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Bellis perennis L., commonly known as the daisy or sun chrysanthemum, belonging to the family Asteraceae, is a perennial herb and is usually used as an ornamental plant worldwide for its vibrant flowers. Simultaneously, B. perennis has been proved to have therapeutic effects used on common colds, wound healing, anti-tumor, anxiolytic and antioxidant (Karakas et al. 2017). In July 2021, typical leaf spot was observed on B. perennis with about 50% disease incidence in Ruyue lake wetland park of Zibo (36.71°N, 118.01°E), Shandong Province, China. We surveyed more than 1000 square meters of planting area, and the diseased leaves were mostly concentrated in the lower location of plants, where the humidity was higher under the forest. Symptoms on the initially diseased leaves appeared as light yellow, round or oval lesions with light or brown borders. With the development of the disease, the area of the lesion gradually expands, the color deepens, and the shape is becoming irregular. To identify the causal pathogen, small pieces of 15 tissues collected from the infected leaves were sterilized with 75% ethanol for 30 s and then 2% sodium hypochlorite (NaClO) for 60 s, finally rinsed with sterile water three times. All the tissues were placed on potato dextrose agar (PDA) and incubated at 25 ℃ in the dark for 5 days (Zhu et al. 2013). A total of 13 isolates were obtained from the above diseased leaves. The cultures were initially grayish white, then a light green halo appeared in the middle of the medium after 5 days, with numerous gray aerial hyphae. For molecular identification, the RNA polymerase II beta subunit (PRB2), Tsr ribosome biogenesis protein, partial coding sequences of chitin synthase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and major allergen Alt a 1 were amplified from genomic DNA extracted from four representative single isolates using the primers PRB2DF/PRB2DR, Tsr1F/Tsr1R, CHSDF1/CHSDR1, GDF1/GDR1, and AltF/AltR (Damn et al. 2019; Lawrence et al. 2013), respectively, and sequenced (GenBank accession nos. OL416000, OL416001, OL416002, OL416003, and OL416004). These genes had more than 99.9% nucleotide identity with the corresponding sequences (KY131957.1, KY131958.1, KY996470.1, MN657411.1, and KY923227.1) of the reference strains of Alternaria alternata in GenBank. For pathogenicity tests, five healthy B. perennis plants each with three living leaves were inoculated with mycelial plugs of A. alternata from a 5-day-old culture grown on PDA. After inoculation, the plants were placed in a greenhouse with 85% relative humidity at 25 ℃ and monitored daily for symptom development. After 3 days, all inoculated leaves with mycelial plugs of A. alternata appeared symptoms similar to those observed in the field previously, while no symptoms appeared on negative controls which were inoculated with sterile PDA plugs. Cultures re-isolated from diseased leaves had the same morphological and molecular results as those isolated in the field, confirming Koch's postulates. The causal agent on B. perennis was confirmed as A. alternata on the basis of morphological and molecular results (Simmons 2007). To our knowledge, this is the first report on the presence of A. alternata affecting B. perennis plants in China. The discovery of this new disease is beneficial to the application and protection of B. perennis, which is a popular landscape and medicinal plant.

Gaillardia pulchella Foug., belonging to the family Asteraceae, is an annual herb commonly seen in tropical America and China. It is often used as ornamental flowers because of its bright color, long flowering period and simple cultivation and management. In June 2021, leaf spot on G. pulchella with ∼40% disease incidence was observed in Laoshan scenic spot of Qingdao, Shandong Province, China. Initial symptoms on leaves appeared as light yellow to brown round or oval spots with dark brown borders, and the lesion area gradually expanded and the color deepened with the development of the disease. Small tissue samples collected from the infected lesions were surface-sterilized with 70% ethanol for 30 s, then rinsed with 2% sodium hypochlorite (NaClO) for 60 s, and finally rinsed with sterilized water three times. All the samples were transferred to potato dextrose agar (PDA) medium and incubated at 25℃ in the dark for 5 days (Zhu et al. 2013). A total of 9 isolates were obtained from the 11 selected tissues of symptomatic leaves. Afterward, all the single spore isolates were transferred onto potato carrot agar (PCA) plates (Mirkova 2003). After 7 to 10 days of incubation on PCA at 25℃ in the dark, colonies had a cottony mycelium with round margins, colored in white to gray. To test pathogenicity, six healthy G. pulchella plants were inoculated with mycelial plugs of the above pure cultures from a 7-day-old culture grown on PCA, while six germfree PCA plugs were served as negative controls. All the inoculated plants were set in greenhouse incubator at 25℃ and 80% relative humidity. Following 5 days incubation, brown spots began to appear on the sites of all inoculated leaves with mycelial plugs, while all the negative controls inoculated with sterile PCA plugs remained healthy. Infected lesions were separated and cultured as the same as those isolated in the field, and the same isolate was again microscopically identified, fulfilling Koch's postulates. 5 isolates were characterized, the colony margins of single spore isolate were round with gray or black aerial mycelia. Conidia were clustered and unbranched with 1 to 4 septa, colored in light or dark brown, shaped in obclavate or ellipsoid with short conical beak at the tip, dimensions varied from 14 to 51 μm (length) × 4.5 to 11 μm (width). The described morphological characteristics were consistent with Alternaria alternata (Simmons 2007). For further identification of molecular characterization, the genes of Chitin synthase (CHSD), RNA polymerase II second largest subunit (PRB2), Tsr1 ribosome biogenesis protein (Tsr1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained by PCR amplification with the primer pairs CHSDF1/CHSDR1, PRB2DF/PRB2DR, Tsr1F/Tsr1R and GAPDHF1/GAPDHR1 (Damn et al. 2019; Lawrence et al. 2013), respectively. The sequenced genes (GenBank accession nos. ON660874, ON660875, ON660876 and ON660877) had more than 99% nucleotide identity with the corresponding genes (GenBank accession nos. KY996470.1, MN304718.1, KY996472.1 and MN158133.1) of the reference strains of A. alternata in GenBank, and the re-inoculated and re-isolated strains have the same results which were repeated three times. The causal agent occurred on G. pulchella was identified as A. alternata based on the morphological and molecular characteristics. To our knowledge, this is the first record causing leaf spot on G. pulchella by A. alternata in China.