First report of postharvest fruit rot disease of Litchi caused by in China.

Litchi (Litchi chinensis Sonn.), a native fruit of southern China valued for its flavor and nutrition, is widely cultivated in subtropical regions (Yao et al. 2021). In May 2024, a postharvest disease was observed on ripe litchi fruits (cv. Guiwei) in Nanning, Guangxi Province, with an incidence of approximately 50% across 200 samples in each of three inspected cartons. Brown circular lesions initially appeared on the exocarp, with corresponding lesions on the endocarp. Within two days, lesions spread across the entire pericarp, typically displaying sparse mycelium at lesion centers. In severe cases, the pericarp cracked and exuded liquid. To isolate the pathogen, tissue samples (5 × 5 mm) were excised from necrotic lesion margins on the pericarps of six symptomatic litchi fruits, surface-disinfected in 1% sodium hypochlorite for 2 minutes, rinsed three times in sterile water, and plated on potato dextrose agar (PDA), and incubated at 28°C for 3 days (12-hour photoperiod). Hyphal tips were transferred to fresh PDA for purification. Ten morphologically similar isolates were obtained, with an 83% isolation frequency. On PDA plates, fungal mycelia were initially grayish-white, turning gray to dark gray, with dense, fluffy aerial hyphae. Conidiogenous cells were smooth, hyaline, cylindrical, and holoblastic. Conidia were ellipsoidal with rounded ends and thick walls; immature conidia were colorless, hyaline, and aseptate, while mature conidia were dark brown, one-septate, measuring 24.2-34.6 × 12.1-17.1 μm (n = 29). The genomic DNA from two randomly selected isolates (LC141 and LC142) was extracted using the cetyltrimethylammonium bromide (CTAB) method (Guo et al. 2000). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), and partial translation elongation factor-1 alpha (TEF-1α), and β-tubulin (TUB) genes were amplified and sequenced using primer pairs ITS1/4 (White et al. 1990), TEF-Las-F/R (AGA CGA TCG AGA AAT TTG AGA AG/GCG AGG TAC CAG TGA TCA TGT TC), and TUB-Las-F/R (TGC CAA AAC ACA CCT GCT CCT GC/TGT AGT GAC CCT TGG CCC AGT TG), respectively. The resulting sequences (ITS: PQ835290-91; TEF-1α: PQ862852-53; TUB: PQ862854-55) showed 99-100% similarity (487/487, 512/516 bp for ITS; 293/300, 303/306 bp for TEF-1α; 416/421, 417/420 bp for TUB) to Lasiodiplodia pseudotheobromae ex-type CBS116459 sequences (EF622077, EF622057, EU673111) (Alves et al. 2008). Phylogenetic analysis of concatenated ITS, TEF-1α, and TUB sequences grouped LC141 and LC142 with L. pseudotheobromae. Based on morphological and molecular characteristics, the isolates were identified as L. pseudotheobromae (Alves et al. 2008).To verify pathogenicity, six healthy litchi fruits (cv. Guiwei) were stab-wounded and inoculated with mycelial fragments of L. pseudotheobromae isolates LC141 and LC142, with three fruits per isolate. Three control fruits were treated with sterile water. Fruits were incubated in transparent plastic boxes at 28°C. Three days post-inoculation, symptoms appeared on inoculated fruits, while controls remained asymptomatic. Experiments were repeated thrice with consistent results. To fulfill Koch's postulates, L. pseudotheobromae was re-isolated from symptomatic tissues and identified by morphology and sequencing; no fungi were isolated from controls. This is the first report of L. pseudotheobromae causing postharvest fruit rot in litchi in China, providing a foundation for developing targeted disease management strategies.