First Report of Fruit Rot Caused by Fusarium graminearum on Ponkan (Citrus reticulata Blanco cv. Ponkan) in China.

Abstract

Ponkan (Citrus reticulata Blanco cv. Ponkan) is a Chinese citrus species with tasty fruit. In November 2021, an unknown postharvest disease of Ponkan fruit caused nearly 15% losses of 2000 fruits in Nanchang, Jiangxi Province (28.68° N, 115.85° E). The initial fruit's surface necrosis was brown (Xu et al. 2022) (Figure 1A). Disease spots spread to the entire fruit, white or grey hyphae appeared, and the fruit rotted. Twenty diseased fruits were surface-disinfested with 2% sodium hypochlorite and 75% ethanol, then rinsed with sterile distilled water to isolate the pathogen. Diseased tissue sections (5 × 3 mm) were incubated on potato dextrose agar (PDA) for 7 days at 25°C. Twelve of 15 monoconidial isolates have similar morphology. On PDA, the isolates produced copious white aerial mycelia. After 5-7 days on straw juice medium, two types of conidia appeared (Rice straw 60 g, Agar 20 g, distilled water 1000 mL) (Figure 1E-I). Macroconidia were abundant, falcate, slender, and slightly curved with 0-8 septa, mostly 4-5 septa (average 41.70 × 3.81 m, n=100) (Figure 1J). Microconidia were globose, oval, or piriform with 0-1 septa, 2.72 to 8.57 × 2.53 to 7.47 m (average 5.49 × 4.52 m, n=50) (Figure 1L), and chlamydospores were not observed. Conidial and colony morphology identified 12 monoconidial isolates as Fusarium graminearum (Fisher et al., 1982; Yulfo-Soto et al., 2021). Genomic DNA was extracted from three isolates using a DNA Extraction Kit (Yeasen, Shanghai, China). The ITS1/4 region combined with partial gene fragments of translation elongation factor-1 alpha (TEF-1α, primer TEF1/2, O'Donnell et al. 1998), RNA polymerase second largest subunit (RPB2, primer fRPB2-5F/7cR, Liu et al. 1999) and β-tubulin (β-tub, primer Bt2a/2b, Li et al. 2013) from the isolates were amplified and sequenced. The three tested isolates showed identical gene sequences. Sequences amplified from one representative isolate (PG16) have been submitted to GenBank. BLAST searches revealed that ITS (OM019317), TEF-1α (OM048103), RPB2 (ON364348), and β-tub (OM048104) had 99 to 100% identity compared with F. graminearum (MH591453.1, KX087136.1, MF662636.1, and MZ078952.1, respectively) in GenBank. The phylogenetic analysis combined ITS - TEF-1α - RPB2 (O'Donnell et al. 2015) concatenated sequences using MEGA7.0 (Mao et al. 2021) showed the isolate was clustered with the F. graminearum clade with 100% bootstrap support (Figure 2). The isolate PG16 was used for pathogenicity tests. Ponkan fruits were surface-disinfested with 75% ethanol and rinsed with sterile distilled water three times. Then, 30 punctured wound fruits (2-mm-diameter, 2-mm-depth) with a sterile needle and 30 unwounded fruits were inoculated with conidial suspension (10 µL, 3.0 × 105 conidia/mL). while the control fruits were inoculated with 10 µL sterile distilled water. All fruits were incubated at 25°C and 90% relative humidity. Two days later, all wounded fruits inoculated with conidial suspension showed disease spots, similar symptoms to the original rotten fruits (Figure 1D). Control and conidial-inoculated unwounded fruits were healthy (Figure 1B-C). The Pathogenicity test was repeated twice, and similar symptoms were observed. Morphologically and molecularly, the re-isolated fungus matched the inoculated isolate. First report of F. graminearum causing Ponkan fruit rot in China. As Ponkan is an important citrus crop with high economic value in China, identification of the causing agent, F. graminearum, for fruit rot allows the development of control measures to manage this disease.