First Report of Lasiodiplodia pseudotheobromae Causing Postharvest Fruit Rot on Indian Jujube in China.

Abstract

Indian jujube (Ziziphus mauritiana Lamarck), is one of the most popular fruit crops in South China. In March 2023, a fruit rot of indian jujube with about 5% disease incidence was observed in two supermarkets of Nanchang City, Jiangxi Province, China. Initially, the symptoms appeared as slightly brown spots on the fruit surface, with disease progression, the lesions gradually expanded and covered with a layer of hyphae. Small pieces (3 to 4 mm2) from the periphery of 15 diseased fruit were surface disinfected using 1% sodium hypochlorite for 30 s, rinsed three times in sterilized distilled water, air dried, and then aseptically placed onto potato dextrose agar (PDA) media and incubated at 25°C for three days. A total of ten single spore isolates with similar morphology were obtained. Colonies of these consisted of initially white, gradually turning gray and eventually becoming black, and aerial hyphae were dense and fluffy. Conidiogenous cells were smooth, hyaline, cylinder-shaped, and holoblastic. Conidia were ellipsoidal, top and base-rounded, and thick-walled, immature conidia were colorless, hyaline, and aseptate, compared with dark brown color of the mature conidia, which were one-septate with longitudinal striations, ranging in size from 22.8 to 31.8 (mean 27.6) µm in length and 12.2 to 20 (mean 14.6) µm in width. The morphological characteristics were consistent with the characteristics of the Lasiodiplodia species (Phillips et al. 2013). To accurately identify the strain, three representative isolates, namely JFRL 03-1147, JFRL 03-1148, and JFRL 03-1149, were selected for further identification. The internal transcribed spacers (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB2) genes/regions were amplified and sequenced using primers ITS1/ITS4, EF1-688F/EF1-1251R, and Bt2a/Bt2b, respectively (Chen et al. 2021). These nucleotide sequences were deposited in GenBank with accession numbers OQ804425-OQ804427 (ITS), OQ818097-OQ818099 (TEF1-α), and OQ818100-OQ818102 (TUB2). A BLASTn homology search for these nucleotides showed 99-100% identity to ITS (EF622077, 487 nt/487 nt), TEF1-α (EF622057, 306 nt/307 nt) and TUB2 (EU673111, 434 nt/434 nt) sequences of Lasiodiplodia pseudotheobromae CBS 116459 (ex-type). The maximum likelihood analyses were performed for the combined ITS, TEF1-α and TUB2 data set using IQtree web server (Trifinopoulos et al. 2016). The phylogenetic tree showed that the three isolates clustered with Lasiodiplodia pseudotheobromae in a clade with 99% bootstrap support. Therefore, the fungus was identified as L. pseudotheobromae based on morphological and molecular characteristics. To evaluate pathogenicity, 4 healthy fruits of indian jujube were surface sterilized with 75% ethanol and wounded by sterile needle, and a 5-mm-diameter agar with 5-days-old mycelium of the isolate JFRL 03-1148 cultured on PDA at 25°C was put on the wound. Another set of 4 fruits was inoculated with sterile agar plugs as controls. The fruits were cultured at 25℃ and 85% relative humidity, and the test was repeated twice. These fruits inoculated with L. pseudotheobromae showed similar rot symptoms after 3 days, while the control group remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as L. pseudotheobromae by morphological and molecular analysis. L. pseudotheobromae has previously been reported causing fruit rot on citrus, mango and papaya (Alam et al. 2021; Chen et al. 2021; Netto et al. 2014). But to our knowledge, this is the first report of L. pseudotheobromae caused postharvest fruit rot on indian jujube in China. Therefore, managers should pay more attention to postharvest fruit rot disease caused by L. pseudotheobromae, and formulate appropriate disease control measures to reduce its losses.