First report of Needle Cast of Cathaya argyrophylla caused by Neofusicoccum parvum in China.

Abstract

Cathaya argyrophylla Chun et Kuang is a first-grade protected tree in China with significant conservation value. In June 2022, needle cast was observed in approximately 27 to 34% of C. argyrophylla (n=200, covering about 1 ha) at Dashahe National Nature Reserve (28.89° N, 107.6° E) in Daozhen County, Guizhou, China. Initial symptoms were small red lesions (3 to 5 mm in diameter) on needles. Over time, these lesions expanded, turned necrotic, and led to needle cast. In addition, the diseased needles exhibit brown spots surrounded by a yellowish discoloured area measuring 0.5 mm2. The disease is most severe in young trees, with over 60% of the needles displaying discoloration in 15% of young trees (n=100). Forty infected conifer tissues were randomly selected, surface sterilized, and incubated on PDA in the dark at 26°C for 5d. Among the 40 samples, 39 exhibited fungi with similar morphology. Single-spore isolation method was used to obtain pure cultures for 9 isolates. The isolates were morphologically identical. On PDA medium, colonies were white with abundant aerial mycelium. After 14 days of growth under light, the colonies became greyish black with septate mycelium. Conidia were hyaline, thin-walled, smooth-surfaced, and ranged from ellipsoid to ovoid in shape, measuring 6.5 to 15.2 ×2.0 to 3.1 μm (n=50). Based on these morphological characteristics, the isolates were identified as belonging to Neofusicoccum spp. (Pavlic et al., 2009). For molecular identification, genomic DNA was extracted from 3 selected isolates. The internal transcribed spacer (ITS) region was amplified using primers ITS1/ITS4 (White et al., 1990). In addition, chitin synthase 1 (CS1) was amplified using primers CT-WK3-S/CT-WK3-A (Zimoch et al., 2003). The ITS (OR710949) and CS1 (OR714767) sequences of isolate JY1-1 were deposited in GenBank, exhibiting a homology of 99% to 100% (537/538, 191/192) with N. parvum MUCC211 (accession numbers EU301017 and EU339495). The reconstructed phylogenetic tree further substantiated the genetic relationship between isolate JY1-1 and N. parvum. Consequently, the isolate associated with needle cast on C. argyrophylla was identified as N. parvum. Isolates JY1-1, JY1-2, and JY1-3 were used to confirm Koch's postulates. The needles of 15 healthy 2-year-old C. argyrophylla saplings were inoculated with a conidial suspension of N. parvum (2.0 × 105 conidia/mL). As a control, the needles of C. argyrophylla saplings were treated with sterile water (n=10). The needles were covered with Ziplock bags, and maintained at humidity levels exceeding 90%. All treatments were placed in a greenhouse at 26℃. After 7 days of inoculation, the needles exhibited a change in color, with reddish-brown symptoms observed on day 30 (disease rate=100%). Furthermore, the lesion expanded, and needles were shed after 4 months. No symptoms were observed in the control group. Pathogens reisolated from all diseased plants exhibited morphological and ITS sequence identity to N. parvum (separation rate=100%), and no pathogens were isolated from control plants. Consistent results were obtained through two repeated experiments. While this pathogen is known to induce foliar disease in various plants (Sun et al., 2020; Shi et al., 2019), our study represents the first report Needle Cast of C. argyrophylla caused by N. parvum in China. These findings provide a foundation for developing effective strategies to monitor and manage C. argyrophylla.