First Report of <i>Coleophoma parafusiformis</i> Causing Leaf Spot on Star Anise ( <i>Illicium verum</i> ) in China

Star anise (Illicium verum) is an important economic plant, and its fruits are widely used as a spice and material in traditional Chinese medicine. In April 2020, a severe leaf spot was widespread in a 1.5-ha I. verum forest in Panzhou county (104°41'23'' E, 25°33'42'' N), Guizhou province, China. The disease incidence varied from 76.5 to 83.5%. Initially, water-soaked spots emerged at the apex or edge of tender leaves and then quickly expanded into large, irregular yellow-brown patches that eventually covered the whole leaf. Ultimately, the leaves withered completely, with dense, black, granular acervuli forming on their surfaces. To identify the pathogen, symptomatic samples were collected from 12 plants. Six samples were cut into small pieces of 2×2 mm, and disinfected with 75% alcohol for 30 s and 0.5% NaClO for 1 min, after being washed five times with sterile water. Then, these pieces were placed onto PDA plates and incubated at 25°C in the dark for 5 days. Three fungal isolates (LB202004-2, LB202004-3 and LB202004-4) were obtained. Colonies were greyish sepia with moderate aerial mycelium, margins lobate to feathery, reaching 27.8 to 28.9 mm (n = 6) in diameter on PDA after 10 days. Paraphyses were intermingled among conidiophores, hyaline, subcylindrical, with 0 to 2 septa, measuring 18.4 to 121.3 × 3.2 to 7.5 μm (n = 30). Conidiophores were branched, smooth, hyaline, with 1 to 3 septa, measuring 12.5 to 46.4 × 3.2 to 5.1 μm. Conidiogenous cells were smooth, hyaline, measuring 10.8 to 24.5 × 2.3 to 6.4μm. Conidia were solitary, hyaline, smooth, cylindrical, curved or straight, measuring 13.1 to 21.4 × 4.2 to 7.7 (average 16.3 × 5.6 μm; n = 30). These isolates were tentatively identified as Coleophoma parafusiformis based on morphological characteristics (Crous et al. 2016). Primer pairs ITS4/ITS5 (White et al. 1990), LSU1Fd/LR5 (Vilgalys & Hester 1990) and Bt-2a/Bt-2b (Glass & Donaldson 1995) were used to amplify internal transcribed spacers of rDNA (ITS), the large subunit of nrDNA (LSU) and beta-tubulin 2 (TUB2), respectively. The obtained sequences were submitted to GenBank (ITS, PP527089 - PP527091; LSU, PP527093 - PP527095; TUB2, PP548173 - PP548175). A phylogenetic analysis based on the combined ITS, LSU, and TUB2 sequences from 18 Coleophoma strains revealed that the three isolates clustered with C. parafusiformis with 99% bootstrap support (ITS with KU728493, 584/584 bp; LSU with KU728533, 848/849 bp; TUB2 with KU728611, 432/434 bp). Pathogenicity tests were performed to evaluate the pathogenicity of the three C. parafusiformis isolates. For each isolate, nine healthy leaves (three per seedling) from three 2-year-old potted star anise seedlings were wounded with sterile needles and inoculated with 2-mm-diameter mycelial disks. Control leaves were treated with sterile PDA disks. Seedlings were kept in a greenhouse at 25℃ and 90% RH under natural light conditions. After 7 days, symptoms developed on all inoculated leaves, whereas no symptoms were observed on controls. The pathogenic isolates were reisolated and identified based on morphological characteristics and sequencing data of ITS, LSU, and TUB2 genes. Previous studies have shown that C. parafusiformis can cause leaf spot on Rhododendron sp. (Crous et al. 2016). As far as we know, this is the first report of I. verum leaf spot caused by C. parafusiformis. The identification of the pathogen is crucial for developing effective strategies to prevent leaf spot outbreaks on star anise in the field.