolates were identified as N. chiangmaiensis. To confirm pathogenicity, three healthy two-year-old seedlings were inoculated with 500 μl of a spore suspension (1 × 10⁵ conidia/ml) on five leaves each. Three control seedlings were inoculated with sterile distilled water. All treated leaves were bagged and maintained in a greenhouse at 25°C and 70% relative humidity. The test was repeated three times. The detailed symptoms observed after inoculation are as follows: Initial symptoms appeared as small, water-soaked, light brown spots at the inoculation sites 3–5 days after inoculation. The spots gradually expanded into circular or irregular necrotic lesions with dark brown margins and grayish-white centers. Under high humidity conditions, black acervuli (asexual fruiting bodies) developed on the lesion surfaces. In severe cases, the lesions coalesced, leading to large areas of leaf necrosis and blight. Control leaves inoculated with sterile water remained asymptomatic throughout the experiment. After ten days, symptoms similar to those observed in the field developed on the inoculated leaves, while no symptoms were observed on the control plants. N. chiangmaiensis was reisolated from symptomatic leaves (100% frequency) and identified based on morphological and molecular characters, fulfilling Koch’s postulates. N. chiangmaiensis has been previously reported on plants such as orchids, roses, and nuts (Guterres et al. 2023; Sun et al. 2023). N. chiangmaiensis was originally described from Thailand and has also been reported in southern China as a saprobe on Pandanaceae plants (Tibpromma et al. 2018). However, this is the first report of N. chiangmaiensis causing leaf spot disease on P. zhennan, representing a new host record in China. This is the first report of this fungus causing leaf blight on P. zhennan in Longquan Mountain, Sichuan Province.