Leaf vein disease in Nelumbo nucifera caused by Nigrospora pyriformis infection in China.

Abstract

Nelumbo nucifera (Nymphaeaceae) has both ornamental and nutritional uses in China. In July 2021, a novel disease was observed on plants in the White Lotus Science and Technology Expo Park in Guangchang county, Fuzhou city, Jiangxi province (26.79° N, 116.31° E). Infection was visible as nearly round black spots on the leaves in the early stages of infection. At later stages, the spots spread along the veins forming reticular necrosis until the entire leaf was infected, and the disease caused approximately 20% of leaves to die. To identify the pathogenic organism, 5×5 mm samples were taken from affected tissue adjoining healthy tissue, sterilized in 75% ethanol for 30 s, and immersed in 0.1% mercury chloride for a further 30 s, before washing in sterile water and transfer to potato glucose agar (PDA) plates. After culturing at 28℃±1℃ for five days, white regularly shaped round colonies were visible that after 10 days turned black with fluffy hyphae. The individual conidia produced by the conidiophore were initially a light-brown color which changed to black as the pigment accumulated; conidia were globose to subglobose, 17.10 (14.77-21.66) ×14.70 (12.08-16.93) μm in size (n=50), with glossy even surfaces lacking in septa, suggestive of Nigrospora. To verify this, PCR amplification was conducted using ITS1/ITS4 primers for the 5.8S rRNA gene. The primers EF1-728F and EF-2 were used for amplifying translation elongation factor 1α (O'Donnell et al. 1998, Carbone and Kohn 1999), and Bt-2a and Bt-2b (Glass and Donaldson 1995) for amplifying β-tubulin. The sequences were found to be 99% to 100% identical to those of Nigrospora pyriformis in GenBank (accession numbers NR153469.1, KY019290.1, and KY019457.1, respectively). The sequences were uploaded to GenBank (accession numbers OK605048, OL362197, and OK662966), with lengths of 522 bp, 475 bp, and 410 bp, respectively. A maximum-likelihood phylogenetic tree was created in MEGA5. Pathogenicity was tested by four isolates hyphal inoculation and conducted in an experimental field of the White Lotus Science and Technology Expo Park. Five-millimeter discs were taken from infected and uninfected PDA plates and inoculated into five 1-month-old healthe lotus leaves using inoculations, three with the pathogen and an uninfected sample as the control every leaf. The discs were covered with moisturized sterile cotton and fixed with transparent tape. The wounds were moistened with sterile water and sealed with adhesive tape. After 14 days, spots were visible at the infected sites while the control sites showed no symptoms. The same pathogen was recovered from the infected leaves, fulfilling Koch's requirements. Leaf spot diseases resulting from N. pyriformis infection or infection with other Nigrospora species include infection of Chenopodium album by N. pyriformis (Chen et al. 2020), Eclipta prostrata by N. sphaerica (Qiu et al. 2022), Nicotiana tabacum by N. oryzae (Wang et al. 2022), and Oxalis corymbosa by N. hainanensis (Zheng et al. 2021). This investigation appears to be the first identification of Nelumbo nucifera vein disease resulting from Nigrospora pyriformis infection. The present results are useful for the management and avoidance of the disease caused by Nigrospora pyriformis.