First Report of Flower Rot Caused by Fusarium luffae and F. asiaticum on White Kiwifruit in China

Abstract

As an important cultivated germplasm, cultivar ‘White’ (Actinidia eriantha) is appreciated by kiwifruit breeders because of its long shelf life, richness in ascorbic acid and peelable skin. In May 2020, about 1% to 3% of cultivated ‘White’ plants displayed typical symptoms of flower rot at farms in Hefei (117°25′E, 31°86′N) and Lujiang (117°27′E, 31°48′N), Anhui Province of China (Fig.1a&b). The infected flowers were yellowish at first, gradually turned brown, withered and shrunk, and finally died without blossoming. Infected flowers were surface sterilized in 70% alcohol for 30 s and 1% NaOCl for 3 min, then washed with double distilled water (ddH2O) for 5 times, and finally incubated in potato dextrose agar at 25 ± 2°C in the dark. Twenty fungal isolates were obtained and their colonies showed slightly raised center with dense and cotton-like mycelium. Colonies from Hefei (HF1~HF9) appeared pale yellow (Fig.1c), and those from Lujiang (LJ1~LJ11) showed purplish-red on PDA (Fig.1d). Two types of colonies grown on oatmeal agar were flat with few aerial hyphae (Fig.1e&f). On carnation leaf agar (CLA), isolate of HF1 produced abundant slightly curved macroconidia with 3 to 6 septa, 4.3-5.5×20.7-42.5 µm in size (n=100) (Fig.1g&h), without microconidia and chlamydospores observed. By contrast, macroconidia derived from LJ1 isolate were straight to slightly curved with 3 to 5 septa, 4.0-6.58×21.70-71.10 µm (n=100) in size (Fig.1i); Its chlamydospores were globose to subglobose (5.1 to 9.5 µm) on CLA (Fig.1j). Pathogenicity tests were performed on A. eriantha cv. ‘White’ flowers. The conidia suspension (105 spore/ml, 30 μL/flower) derived from the HF1 and LJ1 were separately dripped on flowers (n=100). Control flowers were treated with ddH2O. Two-week post-inoculation, all inoculated flowers were turned brown and withered (Fig.1k&m), whereas no symptoms were observed on the controls (Fig.1l&n). This experiment was repeated three times. All isolated and re-isolated pathogens from diseased flowers were subjected to molecular identification. Different molecular markers, including internal transcribed spacers (ITS), translation elongation factor (TEF-1α), calmodulin (CaM), RNA polymerase II subunit 1 (RPB1) gene and RNA polymerase II largest subunit (RPB2) gene, were amplified and sequenced to validate species identification (White et al. 1990; O’Donnell, et al. 1998; O’Donnell, et al. 2012). Based on sequence analysis, the re-isolated strains were identical to the inoculated individuals. Sequences of HF1 and HF2 or LJ1 and LJ2 were deposited in GenBank under accession numbers OK310710 to OK310713 (ITS), OK334291 to OK334294 (TEF-1α), OK412973 to OK412976 (CaM), OK412977 to OK412980 (RPB1), and OK484317 to OK484320 (RPB2), respectively. The BLAST search showed that the sequences of HF1 and HF2 showed 99 to 100% identity with ITS (NR_164594), TEF-1α (MK289601), CaM (MK289698), RPB1 (HM347158), and RPB2 (MK289754) of Fusarium luffae isolates. The sequences of LJ1 and LJ2 also revealed 99 to 100% identity with ITS (NR_121320), TEF-1α (AF212452), RPB1 (JX171459), and RPB2 (MW233412) of F. asiaticum isolates. F. asiaticum species-specific primers were used to detect LJ isolates (Yin et al. 2009), and the correct fragments were amplified (Fig.1o). Phylogenetic trees were constructed based on the tandem nucleotide sequences. Thus, both morphological and molecular criteria supported identification of HF group as F. Luffae (Fig.2a) and LJ group as F. asiaticum (Fig.2b). Fusarium spp. causing flower rot on many hosts have been previously reported (W. Elmer, et al. 2019; Liu, et al. 2021), but this is the first report of F. luffae and F. asiaticum on ‘White’ kiwifruit in China.