First Report of <i>Lasiodiplodia theobromae</i> Causing Trunk Gummosis of Mango ( <i>Mangifera indica</i> ) in South Punjab, Pakistan

The worldwide expansion of mango cultivation has contributed to the emergence of various diseases. Mango trunk gummosis is among the most significant fungal diseases, causing 50–70% losses in orchards (Li et al. 2013). In summer 2022–2023, cankers, necrosis, and gum exudate were observed on trunk bark of mango cultivars collected from 20 orchards in Multan (Multan Sadar, Shujabad, Jalalpur Pirwala) and Muzaffargarh Districts, covering ~100 ha. The most affected variety was Chaunsa, from which disease samples were obtained. Disease incidence was ~40% in surveyed fields. A total of 90 symptomatic cankered, necrotic, gum-exuding bark samples were cut 2–4 mm-wide at lesion margins, surface-disinfested with 70% ethanol for 1 min, rinsed thrice with sterile water, and dried on blotter paper. Samples were transferred to potato dextrose agar (PDA) plates and incubated at 25 ± 2°C for 3–5 days. Fungal cultures with fluffy gray to black mycelium and dense, straight aerial hyphae were consistently isolated. Based on colony morphology, isolates were tentatively placed in Botryosphaeriaceae. Black conidia (n = 50) were initially hyaline, unicellular, ellipsoidal, later developing thick walls, a central septum, and longitudinal striations. Mature conidia measured 24.4–30.5 × 10.7–13.7 µm, consistent with Lasiodiplodia (Timmer et al. 2000). DNA was extracted and ITS amplified with ITS1/ITS4 (White et al. 1990), while tef1-α was amplified with EF1-688F/EF1-1251R primers (Alves et al. 2008) from four isolates. BLASTn searches revealed 99% identity with L. theobromae sequences. Based on morphology and sequencing, gummosis isolates were identified as L. theobromae, and sequences were deposited in GenBank (ITS: ON614647.1, ON614651.1, ON614656.1, ON614659.1; tef1-α: ON624289.1, ON624293.1, ON624298.1, ON624301.1). To confirm pathogenicity, one-year-old Chaunsa mango plants, commonly used as rootstock in Punjab, were inoculated with 3–5-day-old mycelial plugs from all four isolates. Stems were wounded with a sterile scalpel, inoculated with plugs, and sealed with Parafilm. Fifteen plants were used; each isolate was inoculated in triplicate (n = 3), and controls (n = 3) received sterile agar plugs. Plants were incubated at 25 ± 2°C in a greenhouse; the experiment was repeated twice. After 4 weeks, orange-brown gummy exudation and necrotic cankers developed above and below inoculation sites, while controls remained symptomless. The pathogen was re-isolated and matched L. theobromae morphologically and molecularly, fulfilling Koch’s postulates. To our knowledge, this is the first report of L. theobromae causing trunk gummosis in Mangifera indica L. (mango) in South Punjab, Pakistan. This study provides the first evidence of its occurrence in Pakistan, laying the foundation for effective disease management strategies.