Adenia globosa, as an excellent indoor ornamental plant, is planted in Tropical Botanical Museum, Nanjing Zhongshan Botanical Garden, Jiangsu Province, China. In September 2022, a new stem basal rot disease was observed on A. globosa seedlings, being planted here. Stem basal rot were observed on approximately 80% of A. globosa seedlings. The basal stem of cutting seedlings appeared decayed, and stem tip eventually turned dry due to water loss (Figure S1A). To isolate the pathogen, three diseased stems were collected from three cuttings planted in different pots of the Tropical Botanical Museum. The stem sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s and 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in the dark. Pure cultures were obtained by monosporic isolation. Eight isolates were obtained, and all identified as Lasiodiplodia sp.. The colonies morphology of cultures, growing on PDA were cotton-like, the primary mycelia were black gray after 7 days, and the reverse sides of PDA plates were similar to front sides in color (Figure S1B). A representative isolate, QXM1-2 was selected for the further study. Conidia of QXM1-2 were oval or elliptic, with a mean size of 11.6 µm×6.6 µm (n=35). The conidia are colorless and transparent in the early stage, and become dark brown with one-septum in the later stage (Figure S1C). The conidiophores produced conidia after nearly four weeks of cultivation on PDA plate (Figure S1D). The conidiophore was a transparent cylindrical structure, with a size of (6.4-18.2) µm × (2.3-4.5) µm ( n = 35). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The internal transcribed spacer regions (ITS), translation elongation factor 1-alpha (TEF1α) and β-tubulin (TUB) genes (GenBank Accession No.OP905639, No.OP921005, and No.OP921006, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. They had 99.8-100% homology to the ITS (504/505 bp) of Lasiodiplodia theobromae strain NH-1 (MK696029), TEF1α (316/316 bp) of strain PaP-3 (MN840491), and TUB (459/459 bp) of isolate J4-1 (MN172230). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate QXM1-2 clustered in the L. theobromae clade with 100% bootstrap support (Figure S2). To test pathogenicity, three A. globosa cutting seedlings that previously had been wounded with a sterile needle were inoculated with 20 μL conidia suspension (1×106 conidia/mL) on the stem base. The seedlings inoculated with 20 µL sterile water was used as the control. All plants were covered with clear polyethylene bags to keep moisture in a greenhouse (25℃, 80% relative humidity). The experiment was repeated three times. After 7 days post-inoculation, typical stem rot were found on the treated cutting seedlings and the control seedlings did not have any symptoms (Figure S1E-F). The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased tissues of the inoculated stems to complete Koch's postulates. This pathogen has been reported infecting the branch of castor bean (Tang et al. 2021) and root of Citrus (Al-Sadi et al. 2014). For our knowledge, this is the first report of L. theobromae infecting A. globosa in China. This study provides an important reference for the biology, epidemiology of L. theobromae.
Keywords: Adenia globosa; Lasiodiplodia theobromae; molecular identification; morphological characteristics.