Hami melon (Cucumis melo var. saccharinus) is designated as a geographical indication product in China due to its pleasant aroma, crisp texture, sweetness, and coloration (Zhu et al., 2021). In June 2024, approximately 50% (556 out of 1110) post-harvest Hami melons (cv. Xizhoumi 25), cultivated and stored in Turpan, Xinjiang, China (42.88°N, 90.22°E), exhibited initial symptoms of dark brown discoloration after 20 days of storage at 15°C, resulting in fruit rotting accompanied by black-gray fungal growth. To identify the pathogen, segments (5 mm2) from the margins of rotted tissue were excised from five randomly selected symptomatic Hami melons, subjected to surface sterilization twice with 75% ethanol, rinsed three times with sterilized water, and then placed onto potato dextrose agar (PDA) medium. Five morphologically identical single-spore isolates (XJ-1 to XJ-5) were obtained following Leslie and Summerell (2006). On PDA at 25°C in darkness, their average radial mycelial growth rate during the linear growth phase (days 1–3) was 23.67 ± 0.5 mm/day (n = 75; 5 isolates × 5 replicates × 3 time points). Colonies covered the plates by day four and turned dark gray with abundant aerial mycelium by day seven. Conidia from all five isolates (n = 50; 10 conidia per isolate) were dark brown, thick-walled, and exhibited ellipsoid or ovoid shapes with one septum, measuring 19 to 24 × 9 to 13 µm. These morphological features of the five isolates were consistent with Diplodia species (Phillips et al., 2013). For species identification, the internal transcribed spacer (ITS) region, beta-tubulin (TUB), and translational elongation factor 1-alpha (TEF1-α) genes were PCR amplified and DNA sequenced using primer pairs ITS1/ITS4, BT2a/BT2b, and EF1-728F/EF1-968R, respectively (Úrbez-Torres et al., 2008). Multiple sequence alignments with ClustalW revealed that the obtained ITS, TUB, TEF1-α sequences of all five isolates were 100% identical. BLASTn search in GenBank indicated the consensus sequences of the representative isolate XJ-1 (ITS: PQ316093.1; TUB: PQ309646.1; TEF1-α: PV356091.1) shared 98.18 to 100% similarity with reference sequences of Diplodia seriata De Not. (KX464107.1, MT587382.1 for ITS; KX464833.1, MT592548.1 for TUB; KX464597.1, MT592090.1 for TEF1-α). A phylogenetic analysis utilizing the concatenated nucleotide sequences (ITS, TUB, and TEF1-α) grouped all five isolates (XJ-1 to XJ-5) within the D. seriata species complex clade, with a bootstrap support value of 97%. To verify pathogenicity, 20 healthy Hami melon fruits (cv. Xizhoumi 25) were surface-sterilized with 75% ethanol and 2% sodium hypochlorite (NaClO), and then rinsed with sterile distilled water (Zhou et al., 2019). Ten fruits were subsequently wounded with a sterile hole punch and inoculated with mycelium plugs (6 mm in diameter) from a 3-day-old XJ-1 culture. An equal number of fruits inoculated with sterile mycelium-free PDA plugs served as controls. All the inoculated fruits were maintained in an artificial climate chamber at 20°C, with 90% humidity and a 12-h light/12-h dark cycle. After 10 days, all the inoculated fruits exhibited dark brown and water-soaked lesions consistent with those observed on naturally infected fruits, whereas controls remained healthy. The re-isolated fungus from symptomatic fruits matched the original D. seriata isolates in morphology and in ITS, TUB, and TEF1-α sequences, thereby satisfying Koch’s postulates. The pathogenicity test was repeated three times with the same results. To our knowledge, this is the first report of Diplodia seriata causing postharvest fruit rot on Hami melons in China, which will aid in developing future management strategies for this disease in China.
Zheng et al. (Fri,) studied this question.