Leaf Spot caused by Alternaria alternata on Macleaya cordata in China.

Macleaya cordata is a perennial herb that belongs to the Papaveraceae and is typically prescribed as a traditional antibacterial medicine in China (Kosina et al. 2010). The extract from M. cordata has been widely used in the manufacturing of natural growth promoters as an alternative to antibiotic growth promoters in the livestock industry (Liu et al. 2017), and the products are marketed in 70 countries such as Germany, China, etc (Ikezawa et al. 2009). During the summer of 2019, symptoms of leaf spot were observed on M. cordata (cv. HNXN-001) in two commercial fields (approximately 1, 300 m2 and 2, 100 m2) of Xinning county, Shaoyang City, Hunan Province, China, where approximately 2 to 3% of the plants were affected. The initial symptoms were irregular black and brown spots on the leaves. The lesions expanded and coalesced, eventually leading to leaf blight. Six symptomatic basal leaf sections from six plants from two fields were surface disinfested in 0.5% NaClO for 1 min, then 75% ethanol for 20 s, rinsed in sterile water three times, air dried, and placed onto potato dextrose agar (PDA), one dish for samples from a single leaf. Plates were incubated at 26°C in darkness. Nine strains with similar morphological characters were isolated, and one representative isolate ( BLH-YB-08) was used for morphological and molecular characterization. The colonies on PDA were grayish-green with white round margins. Conidia were typically obclavate to obpyriform, brown to dark brown, and 12.0 to 35.0 × 6.0 to 15.0 μm, and with 1 to 5 transverse septa and 0 to 2 longitudinal septa (n=50). Isolates were identified as Alternaria sp. on the basis of mycelial characteristics, color, and conidial morphology. To confirm identity of the pathogen, DNA was extracted from isolate BLH-YB-08 with the DNAsecure Plant Kit (TIANGEN, Biotech, China). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), actin (ACT), 28S nrDNA (LSU), 18S nuclear ribosomal DNA (SSU), histone 3 (HIS3), internal transcribed spacer (ITS) region of ribosomal DNA, and translation elongation factor 1-α (TEF) genes ( Berbee et al. 1999; Carbone and Kohn. 1999; Glass and Donaldson. 1995; White et al. 1990.) were amplified and sequenced. Sequences were deposited into the GenBank database. They were 100% sequence identity of GAPDH (OQ224996) with A. alternata strain AA2-8 (MH65578; 578/578bp), 100% sequence identity of RPB2 (OQ190460) with A. alternata strain SAX-WN-30-2 ( MK605877; 933/933bp), 100% sequence identity of ACT (OQ923292) with A. alternata strain FCBP0352 (OL830257; 939/939 bp), 100% sequence identity of LSU (OQ891167) with A. alternata XL14 (MG839509 ; 908/908 bp), 100% sequence identity of SSU (OQ139544) with A. alternata strain BJ19.4.1(OM736063; 1,067/1,067 bp), 100% sequence identity of HIS3 (MT454856) with A. alternata YJ-CYC-HC2 (OQ116440 ; 442/442 bp), 100% sequence identity of ITS (MT212225) with A. alternata CS-1-3 (OQ947366; 543/543bp), and 100% sequence identity of TEF (OQ190461) with A. alternata strain YZU 221185 (OQ512730; 252/252 bp). To test pathogenicity, the isolate BLH-YB-08 was cultured on PDA for 7 days to prepare conidial suspensions and the spore concentration adjusted to a final concentration of 1×106 spores/ml. The leaves of five potted 45-day-old M. cordata (cv. HNXN-001) plants were sprayed with conidial suspensions, and five control potted plants were wiped with 75% alcohol and washed five times with sterile distilled water. They were then sprayed with sterile distilled water. Plants were placed in a greenhouse at 25 to 30°C with 90% relative humidity. Pathogenicity tests were conducted twice. Fifteen days after inoculation, lesions were found on inoculated leaves, and the symptoms were the same as those in the field, whereas the controls were healthy. A fungus was consistently isolated from the inoculated leaves and identified as A. alternata by DNA sequencing of the GAPDH, ITS, and HIS3 genes, fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on M. cordata caused by A. alternata in China. Understanding its etiology may help to control this fungal pathogen, thus reducing economic losses. Funding: Hunan Provincial Natural Science Foundation General Project (2023JJ30341) Hunan Provincial Natural Science Foundation Youth Fund (2023JJ40367) Seed Industry Innovation Project of Hunan Provincial Science and Technology Department Special project for the construction of Chinese herbal medicine industry technology system in Hunan Province "Xiangjiuwei" Industrial Cluster Project of the Ministry of Agriculture and Rural Affairs.