First report of Epicoccum sorghinum causing leaf spot on Basella alba in Malaysia

Basella alba (family Basellaceae) is a perennial vine that serves as an edible leaf vegetable in Malaysia. In May 2021, red spots were observed on leaf samples of B. alba in Lido, Sabah Province (5°56'39.1"N, 116°04'47.6"E). The disease severity was about 20% with 10% incidence. The spots enlarged and coalesced into larger necrotic spots. Small pieces (5 x 5 mm) of infected leaves were excised from three plants, and then surface disinfected based on Khoo et al. (2022). One fungal isolate (Lido01) was isolated and cultured on potato dextrose agar (PDA) at 25°C. A single isolate with cottony aerial mycelia and pink concentric rings was observed on the upper surface of the culture. Unicellular and multicellular chlamydospores were observed, and measured 7.1 to 14.3. × 17.8 to 74.5 μm. Conidia were unicellular, hyaline, oval, and measured 3.8 to 5.2 x 1.7 to 2.7 μm (n= 20). Pycnidia were spheroid, and measured 66.2 to 114.3 x 44.1 to 86.1 μm (n= 20). Genomic DNA was extracted from fresh mycelia according to the extraction method of Khoo et al. (2022a and 2022b). ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R, and T10/Bt2b primers were used to amplify the internal transcribed spacer (ITS), large subunit (LSU), actin (ACT), and tubulin (TUB) genes, respectively (O'Donnell and Cigelnik, 1997; Chen et al. 2021). PCR products were Sanger sequenced by Apical Scientific Sdn. Bhd. (Serdang, Malaysia). Sequences of isolate Lido01 were deposited in GenBank as OM501130 (ITS), OM501128 (LSU), OM513916 (ACT) and OM513917 (TUB). Respective gene sequences of this isolate showed 100% homology to ITS sequence of isolate BPL01 (OM453926) (507/507 bp), LSU sequence of isolate BPL01 (OM453925) (1328/1328 bp), ACT sequence of isolate CZ01 (MN956831) (275/275 bp) and TUB sequence of isolate BJ-F1 (MF987525) (556/556 bp). The sequences of Lido01 established a supported clade (99% bootstrap value) to the related Epicoccum sorghinum type sequences, according to phylogenetic analysis using maximum likelihood based on the concatenated ITS, ACT, and TUB sequences. Morphological characters also matched the description of E. sorghinum (Li et al. 2020). Koch's postulates were tested as described by Chai et al. (2017) with modification by spray inoculation (106 spores/ml) on the leaves of three healthy one-month-old B. alba, while sterilized distilled water served as the control treatment. Monitoring and incubation were performed in a greenhouse based on Iftikhar et al. (2022). All inoculated leaves developed symptoms as described above by 8 days post-inoculation, whereas no symptoms occurred on controls, thus fulfilling Koch's postulates. The experiment was repeated twice. The reisolated pathogen was morphologically and genetically identical to E. sorghinum. E. sorghinum was reported causing leaf spot on Brassica parachinensis (Yu et al. 2019), Camellia sinensis (Bao et al. 2019), Myrica rubra (Li et al. 2020), Oryza sativa (Liu et al. 2020) and Zea mays (Chen et al. 2021). To our knowledge, this is the first report of E. sorghinum causing leaf spot on B. alba in Malaysia. Our findings have expanded the geographic range and host range of E. sorghinum in Malaysia, though the host range of this isolate is not known.