First Report of Leaf Spot on Kidney Bean Caused by Nigrospora oryzae in China

Abstract

Kidney bean (Phaseolus vulgaris L.) is a legume with high nutritional and economic value. This vegetable crop is widely cultivated in China, providing a year-round supply of young edible pods. In July 2020, a leaf spot disease on kidney bean cultivar 'Dabailong' was observed on a two-hectare field in Longli County (26°16'15.66″ N, 106°48'12″ E), Guizhou Province, China. Disease incidence was estimated to be nearly 50%. Foliar symptoms manifested as black circular spots, surrounded by a yellow halo and accompanied by white mycelium. To identify the pathogen, small portions of tissue (5×5 mm) from margins of leaf spots were cut from 20 symptomatic leaves, surface disinfected with 75% ethanol for 30 s, rinsed two times with sterile distilled water, dried on a sterile filter paper, and incubated on potato dextrose agar (PDA) at 28°C for 3 days. A total of 39 single-spore isolates were obtained. The colonies on PDA were fluffy, changing from white to gray or black with age, and reaching 7-cm diameter in 5 days at 28°C. Conidia were black, globose to subglobose, smooth, solitary, measuring 13.0 to 16.0 × 10.5 to 16.0 µm (n=30). Morphological characteristics were consistent with Nigrospora oryzae. In addition, the rDNA internal transcribed spacer (ITS), large subunit (LSU), β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1) loci were amplified by PCR and sequenced (White et al. 1990, Glass and Donaldson 1995, O'Donnell et al. 1998; Carbone and Kohn 1999). The ITS, LSU, TUB and TEF1 sequences of two isolates, GUCC19-5105 and GUCC19-5192, were submitted to GenBank. BLASTn analysis of these sequences showed >98% homology with those of N. oryzae strain LC 7293 in GenBank (ITS, 99.80% (MZ145361 vs KX985931 - 498/499 bp) and 99.62% (MZ148445 vs KX985931 - 525/527 bp); LSU, 100% (MZ146317 vs KY806236 - 837/837 bp) and 99.76% (MZ148446 vs KY806236 - 847/849 bp); TUB, 98.72% (MZ329335 vs KY019601 - 386/391 bp) and 98.67% (MZ329337 vs KY019601 - 373/378 bp) and TEF1, 98.91% (MZ329336 vs KY019396 - 452/457 bp) and 98.89% (MZ329334 vs KY019396 - 444/449 bp) respectively). The phylogenetic tree of the combined 4 sequences showed that both isolates clustered with N. oryzae. Based on morphological characteristics and the multigene phylogenetic analysis, GUCC19-5105 and GUCC19-5192 isolates were identified as N. oryzae. Pathogenicity tests were performed twice by spraying conidial suspension (1×105 conidia/mL) of the two isolates (GUCC19-5105 and GUCC19-5192) on leaves of ten (five per isolate) healthy 5-week-old kidney bean cultivar 'Dabailong' plants. Two plants sprayed with sterile water served as controls. After inoculation, all the plants were kept moist in plastic bags for 24 hours and incubated in a greenhouse at 25°C for 20 days. Leaf spots similar to those observed in the field were observed 20 days post inoculation, but no lesions were observed on control plants. N. oryzae was reisolated from the infected tissues of inoculated kidney bean plants and the identity of the reisolated pathogen was confirmed as N. oryzae through morphology and sequencing ITS, LSU, TUB and TEF1 loci. In recent years, N. oryzae has been reported to infect a variety of plants such as Aloe vera, Citrullus lanatus and Costus speciosus (Begum et al. 2018; Chen et al. 2019; Sun et al. 2020). To our knowledge, this is the first report of leaf spot disease on kidney bean caused by N. oryzae in the world and provides a basis for diagnosticians and researchers to identify the disease and develop disease management strategies.