First report of Nigrospora chinensis causing dragon fruit (Selenicereus monacanthus) stem spot in China.

Abstract

Dragon fruit (Selenicereus monacanthus), renowned for its economic value and dual utility in both culinary and medicinal applications, is predominantly cultivated in China. In July 2023, a stem spot disease was found on dragon fruit ("Zi honglong" cultivar) plants with 37% incidence, in Huajiang Town (N25°40', E105°39'), Guanling County, Anshun City, Guizhou Province. The symptoms appeared as yellow spots surrounded by watery stains, then the spots expanded to suborbicular, which finally led stem to wither. Twelve symptomatic stem samples were collected in a 1.3-hectare plantation and cut into small pieces (5 mm × 5 mm), sterilized the surface with 75% ethanol for 30 seconds, washed 3 times with disinfected distilled water, moved to potato dextrose agar (PDA) medium, and incubated at 28°C for 5 days. Once the mycelium had developed, they were transferred to another PDA medium and cultured at 28°C for a period of 3-5 days. Totally fifteen identical strains were isolated, their colonies were white and round in shape; hyphae were smooth, hyaline; conidia were globose or subglobose, smooth, aseptate, 5.8-11.9 × 4.2-10.6 μm (av. = 8.8 × 7.4 μm, n = 30), light brown in early stage and gradually turning black over time; sterile cells were terminal on hyphae, pale to dark brown, uhceiform or oval, 17.5-21.6 × 4.6-8.7 μm (av. = 19.4 × 5.8 μm, n = 30). The morphologic characteristics of the isolates matched Nigrospora chinensis described by Wang etal.(2017). The PCR amplification was carried out by 3 primers ITS1/ITS4 (Vilgalys et al. 1990; White et al. 1990), EF1-728/EF2 (O'Donnell et al. 1998; Carbone et al. 1999) and BT2A/BT2B (Glass et al. 1995) belonged to the internal transcribed spacer (ITS), translation elongation factor-1 (TEF1) and β-tubulin (TUB2) gene loci, respectively. The sequences of a representative strain (GUCC 524) had 99.38% (ITS: 483/486 bp, PP391347 vs KX985970), 99.79% (TEF1: 482/483 bp, PP400678 vs KY019427) and 99.73% (TUB2: 372/373 bp, PP400677 vs KY019497) identities with those of N. chinensis (strain LC 3085). The phylogenetic tree constructed by three gene combinations showed that GUCC 524 was significantly clustered with N. chinensis. Ten 6-month-old dragon fruit ("Zi honglong" cultivar) seedlings were engrafted with 10 μL conidial suspension (1×105 conidia/mL), packaged with sealed film, two of them were inoculated with sterile distilled water as controls, and placed in a greenhouse at 28℃ for 10 days, inoculated plants showed yellow spots analogous to field symptoms, no symptoms were found in control plants. This experiment was repeated three times. Morphological character and molecular identification based on 3 gene loci of the strains isolated from the inoculated stems, were consistent with those of the original isolated strains. Therefore, based on morphological identification, phylogenetic analysis and pathogenicity test, the pathogen was identified as N. chinensis. Our study firstly reported N. chinensis as a pathogen causing stem spot disease on dragon fruit. N. chinensis is an important agent resulting for economic losses, previously reported on Camellia sinensis (Wang et al. 2017), Saccharum officinarum (Raza et al. 2019), Aucuba japonica (Qin et al. 2021). This report establishes a pivotal reference point for the progression of scientific strategies in preventing and controlling this disease associated with N. chinensis.