| Abstract |
Manglietia decidua is one of the most endangered Magnoliaceae species in China, whose natural distribution was limited to two isolated localities, Mingyueshan Scenic and Historic Area in Jiangxi and Xiaoxi National Reserve in Hunan (Yi et al. 2022). In late September 2021, a severe leaf spot disease was observed on 3-year-old M. decidua in the nursery in Yichun Research Institute of Forestry in Jiangxi province (28.06°N, 115.24°E), with an estimated incidence of ∼50% of the seedlings. Symptoms usually appeared on the tip or edge of the leaf as a dark brown spot with a thin yellow halo. Twenty symptomatic leaves were randomly sampled, cut into small pieces (5 mm²), dipped in 3% NaClO for 5 min, rinsed three times with sterilized distilled water, placed on potato dextrose agar medium (PDA), and incubated in a biochemical incubator (SPX-160, Ningbo Jiangnan Instrument Factory, China) at 25°C for 7 days. Forty fungal isolates were obtained and 27 isolates were identified as Colletotrichum sp. by morphological characteristics (Weir et al. 2012). Colonies on PDA were light gray to dark gray, reverse grayish cream with a gray-green center. The growth rate was 14 mm day⁻¹ (n = 3). The conidia were hyaline and aseptic with a smooth-walled shape, measuring 12.50 to 17.50 × 3.29 to 5.75 μm (av. 14.70 × 5.32, n = 30). Asci were unitunicate, thin-walled, 62.5 to 68.75 × 10.00 to 12.50 μm (av. 66.75 × 10.52 μm, n = 30). The sausage-like ascospores were hyaline, one-celled, 16.50 to 23.25 × 3.25 to 4.55 μm (av. 20.69 × 3.81 μm, n = 30). For molecular identification, seven genes, ITS, ACT, CHS-1, GAPDH, TUB, CAL, and Apn2/MAT (Liu et al. 2022), were amplified and sequenced. The sequences of isolate HML2-8 were deposited in GenBank (ITS: ON142502; ACT: ON156525; CHS-1: ON156526; GAPDH: ON188684; TUB: ON188683; CAL: ON156527; Apn2/MAT: OP709957), which showed 99.86 to 100% nucleotide identity with reference sequences of several Colletotrichum fructicola. The concatenated multilocus analyses, performed with ITS, ACT, CHS-1, GAPDH TUB, and Apn2/MAT datasets contained 25 taxa and 2, 995 characters composing the matrix. The isolates, including HML2-3, HML2-5, HML2-8, and C. fructicola CPC 28644 clustered in the same clade with a high bootstrap value (96%). Based on morphological-molecular characteristics, the fungus was identified as C. fructicola. To test Koch’s postulates, 10 detached leaves were disinfected with 75% ethanol for 1 min and rinsed three times with distilled sterilized water. For inoculation, a mycelial plug 6 mm in diameter was taken from the margin of an actively growing culture of isolate HML2-8 and placed on the disinfested leaf surface of five plants, each tree with 10 replicates. PDA plugs without mycelium were used as controls. The inoculated leaves displayed necrotic lesions 7 days after inoculation, while the control leaves did not display symptoms. The same results appeared in the field experiment. Twenty strains were reisolated and identified by morphological characters. Moreover, the reisolated HML2-8-A was randomly selected, sequenced, and inferred the phylogenetic relationship. C. fructicola has been reported to cause leaf spots on different plants, such as Annona spp. (Costa et al. 2017), Areca catechu (Cao et al. 2020), and Pyrus spp. (Fu et al. 2019). A leaf spot caused by C. siamense has been reported on M. decidua in China (Wang et al. 2022). To our knowledge, this is the first report of C. fructicola infecting M. decidua in nurseries in China. These results provide a basis for studies on the epidemiology and management of M. decidua, contributing to the knowledge of the host range of C. fructicola.
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