First Report of Alternaria alternata Causing White Leaf Spot on Allium tuberosum in China.

Puding County is the major Allium tuberosum growing area in Guizhou Province of China. In 2019, white leaf spots were observed on Allium tuberosum in Puding County (26.31°N, 105.64°E). The white spots, ranging from elliptic to irregular in shape, first appeared on leaf tips. With disease aggravation, spots gradually coalesced, forming necrotic patches with yellow margins causing leaf necrosis; sometimes there was gray mold on dead leaves. The incidence of the diseased leaf rate was estimated to be 27-48%. To identify the pathogenic agent, 150 leaf tissues (5 mm × 5 mm) were obtained from disease-healthy junctions of 50 diseased leaves. Leaf tissues were disinfected in 75% ethanol for 30 s, soaked in 0.5% sodium hypochlorite for 5 min, and flushed three times with sterile water, before being placed on potato dextrose agar (PDA) in the dark at 25 °C. When colonies appeared, the mycelial tips were picked and placed on new PDA. Purified fungus was obtained after repeating this last step several times. The colonies were grayish-green with white round margins. Conidiophores (2.7-4.5 µm × 27-81 µm) were brown, straight, or flexuous with branches and septa. Conidia (8-34 µm × 5-16 µm) were brown, with 0-5 transverse septa and 0-4 longitudinal septa. The 18S nuclear ribosomal DNA (nrDNA; SSU), 28S nrDNA (LSU), RNA polymerase II second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (TEF-α) (Woudenberg et al. 2013) were amplified and sequenced. The sequences were deposited in GenBank (ITS: OP703616, LSU: OP860684, SSU: OP860685, GAPDH: OP902372, RPB2: OP902373, TEF1-α: OP902374). According to BLAST analysis, the ITS, LSU, GAPDH, RPB2, SSU, and TEF1-α of the straishowed 100% (689 of 731 base pairs; bp), 100% (916 of 938 bp), 100% (579 of 600 bp), 100% (946 of 985 bp), 100% (1093 of 1134 bp), and 100% (240 of 240 bp) sequence identity to those of Alternaria alternata (ITS: LC440581.1, LSU: KX609781.1, GAPDH: MT109295.1, RPB2: MK605900.1, SSU: ON055699.1 and TEF1-α: OM220081.1). A phylogenetic tree was constructed using PAUP4 and the maximum parsimony method with 1000 replicas of bootstrapping for all datasets. According to morphological characteristics and phylogenetic analysis, FJ-1 was identified as Alternaria alternata (Simmons 2007, Woudenberg et al. 2015). The strain was preserved in the Agricultural Culture Collection of China (preservation number: ACC39969). To determine the pathogenicity of Alternaria alternata against Allium tuberosum, wounded healthy leaves were inoculated with a conidial suspension (106 conidial/mL) and round mycelial plugs (4mm). Sterile agar PDA plugs with no mycelium or sterile water were inoculated as negative controls. Three days later, white spots appeared on the wounded leaves inoculated with mycelial plugs or conidial suspension. However, the symptoms caused by conidial suspensions were weaker than those caused by mycelial plugs. No symptoms were observed in the control group. The experimental symptoms were consistent with the phenomena observed in the field. The same fungus was reisolated from necrotic lesions and identified as Alternaria alternata using the method described above. To our knowledge, this is the first report of Alternaria alternata causing white leaf spots on Allium tuberosum in China, a disease seriously affected the yield and quality of Allium tuberosum and caused economic losses to farmers. Reference: Simmons EG (2007) Alternaria: an identification manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands. Woudenberg JHC, Groenewald JZ, Binder M, Crous PW ( 2013) Alternaria redefined. Stud Mycol, 75: 171-212. https://doi.org/10.3114/sim0015. Woudenberg JHC, Seidl MF, Groenewald JZ, Vries M de, Stielow JB, Thomma BPHJ, Crous PW (2015) Alternaria section Alternaria: Species, formae speciales or pathotypes? Stud Mycol, 82:1-21. https://doi.org/10.1016/j.simyco.2015.07.001.