First report of leaf blight on Ligusticum jeholense (Nakai et Kitagawa) Nakai et Kitagawa caused by Alternaria alternata in China.

Abstract

Ligusticum jeholense (Nakai et Kitagawa) Nakai et Kitagawa is one of the sources of Chinese herb "Gao-Ben". It is widely distributed in the Northeastern China. L. jeholense has antipyretic, antibacterial and anti-inflammatory effects (Zhang et al. 2021). In September 2021, a serious leaf blight was found in a 1.2 ha plantation of L. jeholense in Harbin, Heilongjiang Province, and the incidence was about 85%. The foliar symptoms were grayish-brown lesions, surrounded by a yellow margin at the edge of the leaf. In serious cases, the lesions extended into the middle of the leaf, and finally the whole leaf withered. A total of 12 samples (5×5mm) from symptomatic and healthy junction of 12 infected leaves from 6 different plants of L. jeholense with typical symptoms were cut and surface disinfected in 75% ethanol, and with 7% NaClO for 1 min, then rinsed three times with sterilized water. These tissues were placed onto Potato dextrose agar (PDA) plates at 28℃ in the dark. The colonies cultured for 7 days were obtained and transferred onto new PDA and potato carrot agar (PCA) plates by single spore method to further purify. After 7 days, the colonies on PDA were 63 to 75 mm in diameter, circular, grayish, with white aerial hyphae on the edge, the back of the colonies were grayish green. A total of 150 conidia on PCA were single or in chains, ovoid, inverted pear, 2 to 6 transverse septa, 0 to 3 longitudinal or oblique septa, 16.5 to 67.5 × 8.5 to 20.5 μm. The beaks were conical or cylindrical, 2.5 to 25.3 × 2.0 to 3.0 μm. Conidiophores were grayish brown, erect or bent, separated, 57.0 to 137.0 × 5.1 to 13.7 μm. Morphological characteristic showed the 12 isolates were the same fungus and similar to Alternaria sp. (Simmons 2007). Two typical strains (LGB and LGB2) from twelve isolates were randomly selected for molecular identification. Genomic DNA was extracted from mycelia of two isolates on PDA by modified CTAB method, and internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit (RPB2) and Alternaria major allergen (Alt a 1), translation elongation factor 1-alpha (TEF) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified and sequenced with the primers ITS1/ITS4, RPB2-5F2/RPB2-7CR, Alt-F /Alt-R, TEF-F/TEF-R and gpd-F/gpd-R (Woudenberg et al. 2015). The obtained sequences were deposited in GenBank (ITS: OM319506, OM943431; RPB2: OM393721, OM984854; Alt a 1: OM649816, OM984853; TEF: OM238108, OM984852; gpd: OM296228, OM984851). The phylogenetic analysis of maximum-likelihood tree by MEGA7 showed the LGB and LGB2 had 100% identity with A. alternata CBS 916.96. For pathogenicity test, conidial suspension (1 × 106 spores/mL) of the strain LGB and LGB2 was sprayed on 10 healthy 40-day-old L. jenholense plants and five plants with sterile water as control. The plants were incubated at 25℃. After 28 days, grayish withering appeared on the leaves. The test was repeated three times. The same fungi were re-isolated from the inoculated leaves and with the same morphological and molecular characteristics as A. alternata, fulfill the Koch's postulates. No symptoms and fungi were found in the control group. This is the first report of leaf blight on L. jenholense caused by A. alternata. Leaf blight could reduce the yields of L. jenholense. This study provides a reference for the prevention and treatment to the leaf blight of L. jenholense.