Pythium aphanidermatum causing seedling damping-off of Hylocereus megalanthus in China.

Abstract

Hylocereus megalanthus (family Cactaceae), commonly known as bird's nest fruit (Yanwo fruit), was a new tropical plant cultivated commercially in south China because of its high nutritional content and sweet taste. In August 2023, damping-off disease of approximately 60% of seedlings was observed at a nursery in Zhanjiang, Guangdong Province (E110°17'46″ N21°9'2″). Stems of infected seedlings exhibited symptoms of water-soaked tissue which caused collapse at the base of the stem and sloughing of necrotic root cortex tissue was observed (Figure 1). White aerial mycelia were visible on the surface of the stem and soil at a high relative humidity. Diseased tissues about 0.5 cm2 were taken from the infected roots and stems, surface disinfected with 75% ethanol and 3% hydrogen peroxide solution, each for 1 min, subsequently rinsed in sterile water, and placed on potato dextrose agar (PDA). Plates were incubated at 25 to 28℃ in the dark for 3 days. Coenocytic hyphae grew from all infected roots and stems. Hyphal tip transfers were completed twice, and twelve isolates with the same morphological characteristics were obtained. The colony growth on PDA was ample. Main hyphae are up to 9.5 µm wide. Sporangia were terminal, inflated, branched or unbranched. Encysted zoospores were 7.5 µm in diameter. Oogonia were terminal, globose, smooth and of 16.8 to 27.4 µm (average 21.5 µm) diameter. Oospores were typically spherical, thick-walled, yellowish, 19.7 to 26.3 µm (average 21.1 µm) diameter, wall 1 to 2 µm thick. Antheridia were mostly intercalary, sometimes terminal, broadly sac-shaped, 15.0×19.0 µm (Figure 2). The morphological features were very similar to those of Pythium spp. (Toporek and Keinath 2021). For further identification, the LSU and ITS regions of isolate CCAS-YWGCD (stored in Agricultural Culture Collection of China, ACCC 35633) were amplified and sequenced with using primer pairs LROR/LR7 and ITS1/ITS4, respectively (Gao et al. 2017; White et al. 1990). The resulting sequences were deposited in GenBank (ITS: OR775664; LSU: OR775667). BLASTn results showed 100% sequence similarity with reference sequences of Pythium aphanidermatum (AY598622 for ITS and HQ665084 for LSU). Phylogenetic tree generated from maximum likelihood analysis based on combined LSU and ITS sequence data with MEGA 10.1.8, clustered the oomycete in P. aphanidermatum clade with 100% bootstrap support (Figure 3). Therefore, the oomycete was identified as P. aphanidermatum. To confirm Koch's postulates, six three-month-old seedlings of H. megalanthus (height about 15 cm) were transplanted to 15 cm pots. Six-mm-diameter mycelial plugs obtained from 7-day-old cultures at 25℃ in the dark were buried adjacent to the stem of three unwounded healthy seedlings. Another three seedlings inoculated with PDA agar served as controls. The plants were covered with plastic bags, kept at about 30℃, and watered regularly to keep the soil moisture content high. All inoculated seedlings exhibited symptoms of stems rot and damping-off, Symptoms did not develop on the control seedlings. P. aphanidermatum by morphological and molecular analysis was reisolated from the stems. P. aphanidermatum had been reported worldwide causing disease in many agricultural crops (Qi et al. 2021; Kim et al. 2020), but this is the first report causing damping-off of H. megalanthus seedling in China as well as worldwide, and this disease should be monitored in nursery seedlings.