First Report of <i>Lasiodiplodia theobromae</i> Causing Postharvest Fruit Crown Rot on <i>Musa acuminata</i> in Malaysia

Lasiodiplodia theobromae is a destructive pathogen causing postharvest crown rot in many fruits. In September 2024, crown rot symptoms (crown blackening, tissue softening and premature fruit detachment) were observed on Musa acuminata. Ten banana hands were randomly collected from a smallholder plantation in Bangi, Selangor, Malaysia (2°53'19.8"N 101°48'25.2"E). Banana samples were collected and the progression of the disease was observed and became more severe from 3 to 15 days. Three of the crown fruit fragments (∼0.5 cm) from the edge of healthy and diseased tissues were surface disinfected in 70% ethanol for 1 min, followed by 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water and dried on sterile filter paper for 10 min. The fragments were then placed onto potato dextrose agar (PDA) + chloramphenicol (100 mg/liter) and incubated at 28°C in the dark for 5 days to obtain pure culture. Then, monosporic cultures were obtained (isolates UKM1, UKM2 and UKM3). All three isolates showed grayish-white aerial mycelium in culture and, after 10 days, the mycelium turned black. The morphological characteristics were similar to those of Lasiodiplodia theobromae (Alves et al. 2008). For molecular identification, genomic DNA of the isolate UKM1 and UKM2 were extracted using the cetyltrimethylammonium bromide (CTAB) extraction buffer (Madihah et al. 2018). The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1 (tef1) and beta tubulin genes were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), EF1–1018F/EF1–1620R (Stielow et al. 2015) and Bt2a-F/Bt2b-R (Glass & Donaldson 1995) respectively, and sequenced. The sequences from both isolates were deposited in GenBank (ITS: PV833697, PX249735; tef1: PX219869, PX289564; β-tubulin: PX219870, PX289563). Similarity searches showed that isolate UKM1 shared 99.80% (ITS; OR936549), 100% (tef1; OR957987), and 98.52% (β-tubulin; OR957988) sequence identity with Lasiodiplodia theobromae strain SSLTCP1 (Prathibha et al. 2025). Isolate UKM2 showed 98.99% (ITS), 100% (tef1), and 100% (β-tubulin) identity with strains CBS 164.96 (GenBank accession no. NR_111174), SSLTCP1 (GenBank accession no. OR957987) and CBS 164.96 (GenBank accession no. EU673110), respectively (Schoch et al. 2014; Prathibha et al. 2025). Sequence alignment was performed using ClustalW, and phylogenetic analysis in MEGA 12.0 (Kumar et al. 2024) confirmed that the isolates clustered with Lasiodiplodia theobromae. Pathogenicity of isolate UKM1 was tested twice on 10 banana fruits (cv. Williams Cavendish). A 5-mm mycelial disc was placed on the fruit crown without wounding, and fruits were incubated at 28 °C in a humid chamber for 10 days, with uninoculated fruits as controls. UKM1 caused 100% crown and fruit rot, producing symptoms similar to those observed in naturally infected postharvest fruits. The fungus was reisolated from symptomatic tissue and identified as Lasiodiplodia theobromae based on morphology and DNA sequencing (ITS and tef1). To the best of our knowledge, this is the first documented case of Lasiodiplodia theobromae causing crown rot on banana fruits in Malaysia. Effective management strategies covering cultivation, transport, ripening, and storage are needed, as uncontrolled disease could severely reduce banana yield and quality.