First Report of Nigrospora oryzae Causing Brown/Black Spot Disease of Kiwifruit in China

Abstract

China is the world’s leading kiwifruit producer (Belrose 2016). In September 2016, brown/black spot disease was found on leaves and fruit peel in several kiwifruit orchards in Taishun County, Zhejiang Province, and Liupanshui City, Guizhou Province. Diseased leaves and fruits exhibited small irregular brown to black spots (1 to 2 mm), which grew (3 to 5 mm) and darkened with age. The leaves withered in the fall. Eighty trees of 10 kiwifruit orchards in each city were investigated, and approximately 45% of trees were symptomatic in Taishun and Liupanshui. Symptomatic tissues of 20 typical infected leaves and fruits were cut into 4- to 5-mm pieces, surface-sterilized, rinsed three times in sterile distilled water, and cultured on 1.5% potato dextrose agar (PDA) with streptomycin and tetracycline. Petri dishes were incubated at 25°C under a 12-h light/dark cycle. Hyphal tips from the growing edge of colonies cultured for 3 days at 25°C were transferred to PDA to obtain pure cultures. Fungal colonies were white, then gray to black with an unevenly distributed, fast-growing aerial mycelium covering the Petri dish within 5 days at 25°C. The colony turned dark brown when maintained in the dark at 25°C after 7 days, then black/grayish brown upon sporulation after 15 days. Conidia were brown or black, smooth, spherical to subspherical, single-celled (10 to 12 × 14 to 16 μm, average 11.2 to 15.5 μm in diameter, n = 100). Cultural and morphological characteristics were suggestive of Nigrospora oryzae (Ellis 1971; Hudson 1963). To confirm, genomic DNA was extracted from seven isolates. The internal transcribed spacer (ITS) region of the strains’ ribosomal DNA was sequenced using universal ITS4/ITS5 primers. By BLASTn, these ITS sequences (MF198430–36) were 100% homologous with N. oryzae ex-type strain ICMP2736 (GenBank accession no. EU436680), confirmed by molecular phylogenetic trees constructed using MEGA7. The morphological data and nucleotide homology pointed to the isolate being N. oryzae. All isolates were tested for pathogenicity on leaves and fruits of cv. Hongyang. For each isolate, 12 nonwounded/wounded leaves and fruits were inoculated with a conidial suspension (10 μl, 10⁶ conidia/ml) and colonized PDA pieces (5 mm diameter) from 7-day-old cultures of the fungus were placed in Petri dishes, incubated for 48 h, then removed. Sterile distilled water and an agar plug were used as a control. Leaves and fruits were incubated in a controlled environment for 5 days, maintained at 25 ± 0.5°C and 80 ± 5% relative humidity with 12 h of fluorescent white light/day. Five days after inoculation, wounded leaves and fruits showed symptoms like those in the field, whereas control and unwounded leaves and fruits remained symptom-free. N. oryzae was consistently reisolated from affected leaves and fruits but not the controls, fulfilling Koch’s postulates. Pathogenicity tests were carried out twice, with the same results, indicating that N. oryzae is responsible for brown/black spot disease on kiwifruit in China. N. oryzae is a known pathogen for several hosts (Zhai et al. 2013; Zhang et al. 2012), but this is the first report of brown/black spot disease of kiwifruit caused by N. oryzae in China. Geographic distribution and management of the fungus should be further investigated.