First Report of Damping-Off in Actinidia argute (Tara Vine) Caused by Fusarium proliferatum in China

Actinidia argute (tara vine) is a deciduous, fast-growing, twining woody vine typically grown for its attractive foliage and edible fruit and native to Japan, Korea, northern China, and the Russian Far East (Latocha et al. 2013). In March 2019, severe damping-off was observed in a base for seedling cultivation of A. argute located on Taihu farm of Jingzhou (N 111°15′, W 29°26′), Hubei province, China. Disease incidence in variety Longcheng No. 2 was approximately 30%, which resulted in severe seedling loss. At the early stage of the disease, seedlings showed symptoms of light to dark brown lesions on the lower stems near the soil and root necrosis. Finally, the seedlings became wilted and rotted. To isolate the pathogen, stem sections (3 × 2 mm) from 20 individual plants with symptoms were surface sterilized with 75% ethanol for 30 s and 2% NaOCl for 1 min. The sterilized tissue was rinsed four times with sterilized water and then put on potato dextrose agar (PDA) amended with 50 μg/ml of ampicillin and kanamycin to incubate at 25°C in the dark for 5 days. Isolated colonies were subcultured by the hyphal tip transferring method. Sixteen fungal isolates were obtained from 16 sterilized tissues, except for four tissues. Through the preliminary inoculation tests, we found that 13 isolates showed damping-off symptoms, except for three isolates. These 13 isolates with pathogenicity showed similar morphological characteristics with white to salmon pink colonies on PDA and abundant white aerial hyphae. Mung bean soup (MBS) medium was used to stimulate conidia production. Macroconidia were long, sickle shaped, three to four septa, 37.29 ± 6.03 × 3.56 ± 0.40 μm (n = 50), and microconidia were short oval, zero to one septa, 8.92 ± 1.74 × 2.88 ± 0.36 μm (n = 50). The isolation was identified as Fusarium sp. based on morphological characteristics (Leslie and Summerell 2006). Total genomic DNA of 13 isolates was extracted with the CTAB method (Stenglein and Balatti 2006). The ITS gene of all were sequenced using primers ITS4/ITS5 (White et al. 1990) and differed by only 1 or 2 bp of 466 nt total. A representative isolate, RZ1901, was used for further characterization using translation elongation factor 1α (EF-1α) (EF-728F/EF-986R) (Carbone and Kohn 1999) and β-tubulin genes (BT2-a/BT2-b) (Glass and Donaldson 1995), respectively. ITS (accession no. MN095191), EF-1α (MN227186), and β-tubulin (MN103540) sequences were deposited in GenBank. Three sequences were analyzed via FUSARIUM-ID (http://fusariumdb.org/index.php) and Fusarium MLST (http://www.wi.knaw.nl/fusarium/) and showed at least 99.57% identity to F. proliferatum (CBS130179, CBS 217.76, and NRRL 22109). For the pathogenicity test, isolate RZ1901 was cultured in MBS for 1 week to get spore suspension. Stems and main roots of 20 healthy 2.5-month-old A. argute seedlings were wounded by sterilized insect needle and soaked in RZ1901 conidial suspension (10⁶ spores/ml) for 60 min, and 10 seedlings with sterile distilled water were used as a control. All plants were transplanted to pots filled with autoclaved (121°C) peat moss and placed in a greenhouse (25 ± 2°C, 65% relative humidity). After 3 weeks, damping-off symptoms were observed on inoculated seedlings, similar to the diseased seedlings in the field. But there were no symptoms for the controls. The experiment was repeated twice. Reisolation was performed with surface-sterilized tissue from symptomatic seedlings in one experiment. Fungal isolates were confirmed as F. proliferatum based on morphological and molecular characters. F. proliferatum, a soilborne pathogen with a wide host range, has been reported to cause soybean damping-off in the United States (Díaz Arias et al. 2011). To our knowledge, this is the first report of damping-off by F. proliferatum on A. argute in China. The intensity and crop loss of this disease will be carefully monitored to determine if management strategies are justified for control.