First report of leaf spot on Basella alba caused by Dichotomophthora basellae in Taiwan.

Abstract

Malabar spinach (Basella alba) is a widely cultivated leafy vegetable in Asia. In July 2023, a severe leaf spot outbreak with 100% incidence occurred in multiple net houses on an organic farm in Miaoli county, Taiwan. Initial symptoms were small pale brown necrotic lesions, often with a reddish halo. As the disease progressed, the lesions enlarged, forming brown spots with concentric rings and abundant sporulation. The pathogen was isolated using two methods: tissue isolation from small non-sporulating lesions after surface-sterilization, and single conidium isolation from sporulating lesions. Four isolates, BaDpO-1, BaDpO-2, BaDpY-1, and BaDpY-2, exhibiting similar morphology were obtained from both isolation techniques. Conidiophores of these isolates were macronematous, unbranched or irregularly branched, with a swollen head at the apex. Heads were repeatedly dichotomously or trichotomously lobed, pale brown to brown, and measured 17-58.1 μm wide (n=40). Conidiogenous cells were terminally lobed and polytretic. Conidia were solitary, ellipsoidal to cylindrical rounded at ends, subhyaline to yellow brown, 2-4-distoseptate, and measured 35.8-66.2 × 11.9-18 μm (n=50). Sclerotia were ovoid to subglobose, dark brown to black, and measured 86-461 × 85-387 μm (n=50). Colonies on PDA displayed a black center with concentric sporulation and diffused yellow pigment. Genomic DNA from the four isolates was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), and RNA polymerase II second largest subunit gene (RPB2) with the primers V9G/ITS4, gpd1/gpd2, and RPB2-5F2/fRPB2-7cR, respectively (Marín-Felix et al., 2019). The sequences were deposited in GenBank (ITS: PV336058 to PV336061; GAPDH: PV345458 to PV345461; RPB2: PV345454 to PV345457) and showed 100% identity with the ex-type strain CPC 33016 of Dichotomophthora basellae Hern.-Restr., Cheew. & Crous. Phylogenetic analysis inferred from concatenated sequences of the three genes revealed that all four isolates clustered with D. basellae CPC 33016, supported by a 100% bootstrap value. Pathogenicity tests were conducted by point-inoculating conidium suspension (10⁵ conidia/mL) of each isolate to four leaves of 1-month-old B. alba plants without wounding at 25±3°C. Control leaves were treated with sterile water. The four isolates caused expanded necrotic lesions at one day post-inoculation (dpi). Later, abundant conidiophores and conidia produced on lesions were morphologically matched the original isolates. Additionally, a conidium suspension (10⁴ conidia/mL) of isolate BaDpY-2 was sprayed onto three 2-month-old plants, while three control plants received sterile water. Numerous necrotic lesions appeared on leaves without sporulation at one dpi. The pathogens were successfully re-isolated from symptomatic tissues and matched the original isolates, fulfilling Koch's postulates. Based on molecular, morphological, and pathogenicity analyses, D. basellae was confirmed as the causal agent of B. alba leaf spot, marking its first report in Taiwan. This disease was initially recorded in Thailand (Crous et al., 2019; Marín-Felix et al., 2019), with subsequent reports in Bangladesh (Kader et al., 2024). It has caused significant damage to Malabar spinach wherever it occurs, underscoring the importance of early identification and effective management strategies.