First Report of Vacuiphoma oculihominis Causing Fruit Rot on Navel Orange (Citrus sinensisi) in China

Field: Agricultural and Biological Sciences (Life Sciences)

Newhall navel orange [Citrus sinensis (L.) Osbeck] is an economically important agricultural product in China. In February 2022, a rare lesion symptom was observed on Newhall navel oranges that were harvested from an orchard Ganzhou city, Jiangxi province, China (25.53° N, 114.79° E) and stored for 90 days (18±2℃, 80 to 90% RH) at the Jiangxi Key Laboratory for Postharvest Technology and Non-destructive Testing of Fruits and Vegetables (28.68° N, 115.85° E). Approximately 2% (15/750) of the oranges exhibited symptoms, with normal appearance but ink-black flesh and juice, yellowish lesions on edges of the symptoms, and no unusual odor. To isolate the pathogen, three 5 × 5 mm pieces of symptomatic tissue from a diseased orange were disinfected in 75% ethanol for 30 s, rinsed three times with sterile water, and inoculated on potato dextrose agar (PDA) at 25±1℃ and a 12:12 h photoperiod for 7 days. A pure isolate named ND-hsp was obtained. The colony was light yellow center with pale edge on the top and brown on the bottom. Conidia and pycnidia were observed on PDA medium after 2 months. Conidia were long oval, no septa, 2.9 × 3.4 μm (n = 50), and pycnidia were solitary, 39.4 × 43.9 μm (n = 20), with one or no orifice, brown to dark brown. The morphological characteristics of ND-hsp strain on PDA, oatmeal agar and malt extract agar were similar to those of the Didymellaceae (Aveskamp et al. 2010). Ulteriorly, the genomic DNA of the ND-hsp isolate was extracted from its mycelia using a fungal genomic DNA extraction kit (Solarbio, Beijing, China) for subsequent phylogenetic analyses. Four primer sets, LR0R (Rehner and Samuels 1994) /LR7 (Vilgalys and Hester 1990), V9G (Hoog and Gerrits 1998) /ITS4 (White et al. 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RPB2-5F2 (Sung et al. 2007)/RPB2-7cR (Liu et al. 1999) were used to amplify the corresponding DNA fragments of large subunit ribosomal RNA (LSU), internal transcribed spacer region (ITS), beta-tubulin gene (TUB2) and RNA polymerase Ⅱ second largest subunit (RPB2), respectively. The obtained sequences were assigned GenBank accession numbers and showed 99 to 100% identity with their counterparts of Vacuiphoma oculihominis UTHSC DI16-308. A phylogenetic tree was constructed in MEGA 7.0 using the concatenated sequences, placing the isolate within the V. oculihominis clade by 100% bootstrap support. Pathogenicity experiments were performed in triplicate. Ten Newhall navel oranges were surface sterilized with 75% ethanol and inoculated with 15μL of a spore suspension (2×106 spores/ml) into a 3 mm-diameter wound on the equator. The control group received sterile water instead of the spore suspension. Treated and control oranges were incubated at 25±1 ℃ and about 90% relative humidity for 20 days. All oranges were cut longitudinally or transversely through the inoculated wound and examined internally. The oranges inoculated with ND-hsp exhibited ink-black flesh and juice symptoms consistent with the initial oranges. The control oranges remained asymptomatic. Under the Koch's rule, V. oculihominis was reisolated from diseased oranges and kept in Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province. GenBank database analysis confirms that V. oculihominis has been found in human eye secretions and decayed trees. This is the first report of V. oculihominis as a pathogen on navel oranges in China. Our findings contribute to understanding of citrus fruit pathogens.