<i>Globisporangium</i> ( <i>Pythium</i> ) <i>spinosum</i> Causing Damping-Off and Rhizome Rot on <i>Zingiber officinale</i> in China

Ginger (Zingiber officinale Roscoe) is cultivated worldwide primarily for its physiological benefits (Dalsasso et al. 2022). In China, the cultivation area of ginger has reached 63,806 hectares, with a production of 672,914.37 tons (FAOSTAT 2023). In August 2023, ginger plants displaying yellowing and damping-off were sampled from four commercial fields of approximately 5,800 m2 in Anqiu (around 36.5°N, 119.2°E) and Yishui (around 35.8°N, 118.6°E), Shandong Province, with disease incidence ranging from 20 to 40%. Stems exhibited symptoms of water-soaked constriction at the base. White aerial mycelia were visible on the surface of the browning rhizomes. Typical symptomatic tissues (about 0.5 cm2) of the infected rhizomes were surface disinfected with 75% ethanol for 1 min, subsequently rinsed three times with sterilized distilled water, then placed on oatmeal agar (OA) medium, and incubated at 25℃ in the dark for 5 days. Sixteen morphologically similar oomycete-like colonies were obtained and produced white, cottony and dense aerial mycelium. Mycelium was aseptate and hyphal swellings were globose, thin walled, and 13.0 to 19.7 µm (average 15.3 ± 1.6 µm, n = 26) in diameter. Oogonia were globose, thick walled, with a varying number of blunt, digitate projections, and 23.0 to 30.8 µm (average 26.7 ± 2.3 µm, n = 26) in diameter. Oospores were plerotic, rarely aplerotic, spherical, and 16.0 to 21.3 µm (average 17.9 ± 1.8 µm, n = 34) in diameter. Antheridia were clavate or curved, and making apical contact with the oogonia. The morphological features were very similar to those of Pythium spp. (Uzuhashi et al. 2010). The internal transcribed spacer (ITS) (White et al. 1990) and cytochrome c oxidase subunit I (COI) (Robideau et al. 2011) of the isolates SAAS-ZO-P7, SAAS-ZO-P10 and SAAS-ZO-P14 were amplified. The resulting sequences were deposited in GenBank (accession nos. PV390628, PV390629, and PV390630 for ITS and PV391154, PV391155, and PV391156 for COI). BLASTn analysis of the ITS sequence showed 99.89% identity (919/928 bp) with the Globisporangium spinosum (Synonymy: Pythium spinosum) isolate SQU14064 (KY684283.1). COI sequences showed 99.86% identity (696/695 bp) with the G. spinosum isolate UPF19-OM02 (MW605192.1). Phylogenetic analysis showed the three selected isolates clustered with four published isolates from P. spinosum on the same evolutionary branch. Koch's postulates were fulfilled using two different assays on ginger (cv. 'Laiwu Dajiang'). All 16 isolates obtained were tested for pathogenicity separately. In a disk assay, surface-sterilized rhizomes were inoculated with 5-mm OA discs with mycelium of each isolate. Rhizomes inoculated with sterile OA discs served as controls. In a separate pot experiment, each 2-month-old ginger plant was inoculated with 3-ml V8 broth of 1 × 106 fragments/ml mycelial suspension by the root-drenching method. Plants inoculated with sterile V8 broth served as controls. Rhizomes (three rhizomes/isolate) and plants (three plants/isolate) were incubated at 25℃ under a 16 h photoperiod with regular watering. Seven days later, inoculated rhizomes displayed browning and rot with dense white aerial mycelia on the surface, which were identical to those observed in the fields, whereas the control rhizomes remained healthy. After 14 days, inoculated plants showed symptoms of yellowing and damping-off like those observed in the field, while no symptoms were observed on the non-inoculated plants. For both assays, the pathogen was re-isolated from symptomatic tissues and confirmed as G. spinosum by morphological and molecular analyses described above. Globisporangium spinosum (P. spinosum) has been reported causing rhizome rot on ginger in Australia and Japan (Le et al. 2016; van der Plaats-Niterink et al. 1981). To our knowledge, this is the first report of G. spinosum causing damping-off and rhizome rot on ginger in China. This finding is critical for developing specific diagnostic tools and effective agrochemicals to control ginger rhizome rot in the field.