First Report of Penicillium digitatum causing Postharvest rot of Citron Fruit in Kunming, China

Abstract

Citron (Citrus medica L.) is a perennial evergreen woody tree of Rutaceae family and Genus of Citrus. The citron is cultivated for its economic, medicinal and ornamental values in the south of China. (Yang et al., 2015). The shapes range from spherical to ovate and the sizes range from 3 to 5 kg (Klein et al., 2016). In June 2021, some postharvest citron fruits (Citrus medica var. medica) were found to have decay with a green or greyish mycelium on part or whole citron in 2 farmer’s markets in Kunming city, Yunnan Province (N 25°02′; E 102°42′), southwest China. Initial symptoms appeared as white, brown, and irregular necrotic spots in the pericarp. The lesions enlarged gradually and developed into green, water-soaked areas which extend rapidly. Eventually, the diseased fruits were rotten, soften, and the green spore masses confined to the surface (Fig. 1A). The incidence of this disease in postharvest citron fruits ranges from 15 % to 35 %, which is extremely destructive to the fruit of Rutaceae family plants (Chen et al., 2019). Small pieces (5 mm2) of symptomatic citron fruits were surface disinfected in 75 % ethanol and 0.3 % NaClO for 30 s and 2 min respectively, rinsed with distilled water for three times, blotted dry, placed onto potato dextrose agar (PDA) medium aseptically and incubated in a growth chamber at 25 ± 1 ℃, after 7 days, different colonies grew on PDA plates that were isolated and purified on new PDA medium at 25 ± 1 ℃ for 7 days. Inoculating repeatedly until six single-strain (XY01 to XY06) were obtained, and these isolates were stored in 15 % glycerol at –80 ℃ in a refrigerator in the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan Agricultural University. The selected pathogens (XY01 to XY06) were inoculated on PDA medium, incubated at 25 ± 1 ℃. After 7 days, colonies of the isolate obverse are olive green, the white margin and greyish-green spores on the surface, and the reverse colorless to cream yellow or pale dull brown. Colonies texture was velutinous, with a special fragrance. The conidia structure was very fragile and break up easily into many cellular elements. Conidiophores were terverticillate, produced by subsurface or aerial hyphae, irregularly branched and composed of short stipes with few metulae and branches that terminate in whorls of three to six phialides, which are often solitary, cylindrical with a short neck. Conidia are hyaline to pale green, smooth-walled, without septate, partially ellipsoidal, or obovate (4.9 to11.9× 4.3 to 8.9 μm). Partial cylindrical (8.2 to 10.5× 2.7 to 5.3 μm), there are some small conidia, which were ellipsoidal or spherical (3.9 to 5.2× 2.7 to 5.2 μm). According to morphological characteristics, the fungus was identified as Penicillium digitatum (Pers.) Sacc. Isolate XY01 and XY02 were used for molecular identification and genomic DNA was extracted using the CTAB method (Aboul-Maaty & Oraby, 2019). The universal primers ITS1 and ITS4 were used to amplify and sequence the ITS1, 5.8S, and ITS2 rDNA region. Using NCBI’s BLASTn tools, the nucleotide sequences of XY01 and XY02 (Gen-Bank accessions MZ976843 and OK513274) show 100 % identity to MK450692 (P. digitatum strain CMV010G4). Pathogenicity tests have used the fruits (Citrus medica), which maturity was more than 80%. The pathogens (XY01, XY02) were cultured for 7 days on PDA medium, washed with sterilized water the resulting spore suspensions diluted to 1.0 × 106 spores/ml. Wounds (0.5 × 0.5 cm) were made on the surface of citron fruits by scraping with a sterile scalpel and then treated with 200 µl of spore suspension (Wild, 1994). Control citron fruits were treated with sterile water. citron fruits were incubated at 24-26 °C. Each treatment was performed in triplicate with 6 citron fruits. After 3 days, all fruits had developed lesions, in a water-stained, pale brown, and rapidly formed white hyphae, white mold layer was observed with a length of 1.5-2.5 cm and a width of 1-2 cm (Fig.1C), but control did induce infection. After 7 days, decay developed more quickly, the hyphae rapidly expanded on the surface of the pericarp, with vague and irregular edges, then a green mold layer was formed, the whole fruit was observed to rot and soften, When the citron was cut, the white flesh inside turned black and rotted (Fig.1B). P. digitatum was consistently reisolated from the inoculated plants but not from the controls. No symptoms developed on the control (Fig.1D). According to Koch’s postulates, the inoculated strains of XY01 and XY02 were the isolates causing citron decay disease. Based on symptoms, morphological characteristics, rDNA-ITS sequence analysis, and pathogenicity, this fungus was identified as P. digitatum. To our knowledge, this is the first report of the distribution of P. digitatum on Citron (Citrus medica) in China.