The identification and first report of Alternaria alternata causing leaf spot on Gaillardia pulchella Foug. in Shandong province of China.

Abstract

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.