First Report of <i>Lasiodiplodia pseudotheobromae</i> Causing Leaf Spot on <i>Millettia speciosa</i> in China

Millettia speciosa Champ (M. speciosa), a member of the Leguminosae family, is extensively cultivated in Guangxi, China, with planting area exceeding 8,000 hectares. The edible tubers of this plant are valued for their antibacterial and antioxidant properties (Luo et al. 2023; Shu et al. 2020). In July 2024, leaf spot symptoms were observed in a plantation in Guilin, Guangxi, China (25°93'N; 111°09'E), affecting over 40% of plants across a 78,230 m2 area. Symptoms began as small, circular, pale-yellow spots that enlarged into irregular, light brown lesions, eventually causing leaf wilt and defoliation. A total of six symptomatic leaves were collected, surface-disinfected with 75% ethanol for 30 s and 1% NaClO for 2 min, rinsed with sterile distilled water (SDW), and cultured on potato dextrose agar (PDA) at 28°C in darkness for 7 days. Seven isolates were obtained through single-spore isolation. Colonies were initially white, becoming greyish-white with dense, fluffy aerial hyphae. Immature conidia were hyaline, ellipsoid and aseptate, while mature conidia were dark brown, one-septate, and had longitudinal striations, measuring 22.8-29.5 µm × 11.2-15.6 µm (n = 50). The morphological features were consistent with Lasiodiplodia sp. (Zhang et al. 2024). To confirm the pathogen, three representative isolates (including N2-1-1, which yielded identical sequences to the other two for the targeted loci) were sequenced for the internal transcribed spacer (ITS), β-tubulin (BT), and translation elongation factor 1-alpha (EF1-α) regions (Wang et al. 2024). The ITS, BT and EF1-α sequences (Genebank accessions nos. PQ637400, PQ654012 and PQ654013) showed high identity to the ex-type strain of Lasiodiplodia pseudotheobromae (CBS 116459), with identities of [ITS: 528/528 bp (100%)] (cf. EF622077), [BT: 452/460 bp (99%)] (cf. EU673111), and [EF1-α: 299/300 bp (99%)] (cf. EF622057), respectively). Based on morphological and molecular data, the pathogen was identified as L. pseudotheobromae. Pathogenicity was tested on one-year-old M. speciosa seedlings by inoculating leaves with 5-mm mycelial plugs, while control leaves received sterile PDA plugs. The experiment was performed with three biological replicates. Plants were maintained in a greenhouse (12/12h light/dark cycle, 25 ± 2°C, 90% humidity). After 7 d, inoculated leaves developed symptoms identical to those observed in the field, while controls remained symptom-free. The fungus was consistently reisolated, fulfilling Koch's postulates. L. pseudotheobromae has previously been reported to cause diseases in strawberry in Florida, plum and Peanut in China (Zhang et al. 2024; Wang et al. 2024, Zhang et al. 2022), but this is the first report of L. pseudotheobromae causing leaf spot on Millettia speciossa in China. This finding provides a basis for further studies on disease management and biology.