First report of Pestalotiopsis lushanensis causing leaf spot of Manglietia decidua (Magnoliaceae) in China.

Abstract

Manglietia decidua is an extremely endangered species, known for its limited population and a narrow distribution range restricted to China (Yu 1994). In October 2021, a leaf disease affecting the foliage of 3-year-old M. decidua was observed at the nursery garden of the Yichun Forestry Institute of Jiangxi Province (27°55'52.7"N, 114°17'36.4"E), with approximately 60% disease incidence. Initially, small reddish-brown specks with round or oval-shaped spots appeared. These spots gradually expanded, resulting in large irregular lesions with grayish-white centers. Furthermore, the affected areas exhibited desiccation and curling, with lesion diameters exceeding two cm. Fifteen diseased leaves were collected to isolate the pathogen using the method described by Zhang et al. (2021). Out of 40 fungal isolates obtained, 28 exhibited consistent culture characteristics and were identified as Pestalotiopsis sp.. We chose HML2-1 as the representative for morphological research. Colonies on Potato Dextrose Agar (PDA) reached a diameter of 80 mm after seven days at 25°C, displaying a pale honey-colored appearance with fluffy aerial mycelia arranged in a concentric-pattern, while the back of the colony appeared orange. Conidia were fusoid and ellipsoidappearing straight to slightly curved, composed of five cells, with four septa at which were slightly constricted, and measured 17.5-22.5 × 5-7.5 µm. The 3 median cells exhibited a brown color; the apical cells were conical and colorless, featuring 2-3 branches at the top as flagella; the basal cells were conical, transparent, and smooth. For further identification, three isolates were selected for the amplification and sequencing of three loci: the internal transcribed spacer (ITS), the partial translation elongation factor1-alpha (TEF1) and beta-tubulin (TUB2). The sequences were deposited in GenBank (ITS: OR415306-OR415308; TEF1: OR420799, PP278043, PP278044; TUB2: OR420798, PP278045, PP278046). All three DNA sequences of HML2-1 had a high identity to P. lushanensis (the ex-type isolate LC4344) with the accession numbers KX895005 (100%), KX895223 (100%) and KX895337 (99.34%), respectively. The multi-locus phylogenetic tree employing W-IQ-TREE (Trifinopoulos et al. 2016), revealed that the studied three isolates clustered in P. lushanensis clade with strong support. Based on morphological and molecular evidence, the isolate HML2-1 was identified as P. lushanensis, despite the fact that conidia sizes are smaller than those of type strain (Liu et al. 2017). To verify pathogenicity, a 20 µL conidial suspension (5×105 conidia/mL) of isolate HML2-1 was added to each leaf from an 8-year-old tree in the field, with 10 leaves punctured and 10 leaves left intact. Control including wounded or unwounded leaves were treated with sterile water in another tree. The treated leaves were wrapped in plastic bags to maintain moisture. After three to seven days, field-inoculated leaves exhibited typical reddish-brown lesions with grayish-white centers, primarily on wounded leaves, while the lesions on unwounded inoculated leaves were smaller. The control remained symptomless. The re-isolated HML2-1A was identified as P. lushanensis by ITS sequence. At present, the pathogens of M. decidua include Calonectria ilicicola (Yi et al. 2022), Sclerotium rolfsii (Yi et al. 2021) and Colletotrichum fructicola (Liu et al. 2023), while this study expands understanding of M. decidua's diseases.