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Presence of Aspergillus tubingensis Causing Leaf Spot Disease of Helleborus Species in Shanghai, China

Liaquat, F., Munis, M. F. H., Arif, S., Che, S., Liu, Q.
Plant disease 2019 v.103 no.4 pp. 766
Aspergillus tubingensis, DNA, Helleborus niger, Helleborus thibetanus, calmodulin, canopy, conidia, culture media, cut flowers, ethanol, fungi, genes, genetic databases, greenhouses, internal transcribed spacers, leaf spot, leaves, medicinal plants, ornamental plants, pathogenicity, pathogens, plant spines, sequence analysis, sodium hypochlorite, tubulin, China, Europe, United States, West Asia
Genus Helleborus is well known for its medicinal plants and ornamental cut flowers in different parts of the world. This genus belongs to the family Ranunculaceae, and about 22 species are present in Europe, West Asia, and the United States (Shiraishi et al. 2011). In February 2018, brown leaf spot symptoms were detected on leaves of two species of hellebores, namely, Helleborus thibetanus and H. niger, in the greenhouse in Shanghai, China (N31°02′53.99″, E121°29′30.4″). Disease distribution was observed on 80% of the plant population. The brown spots were more prevalent on middle and upper leaves of plant canopy. Early symptoms appeared in the form of small brown spots (1 to 3 mm), and later, the leaves withered and dropped off the plant. Symptomatic leaves were collected, washed with sterile water, surface sterilized in 75% ethanol for 30 s and 1% sodium hypochlorite for 3 to 5 min, and washed again with sterile water three times. Leaf fragments (approximately 5 × 5 mm) were aseptically cut from the margins of the lesions, placed onto potato dextrose agar (PDA), and incubated at 25 ± 1°C. A colony about 6 to 7 cm in diameter appeared on PDA after 5 days, and it was black at the center and white at the edges. Under a compound microscope, each conidiophore appeared yellow, and the vesicles were not septate. The globose and yellow vesicle was covered by a layer of phialides, which were 34 to 36 μm in diameter. The phialides were greenish and 22 to 34 μm long. The conidia, which were 2.4 to 4.8 μm in diameter on each phialide, were globose and dark brown, and their surface was smooth initially and gradually produced some thorns. The sizes of the vesicles and conidia were measured from 50 random samples of each. Identity of the fungus was further investigated by extracting DNA from one of the isolates, named BSZ-6(2), and sequence comparison of the internal transcribed spacer (ITS) gene (primers ITS1/ITS4), the β-tubulin gene (primers Bt2a/Bt2b), and the calmodulin (CaM) gene (primers CMD5/CMD6) was performed. BLASTn analysis of the ITS gene (MF379660.1), β-tubulin gene (KY608858.1), and CaM gene (FR751422.1), obtained with cognate sequences available in GenBank database, revealed 100% sequence identity to Aspergillus tubingensis. These morphological and molecular characteristics of the isolate AT_01 were consistent with those of A. tubingensis (Guo et al. 2017; Shiraishi et al. 2011). Pathogenicity of the isolate was confirmed by placing PDA plugs from 15-day-old cultures on eight wounded and eight unwounded leaflets in each of six Helleborus samplings. As a control, uncolonized PDA plugs were placed onto an equal number of wounded and unwounded leaflets. All the inoculated leaflets presented symptoms, whereas control leaflets remained healthy. The pathogen was recovered from symptomatic leaflets and was found to be morphologically identical to the inoculated one. To our knowledge, this is the first report of A. tubingensis casing leaf spot disease on any Helleborus species. This leaf spot disease can significantly affect the growth of this economically important medicinal and ornamental plant.