First Report of a New Postharvest Disease of Pear Fruit Caused by Ceratocystis fimbriata in Kunming, China.

Abstract

Pear (Pyrus pyrifolia (Burm.f.) Nakai) is widely planted in China and plays a key role in economy. In the autumn of 2016, five pear fruits showing symptoms of brown rot (Fig. 1A) were found in a Suancun farmer market in Kunming, Yunnan Province, China (25°02' N; 102°42' E). The incidence of this disease in postharvest pear fruits ranged from 2 % to 5 % in this city. Three fruit samples were taken to run further tests. The decayed area of the fruit was soft, brown, slightly sunken, and circular. Carrot baiting was used to isolate the pathogen from symptomatic tissue (Moller et al. 1968). Primary isolates were made by transferring ascospore drops from the tips of the perithecia formed on the carrot discs onto PDA plates. Single ascospore cultures were generated by transferring single ascospores to potatoe dextrose agar (PDA) plates. Cultures were incubated 7 days at 25°C with a 12-h light/12-h dark cycle. In culture, mycelium was initially white, turned to a shallow celadon and gradually to grey-greenish later. Measurements were made 10 days after the formation of perithecia. Six pure cultures (lik-1~lik-6) were stored at -80 °C in 15% glycerol and stored at the State Key Laboratory for Conservation and Utilization of Bio-Resources of Yunnan Agricultural University. Four isolates (lik-1~lik-4) produced ascomatal bases that were submerged in the agar. Bases (Fig. 1E) were globose, black, 192.15 to 250.81 µm wide, 192.94 to 251.31 µm long, and had straight necks terminating in ostiolar hyphae (Fig. 1F) that were divergent, hyaline, and 74.19 to 116.33 µm long. Asci were not observed. Ascospores (Fig. 1I) were ovoid, hat-shaped (dimensions 3.2 to 5.1 × 2.3 to 4.6 µm). Conidiogenous cells were with enteroblastic conidium ontogeny, flask-shaped or tubular, 65.3 to 130.6 μm long, and produced cylindrical, straight aseptate conidia (8.5 to 18 × 2.5 to 3.5 µm) (Fig.1 G). All isolates produced dark brown, 10.07 to 13.08 ×8.51 to 11.64 µm aleurioconidia (Fig. 1H). Two (lik-1, lik-3) of six isolates were used for molecular identification and genomic DNA was extracted using the CTAB method (Lee & Taylor 1990). The primers ITS1 and ITS4, EF1F and EF2R were used to amplify and sequence the rDNA-ITS and TEF-1α regions (Thorpe et al. 2005; Jacobs et al. 2004). The sequences of rDNA-ITS of the isolates lik-1 and lik-3 (GenBank Accession Nos: MF153994, MF153993) showed 99.49% similarity to AF395679 (C. fimbriata isolate CMW2219). Additionally, the TEF-1α sequences of isolates lik-1 and lik-3 (GenBank Accession Nos: KY708912, KY708915) showed 100% identify to MF347676 (C. fimbriata isolate CM18). Based on symptoms, morphological characteristics, rDNA-ITS and TEF1-α sequence analysis and pathogenicity, this fungus was identified as C. fimbriata. Pathogenicity tests were conducted using 2 isolates (lik-1, lik-3) and repeated three times. Three fresh pear fruits were disinfected with 75% alcohol, then they were wounded with a 2 mm hole punch and inoculated with 200 μL conidia suspension of the fungus (approximately 2.0 × 106 conidia / mL) on the fruit surface. After inoculation pear fruits were incubated in boxes at 25°C with a relative humidity of 80% and a 12-h light / 12-h dark cycle. Three pear fruits that served as controls were wounded by punching a 2 mm hole into the skin and inoculated with 200 μL sterile distilled water. Symptoms of rot were observed one week after inoculation (Fig.1 B). The diameter of the external lesion varied from 1.5 to 2.5 cm, on average 1.9 cm. When pears were cut, the white pulp had turned black and was rotting (Fig.1 C, D). The pathogen re-isolated from all inoculated symptomatic tissue was identical to the isolates originally obtained from the pear fruits at the market by morphology and ITS analysis. No symptoms developed on the control. The pathogenicity assay showed that C. fimbriata was pathogenic on pears. To our knowledge, this is the first report of C. fimbriata on pear in China. The spread of this disease may pose a threat to pear quality in China and further studies could be performed to determine effective disease management strategies.