First report of anthracnose caused by Colletotrichum kahawae on Hypericum chinensis in China

Abstract

Hypericum chinensis is growing in popularity amongst consumers in cut-flower and pop-flower market as an ornamental woody plant for its florid berry and colorful flower. In August 2019, a new leaf spot disease was observed on H. chinensis in three commercial nurseries in Kunming (25°05′N, 102°72′E), Yunnian province, China. Disease symptoms were observed on approximately 40% of the plants one year after planting and 30% of the leaves were infected. Leaf symptoms began as small, water-soaked lesions on young leaves which later became larger, dark brown and necrotic. The lesion size ranged from 0.2 to 2.8 cm in diameter. For pathogen isolation, three samples of symptomatic leaves were collected from four different nurseries. The leaves were cut into 0.5 mm pieces, surface sterilized using 70% ethanol for 30 s, and 3% NaOCl for 5 min, rinsed three times in sterilized distilled water and plated on potato dextrose agar (PDA) (Zhou et al. 2023). The plates were incubated at 26°C in the dark for 3 days. Eight isolates with comparable morphological characteristics were obtained. Initially, colonies produced pale gray to white aerial mycelia, turning dark gray after 5 days. The isolates produced hyaline, single celled, straight and cylindrical conidia, with mean size 9.7 to 14.8 μm long × 3.7 to 5.6 μm wide (n = 100). Morphological characteristics were consistent with Colletotrichum sp. (Bailey and Jeger 1992). For molecular analysis, genomic DNA was extracted from three representative isolates (XSD1, XSD3 and XSD5), amplified using the primers ITS1/ITS4 (Yin et al. 2012) and T1/Bt2b (Glass and Donaldson 1995) and submitted to sequencing (Weir et al. 2012). DNA sequences of the isolates XSD2, XSD3 and XSD8 were identical. DNA sequences of a representative isolate XSD2 were deposited in GenBank (accession no. MW202334 for ITS, and OR347007 for TUB 2). MegaBLAST analysis of the ITS and TUB2 sequences showed 99.5% and 99.3% similarity with C. kahawae strain ICMP 18539 (accession no. NR_120138.1 for ITS) and strain IMI319418 (JX145227.1 for TUB 2). Pathogenicity tests were conducted by inoculating the pathogen on healthy mature leaves of H. chinensis in the field. Ten leaves (two leaves/plant) were inoculated by spraying conidial suspension (106 spores/ml) of isolates XSD1, XSD3 and XSD5, and covered with plastic bags to maintain high humidity for 48 hours, respectively. Leaves treated with sterile distilled water served as a control. All inoculated leaves showed symptoms similar to those observed in the field at 23±5°C 10 days after inoculation. No symptoms developed on non-inoculated leaves. The pathogen was re-isolated from inoculated diseased leaves and identified as C. kahawae based on morphological and molecular characters. C. kahawae has been reported to cause leaf spot on cultivated rocket in Italy (Garibaldi et al. 2016), and anthracnose disease on tree tomato in Colombia (Rojas et al. 2018), to our knowledge, this is the first report of C. kahawae causing anthracnose on H. chinensis worldwide. Due to important ornamental and economic value of H. chinensis, the distribution of C. kahawae needs to be investigated and monitored for effective disease management strategies to be developed.