First Report of Leaf Spot on Chaste Tree Caused by Alternaria alternata in China

Abstract

Chaste-tree (Vitex agnus-castus Linn.) is a perennial ornamental shrub that is native to Europe, which has been widely distributed in China. Since 2021, a serious leaf spot on chaste-tree leaves was observed in Nanjing Botanical Garden, Jiangsu Province, China (31°14′6″N, 118°22′12″E). The disease incidence on the leaves ranged from 20 to 40%. The disease symptom initially appeared as irregular small gray spots on leaves that gradually coalesced into larger lesions with diseased leaves turning black and withering. From August of 2021 to 2022, small pieces of leaf tissues (5×5mm) from the necrotic borders of five typical symptomatic infected leaves were collected and surface sterilized (with 75% ethanol), then incubated in darkness at 25°C for 7 days. A total of fifteen isolates were obtained by monosporic isolation (isolation frequency of 76%). The fungal colonies were initially grayish-white and turned into dark gray with abundant cotton-like aerial hyphae. Microscopic observations revealed light-brown conidia that were obclavate or obpyriform (inversely pear-shaped) with length between 10 and 20 µm (mean 13.3 ± 2.4 µm) and widths between 5 and 10 µm (mean 7.8 ± 1.2 µm), 2 to 4 transverse septa and 0 to 2 longitudinal septa per conidium (n=30) were observed. The fungus was identified as Alternaria alternata based on the colony characteristics (Simmons 2007) and the representative isolate Aa1 was used for further studies. To further identify Aa1, the region of internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (EF-1a) and RNA polymerase second largest subunit (RPB2) genes were amplified from genomic DNA and sequenced with the primer pairs ITS1/ITS4 (Jayawardena et al. 2019), EF-728F/EF-986R (Carbone and Kohn 1999), Gpd1/Gpd2 (Berbee et al. 1999) and RPB2-5F/RPB2-7cR (Liu et al. 1999) respectively. Sequences were deposited into GenBank (Accession No. OQ626644 and OQ630494-OQ630496), which showed 99.2 to 100% sequence homology with those A. alternata strains in GeneBank (ITS, MN394880; GAPDH, MN410920; EF-1a, MN410916; RPB2, MN410918). The multigenes phylogenetic analysis revealed that isolate Aa1 and Alternaria alternata TCS3002 + CBS 916.96 clustered within the same clade with 99% bootstrap support. To test pathogenicity, conidial suspension (1×106 spores/ml) of Aa1 was sprayed uniformly across the leaves of three 1-year-old healthy chaste-tree seedlings; sterilized distilled water sprayed on other trees were used as negative control and the experiment was repeated three times. All inoculated plants were kept in same condition (25°C, under a 16 h/8 h photoperiod and 70% relative humidity). One week later, black/gray spots were observed on the leaves of inoculated plants, similar to the symptoms that were observed on the original diseased plants, while controls remained asymptomatic. Cultures were re-isolated from the infected leaves and were again identified as Aa1 by both morphological characteristics and DNA sequence analysis. The pathogen reported here has a broad host range, and has also been reported on Magnolia grandiflora L. (Liu et al. 2019), Kalanchoe pinnata (Sanahuja et al. 2018) and Kadsura coccinea (Zhang et al. 2020) to cause leaf spot. To our knowledge, this is the first report of A. alternata causing leaf spot disease on chaste-tree and provides an important reference for further biology and epidemiology research.