First Report of Neofusicoccum parvum Causing Dieback on Cinnamomum daphnoides in China

Cinnamomum daphnoides Sieb. et Zucc. (Lauraceae), a newly recorded species discovered in 2014 in Ningbo, Zhejiang, China (Zhang et al. 2014), was found to be affected by a destructive branch blight disease in June 2025. The disease primarily damages branches and leaves, causing chlorosis and yellowing of foliage, leading to partial dieback or even complete wilting of the plant. To identify the causal agent, longitudinal sections of infected branches reveal brown necrotic lesions, which were cut into pieces, surface sterilized with 1% NaClO for 1 min, followed by 75% ethyl alcohol for 2 min, and rinsed in sterile distilled water (SDW) three times (Zhou et al. 2019). The tissue fragments were then placed on PDA medium supplemented with streptomycin sulfate (100 μg/mL) and incubated at 25 °C for 4 days. Pure cultures were obtained by transferring hyphal tips onto new PDA plates. Three fungal isolates (Np-1~3) were obtained and formed colonies with smooth margins. Initially, the aerial mycelium appeared white, and the colonies exhibited relatively rapid growth. After 5 days, the mycelium turned gray-black to black, and no spore production was observed even after 30 days of colony growth on PDA, CzA, or SNA medium, even at the sites of inoculation of branches. To identify the isolates Np-1~3, the internal transcribed spacer (ITS), translational elongation factor 1-alpha (TEF1-α), and β-tubulin (TUB2) genes were amplified and cloned from the isolates, which were amplified using primers ITS1/ITS4, EF1/EF2 and BT-2a/2b (White et al. 1990; Carbone and Kohn 1999; Glass et al. 1995), respectively. The isolates Np-1~3 had 100% identical DNA sequences at each of the three loci; therefore, only the DNA sequences from strain Np-1 were deposited in GenBank. Sequence analysis showed that the ITS (579 bp, GenBank Acc No. PX210504), TEF1-α (291 bp, GenBank Acc No. PX584295) and TUB2 (459 bp, GenBank Acc No. PX584296) of isolate Np-1 were 100%, 100% and 100% identical to those of N. parvum strains CMW 24571 (ITS, KF766152), LSCKS8-4 (TEF1-α, ON773425) and AH-3-1-01s (TUB2, JX462257), respectively. A multilocus phylogenetic analysis was performed based on ITS-TEF1-α-TUB2 sequences of Np-1~3 and other strains. The result showed that Np-1~3 clustered together with N. parvum. Thus, both colony morphology and molecular characterizations supported the strains Np-1~3 as N. parvum. To confirm the pathogenicity, mycelium agar plugs (MAPs, 6 mm in diameter) removed from the colony margin of a 3-day-old culture of strain Np-1 were used to inoculate the detached branches of C. daphnoides. Before inoculation, healthy detached branches were selected, soaked in 2% NaClO solution for 2 min, and washed in SDW. The branches were wounded with a sterile needle, and inoculated by placing MAPs on them, and mock inoculation with mycelium-free PDA plugs was used as a control. Four branches were used in each treatment. The inoculated and mock-inoculated branches were incubated at 25°C with high relative humidity. Black spots could be observed on the inoculated branches 6 dpi, whereas the mock-inoculated branches remained symptomless. The fungus reisolated from the diseased branches resembled the colony morphology of the original isolate. The experiment was conducted three times and produced the same results. To our knowledge, this is the first report of N. parvum causing dieback on C. daphnoides in China. This report will provide a reference for the prevention and control of this disease of C. daphnoides.