First Report of Alternaria alternata Causing Leaf Blight on Clematis terniflora var. mandshurica in China.

Abstract

Clematis terniflora var. mandshurica (Rupr.) Ohwi, a traditional Chinese medicinal plant, is widely distributed in northeast China, such as Heilongjiang, Jilin and Liaoning provinces. In August 2019, C. terniflora var. mandshurica (Rupr.) Ohwi leaf blight was found in Harbin (45.72°N, 126.68°E), Heilongjiang, China. The incidence was up to 90% on the 0.4 ha plantation, and almost every plant leaf was withered. Initial symptoms were chlorosis at the leaf apex or margin, and the leaf apex gradually formed brown lesions. As the infection progressed, the lesions expanded and leaves withered. Ten symptomatic leaves were randomly collected from ten plants in Clematis plantation. The leaves (5mm×5mm) between symptomatic and healthy tissue were cut and disinfected with 75% ethanol for 1 min and 1% NaClO for 2 min, rinsed three times in distilled water, plated on potato dextrose agar (PDA: the diameter of Petri dish is 90 mm) and incubated for 7 days at 25℃, further purified by single-spore isolation method. The morphology of all the ten isolates was similar. The colonies were round or sub round, 60-68 mm diameter in size, velvety, brownish green with distinct concentric rings. Conidiophores were solitary or clustered, suberect, brown, 30.3-82.4×2.5-5 μm (n=150) in size, and with septum. Conidia were solitary or in chains, smooth surface, obclavate, ellipsoid, brown, 12.5-46.25×10-16.25 μm (n=150) in size, with 2-7 transverse septa, 0-3 longitudinal septa. Conidia were produced continuously and extended in a synaxial manner. Based on the morphological characteristics, the isolates were identified as Alternaria alternata The morphological characters matched those of Alternaria alternata (Simmons 2007). Two representative strains TXL0816 and TXL0916 were used for molecular identification. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (tef1), RNA polymerase second largest subunit (RPB2), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS1/ITS4 (Iturrieta-González et al. 2020), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), Alt-for/Alt-rev (Hong et al. 2005), gpd1/gpd2 and EF1-728F/EF1-986R (Nishikawa and Nakashima 2020). The obtained sequences were deposited in the GenBank (ITS: MW403865 and MT508666, GAPDH: MZ298638 and MZ298639, tef1: MZ298640 and MZ298641, RPB2: MW432545 and MT510746, Alt a 1: MW432544 and MT501719). The phylogenetic tree of combined sequences showed that isolates TXL0816, TXL0916 and A. alternata CBS916.96 were clustered together with 100% bootstrap values. clustered into one branch supported with 100% bootstrap values. To verify pathogenicity, twenty leaves of eight plants were sprayed with spore suspensions (1×106 spores /mL) of the 7 days-old isolates TXL0816 and TXL0916 (four plants were used as replicates for each isolate)each isolate infected four plants). As a control, four plants were sprayed with sterile distilled water. The plants were incubated at 25℃ in a greenhouse. Twenty days after inoculation, brown lesions appeared on the leaf apex. The pathogenicity test was repeated three times. The same fungi were re-isolated from the inoculated leaves, The fungi were re-isolated from inoculated leaves with the same morphological and molecular traits as A. alternata, fulfilling Koch's postulates. No fungi were isolated from the control group. To our knowledge, this is the first report of A. alternata causing leaf blight on C. terniflora var. mandshurica (Rupr.) Ohwi. This study provides a basis for the pathogenesis of the leaf blight on C. terniflora var. mandshurica (Rupr.) Ohwi, and helps to formulate control measures to reduce future economic losses.