Kiwifruit (Actinidia spp.) has been extensively cultivated (about 165728 hm2 recorded in 2017) and postharvest rot diseases have caused severe losses to the industry in China. In October 2019, fruit (n=60) of cv. Xuxiang (A. deliciosa) were obtained from a farm (120.62°E, 28.92°N) in Pan’an county, Zhejiang province, China. After the fruit were stored at 24 °C and 70% relative humidity (RH) for 10 days, soft lesions (20 to 45 mm in diameter) with sour odor and white mycelium were observed on ~20% of fruits (Fig. 1a). Irregular lesions were produced on the mesocarp were off-white to pale yellow (Fig. 1b). Small pieces (4×4 mm) from the lesion margins were excised, surface disinfested in 70% ethanol for 1 min and 10% NaOCl for 5 min, washed, dried, plated on PDA and incubated at 25°C for 7 days. A total of seven pure fungal colonies were obtained, and included two isolates of Nigrospora sphaerica (Li et al. 2018) and five unknown isolates. The remaining five isolates produced thin, flat, white to cream and feathery (Fig.1c & d) mycelium. Hyphae were hyaline, septate, dichotomously branched and break into chains of subglobose to cylindrical arthrospores (Fig. 1e, f & g). The dimension of arthrospores varies from 4.11 to 12.55 × 2.54 to 5.84 µm (n=100) (Fig. 1h). To identify these isolates to species, the internal transcribed spacer (ITS), 26S rDNA, translation elongation factor-1 alpha regions (TEF-1α) were amplified and sequenced (Ma et al. 2018). Sequence analysis indicated no differences in 26S rDNA and TEF-1α, but the ITS that placed the isolates into two phylogenetic groups. Isolates gx2-2 and gx3-1 representing group one (gx2-1, gx2-2, and gx5-1) and group two (gx3-1 and gx4-1) respectively, were employed for further studies. Based on BLASTn analysis, ITS sequences for gx2-2 (MT946912) and gx3-1(MT946913) isolates had 100% and 98.40% identity respectively, with G. candidum accessions KY103452 and KY103455. Nevertheless, 26S rDNA sequences (MT981194; MT981195) showed 99.82% identity with G. candidum accessions JN974268 and KF112070. Consistently, the TEF-1α (MT981184; MT981185) had 100% identity with G. candidum accession MT346370. Phylogenetic trees were constructed using the Neighbor-Joining method with a dataset of ITS (Fig. 2a) and 26S rDNA sequences (Fig. 2b), respectively. Based on morphology and phylogenetic analysis, the pathogens gx2-2 and gx3-1 were identified as Geotrichum candidum (De Hoog et al. 1986). To determine pathogenicity, healthy and mature kiwis cv. Xuxiang were surface sterilized. Wounded and unwounded fruits were inoculated with each conidial suspension derived from the two isolates (107 conidia/mL, 30 μL for each fruit) and stored at 24 °C under 90% RH. Control fruit were treated with sterile distilled water. Each treatment consisted of 20 fruit was evaluated daily for 10 days and repeated once. The symptom was mimic the naturally infected fruits (Fig.1i, m & n). The pathogen could develop into inner pericarp after 7 days while cv. Jinyan (A. chinensis) was used as host (Fig. 1k & l). However, control group remained disease-free (Fig. 1j, o & p). The fungus could penetrate into fruit peel and produce spores that were visualized by scanning electron microscope (Fig.1q & r). For both isolates, the incidence of wounded fruit were 100%, and the incidence of unwounded fruit was 80%. The fungi were re-isolated from diseased tissues and re-identified as G. candidum based on morphology and sequences analyses. G. candidum causes sour rot on many hosts and similar symptom have been previously reported in other regions(Pennycook et al.1989; Horita et al. 2016; Ma, et al. 2018; Zhang et al. 2018; Khan et al. 2019; Halfeld-Vieira et al. 2020), but this is the first report of G. candidum on kiwifruit in China.
Bioethanol-Producing Yeast Isolated from Fermented Cocoa