First Report of Leaf Spot on Basella rubra Caused by Fusarium proliferatum in Malaysia

Basella rubra (family Basellaceae), locally known as 'Remayong Merah', is the edible perennial vine served as leafy vegetable in Malaysia. In May 2021, B. rubra's leaves with circular to subcircular purple spots (ranging from 1-10 mm wide) were collected in Lido (5°56'44.6"N 116°04'46.5"E), Sabah province. The disease severity was about 60% with 20% disease incidence on fifty plants. As disease developed, the spots grew larger and necrosis were formed within the purple spots. Small pieces (5 x 5 mm) of five diseased spots were excised, and then surface sterilized based on Khoo et al. (2022b) before plating on water agar at 25°C. Once obtained the pure cultures from all diseased spots, they were incubated on potato dextrose agar at 25°C. After 7 days, white aerial mycelium with light violet pigmentation on lower side were observed on PDA. Then, the fungi were cultured on Carnation leaf agar (CLA) at 25°C and 12-h light/dark photoperiod for 10 days. Thin-walled slender and slightly curved macroconidia (n= 20) with 3 to 5 septa were ranged from 2.3 to 2.6 µm wide by 26.8 to 44.5 µm long in size. Oval microconidia (n= 20) with no septa were 2 to 2.2 µm wide by 9.5 to 15 µm long in size. Chlamydospores were absent. Monophialids with false head were observed. Isolate Lido and Lido02 were kept in the Laboratory of Genetics, Faculty of Science and Natural Resources, Universiti Malaysia Sabah for public request. Their genomic DNA were extracted from fresh mycelia of isolates based on Khoo et al. (2022a). EF1/EF2, RPB1-Fa/RPB1-G2R and RPB2-5f2/RPB2-7cr (Jiang et al. 2021) were used to amplify the translation elongation factor 1-α (TEF1) region, RNA polymerase largest subunit gene (RPB1) and RNA polymerase second largest subunit gene (RPB2) based on PCR condition in Khoo et al. (2022b). The isolate's sequences were deposited in GenBank as OM048109, OM634654 (TEF1), OM634655, OM634657 (RPB1) and OM634656, OM634658 (RPB2). They were 99 to 100% homology to TEF1 of isolate DPCT0102-2 (LC581453) (657/657 bp), RPB1 of strain ZJ05 (MT560605) (1558/1558 bp) and RPB2 of isolate GR_FP248 (MT305154) (1867/1869 bp) sequences. These sequences were polyphasic identified at the Fusarium MLST (https://fusarium.mycobank.org/), and were more than 99% similarity to Gibberella fujikuroi species complex (NRRL 25200). Gibberella fujikuroi and Fusarium fujikuroi are synonymous with Fusarium proliferatum (Leslie and Summerell 2006). The pathogen was identified as F. proliferatum based on morphological characterization, molecular data and phylogenetic analysis. Two non-wounded leaves of three one-month-old B. rubra seedlings were inoculated with mycelium plug (10 x 10 mm). Additional three B. rubra seedlings received sterile PDA agar plug (10 x 10 mm) to serve as controls. They were incubated in a glasshouse at room temperature 25°C with a relative humidity of 80 to 90%. After 8 days of inoculation, all inoculated leaves exhibited the symptoms as observed in the field, while the controls showed no symptoms, thus confirming the Koch's postulates. The experiment was repeated two more times. The reisolated pathogens were identified as F. proliferatum via PDA macroscopically, CLA microscopically and PCR amplification. F. proliferatum was reported previously causing leaf spot disease on Cymbidium orchids (Wang et al. 2018), tobacco (Li et al. 2017) and tomato (Gao et al. 2017). To our knowledge, this is the first report of F. proliferatum causing leaf spot on B. rubra in Malaysia. Infections of leaves reduce plant vigor and marketability. The identification of leaf spot caused by F. proliferatun will enable plant health authorities and farmers to identify practices to minimize disease on this important crop.