First Report ofNigrospora sphaericaCausing Kiwifruit Postharvest Rot Disease in China

Abstract

Postharvest rot disease of kiwifruit (Actinidia sp.) has recently become a serious problem in China, the world’s largest kiwifruit cultivating and exporting country. Between September and December 2016, we collected 357 kiwifruit of cv. Hongyang from Taishun County of Zhejiang Province and Liupanshui City of Guizhou Province. After 2 months’ cold storage (4°C, 2% O₂, 5% CO₂), 68 fruits developed soft rot symptoms. The infected fruit skin was softer and slightly lighter than adjacent healthy tissues. Beneath the skin, flesh was soggy and milk-white in color. Severely decayed fruits smelled sour or fermented. A small portion (<1 cm²) of symptomatic fruit tissue was surface-sterilized with 1% sodium hypochlorite for 90 s and 75% ethanol for 30 s, rinsed three times in sterile distilled water, blotted dry, and plated aseptically onto 1.5% potato dextrose agar (PDA). A hyphal-tip from the growing edge of colonies cultured for 3 days at 25°C was transferred to PDA to obtain pure cultures. Most isolated strains were identified morphologically and molecularly as Phomopsis sp., Pestalotiopsis sp., Botryosphaeria dothidea, and Alternaria alternata, previously reported to cause rot in stored kiwifruits in China (Li et al. 2017). However, five previously unidentified morphologically identical fungal isolates were recovered on PDA and characterized by confocal optical microscopy at 400× magnification. Fungal colonies were initially flat and white, then developed into dark gray. Conidia were brown or black, smooth, spherical to subspherical, single-celled, 12.1 to 14.8 × 16.6 to 18.2 μm in diameter, n = 100. Morphological characteristics of the five isolates were consistent with Nigrospora sphaerica (Ellis 1971). To confirm this, the internal transcribed spacer (ITS) region of the ribosomal DNA was sequenced using universal ITS4/ITS5 primers. By BLASTn, these ITS sequences (MG603658–MG603662) were 100% homologous with N. sphaerica ex-type strain Ns-12 (GenBank accession no. KX256179). Molecular phylogenetic analysis confirmed five strains were N. sphaerica. All isolates were tested for pathogenicity. Near-ripe healthy fruits (cv. Hongyang) were surface-sterilized with 70% ethanol for 2 min, rinsed in sterile water, and air dried. For each isolate, 12 nonwounded and wounded (2 to 3 mm deep with a sterile pin) fruits were inoculated with conidial suspension (10 μl, 10⁶ conidia/ml), and equal numbers of fruits were inoculated with colonized PDA pieces (5 mm diameter) from 7-day-old cultures of the fungus in Petri dishes. Control fruits were treated with sterile distilled water and agar pieces. These fruits were incubated in a controlled environment chamber at 25°C with 80 to 85% humidity. The test was performed twice. Soft decay was observed in the wounded, infected flesh below the skin 5 days after inoculation, but control and unwounded fruits remained healthy. N. sphaerica was consistently reisolated from the artificially inoculated experimental fruits, but not from the tissue from noninoculated control fruits, thus fulfilling Koch’s postulates. The results indicate that N. sphaerica is a causal agent of kiwifruit postharvest rot disease in China. N. sphaerica was reported as a leaf pathogen on kiwifruit (Chen et al. 2016), but this is the first known report showing N. sphaerica to be the causal agent of postharvest rot disease on kiwifruit fruits in China. The fungus likely infected kiwifruit plants in the field through wounds caused by insects or frost after wet conditions. Future research will focus on managing the disease.