First Report of Anthracnose Caused by Collectotrichum higginsianum on Rumex crispus in China.

Abstract

Rumex crispus L. is a perennial herb with medicinal properties derived from its roots and whole plant (Bhandari et al. 2022). In December 2022, symptoms of anthracnose were observed in approximately 40% of naturally occurring R. crispus plants in Longquan Reservior, Nanchang city (115°53' N, 28°43' E), Jiangxi Province, China. Initially, red lesions appeared randomly on various parts of the leaf blade, which gradually became dry and brown at the center, eventually leading to leaf death. To isolate the fungal pathogen responsible, ten symptomatic leaves were randomly collected and their lesions were cut into small pieces (4 × 4 mm). The leaf fragments were surface-sterilized in 70% ethanol for 45 s and then in 1% NaClO for 45 s. The leaf pieces were rinsed three times with sterile distilled water. The surface-sterilized leaf pieces were then placed onto potato dextrose agar (PDA) and incubated at 28 ℃, dark condition for 3 days. Twelve isolates were obtained, characterized by a milky white and uneven growth pattern with a white root-like structure branching out at the edge, along with scattered black deposits on the bottom of the plate. Conidiogenous cells cylindrical, smooth-walled, hyaline, 9.3-23.2 × 3.6-4.2 μm. Conidia elliptical, aseptate, smooth-walled, with one end blunt and the other truncate, ranging in size from 10.4 to 22.3 (mean 16.7) μm in length and 3.2 to 5.0 (mean 4.1) μm in width (n = 50), which are consistent with the characteristics of the members of Colletotrichum destructivum species complex (Damm et al. 2014). To accurately identify the strain, three representative isolates, namely JFRL 03-930, JFRL 03-931, and JFRL 03-935, were selected for further identification. The internal transcribed spacer (ITS) region, actin (ACT), chitin synthase (CHS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GADPH), histone3 (HIS3), and beta-tubulin (TUB2) genes were amplified and sequenced using specific primer pairs, including ITS5/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, GDF1/GDR1, GYLH3F/CYLH3R, and T1/Bt2b (Damm et al. 2014). All sequences were deposited in GenBank with accession numbers OQ560476-OQ560478 (ITS), OQ576154-OQ576156 (ACT), OQ576157-OQ576159 (CHS), OQ576160-OQ576162 (HIS3), OQ576163-OQ576165 (GADPH), and OQ576166-OQ576168 (TUB2). A maximum likelihood phylogenetic tree was constructed using IQtree v1.5.6 based on the combined ITS, ACT, CHS, GAPDH, HIS3 and TUB2 data set (Nguyen et al. 2015), The phylogenetic tree showed that the three isolates clustered with C. higginsianum in a clade with 91% bootstrap support. Based on morphology and molecular characters, the isolates were identified as C. higginsianum of the C. destructivum species complex. To confirm the pathogenicity, One-year-old R. crispus were collected from the wild and potted in an climate chamber. Six healthy leaves of R. crispus were surface sterilized with 70% ethanol and wounded by sterile needle, and a 20-μl conidial suspension (3×105 conidia/ml) of the isolate JFRL 03-931 was inoculated on the wound. Another set of six leaves of R. crispus was inoculated with distilled water as controls. The potted plants were incubated under conditions of 25 ℃ and 80% humidity. After 10 days, reddish brown spots were observed on all inoculated leaves, while the control leaves remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated leaves and confirmed as C. higginsianum by morphological and molecular analysis. It has been reported that C. higginsianum caused anthracnose disease on several cruciferous vegetables, Boehmeria nivea and Rumex acetosa in China (Damm et al. 2014; Wang et al. 2011; Patel et al. 2014; Zhang et al. 2018). But to our knowledge, this is the first report of C. higginsianum casued anthracnose on Rumex crispus in China. Therefore, we should pay more attention to this pathogen and develop appropriate control strategies.