First Report of Leaf Spot Caused by Alternaria alternata on Artemisia stolonifera (Maxim) Komar. in China.

Abstract

Artemisia stolonifera (Maxim.) Komar., also known as the 'elite of Artemisia', is currently widely planted in Jiangxi, Hubei, and other provinces in China. Since April 2023, a leaf spot on Ar. stolonifera was observed in the total cultivation area of 33.33 ha in Zhangshu City, Jiangxi Province. The incidence was 50% to 60%. Small irregular blackish-brown spots first appeared on the leaves, some with chlorotic margins, which later joined into clumps. In severe cases, the leaves were completely necrotic, seriously affected the growth and quality of Ar. stolonifera. Small pieces (3 to 4 mm) were excised from the necrotic borders of 20 typical symptomatic infected leaves, disinfected with 2.5% sodium hypochlorite solution for 3 min, rinsed five times with sterile water, and placed on potato dextrose agar (PDA) medium, incubated at 25℃ for 5 days. 15 purified isolates with similar morphological characteristics were obtained by transferring hyphal tips (isolation frequency of 75%). The colonies on PDA initially were white, and then became olive green with a white rim. After 10 days of culture on potato carrot agar medium (PCA), the conidia were septate, light brown in color, with dimensions ranging from 10.82 to 30.59 × 7.12 to 11.97 μm (n=50). The culture and morphological characteristics corresponded to Alternaria spp. (Simmons 2007), and the representative isolates JNC01 and JNC02 were used for further identification. To further identify the isolates, the RNA polymerase II second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen (Alt a1), translation elongation factor 1-alpha (TEF-α) and histone 3 (HIS3) were amplified using primers RPB2-5F2/RPB2-7CR, ITS1/ITS4, gpd1/gpd2, Alt a1-F/Alt a1-R , EF1-728F/EF1-986R and H3-1a/1b, respectively (Glass and Donaldson 1995; Lawrence et al. 2013; Berbee et al. 1999; Hong et al. 2005). The subsequent sequences were deposited in GenBank (accession nos. PP101993 and PQ458469; PP350760 and PQ45545; PP409573 and PQ474679; PP746506 and PQ474680; PP375820 and PQ458468; PQ001738 and PQ458467 for JNCO1 and JNC02 respectively. NCBI BLASTn sequence analysis revealed that all sequences, except for HIS3 had 100% homology with those of the Al. alternata strain CBS 612.72 (RPB2: KP124777.1, ITS: KP124308.1, GAPDH: KP124165.1, Alt a1: KP123861.1, TEF-α: KP125084.1), meanwhile, HIS3 had 100% identity with the Al. alternata strain MRY1 (MK210171.1). Phylogenetic trees, constructed using concatenated sequences based on ITS, GAPDH, Alt a1, TEF and RPB2 genes, as well as one built solely with HIS3, both placed JNC01 and JNC02 in the Al. alternata clade with high confidence. To test pathogenicity, about 5 mL of conidia suspension (1×106 conidia/mL) of JNC01 was sprayed on five 1-year-old healthy seedlings, while five seedlings were sprayed with sterile distilled water as a control. All plants were covered with plastic bags and placed in a greenhouse at 25 ± 2℃. After 7 days, brown spots, some with chlorotic margins, were observed, similar to those observed in the field. The controls remained symptom-free. The pathogens reisolated from the diseased tissue were again identified as Al. alternata by both morphological and molecular identification based on ITS and HIS3. This study is the first report of Al. alternata causing leaf spot on Ar. stolonifera in China and provides vital information on the pathogen for further diagnosis and management of the disease.