First Report of Root Rot caused by Coniella fragariae on Paeoniae radix Rubra in China.

Abstract

Paeoniae radix Rubra is a traditional Chinese herbal medicine, which has the effect of clearing heat and cooling blood, activating blood and removing stasis. It has become popular in the Chinese market in recent years due to its extremely high medicinal value and showy flower color. In May 2021, typical symptoms of root rot were observed in a field (35°7'12″ N, 103°58'48″ E) in Dingxi, Gansu province, China. Approximately 10% of the plants in the field had typical root rot symptoms, and the root of each affected plant is at least 5% severe. The roots of the naturally infected plants in the field discolored and decayed with black brown spots on the surface of the root bark, the root bark detached from the phloem，and some leaves were chlorosis, shrunken and smaller, and the branches were dead and underdeveloped. In the transverse section, the xylem was black diffusion and abnormal odor. Three diseased plants with typical symptoms were chosen at random and brought back to the lab. Small pieces cut from the margins of lesions were surface disinfested with 75% ethanol for 15 s, and 0.5% NaClO solution for 30 s, rinsed three times in sterile distilled water, dried on sterile filter paper, plaed onto potato dextrose agar (PDA), and incubated at 25 ± 1℃ for 7 days in the dark. The pure cultures were obtained by single-spore isolation. All isolates produced wavy on the surface, radial from the inside out, initially white or milky white to orange colonies with abundant black brown oily conidiomata pycnidia on PDA at 25 ± 1℃ after 15 days in the dark. The conidiomata pycnidia is spherical to irregularly spherical, 231.5 to 512.4 μm, initially transparent with age turning brown, with a dark brown internal conidial mass inside, and with a 13.1 to 45.4 μ m wide ostiole central. Young conidia (n=100) developed from conidiogenous cells, which were simple, tapering, hyaline, smooth, and 12.3 to 18.0 × 2.5 to 4.6 μm, 1.0 to 1.5 µm wide at apex. Mature conidia (n=100) were ellipsoid, apices tapering, subobtusely rounded, brown, and 6.5 to 11.0 × 4.1 to 7.5 µm. The morphological characteristics of the isolates were consistent with previous descriptions of the genus Coniella (Crous et al., 2014). A representation isolate CS-1 was deposited in the Institute of Plant Protection, Gansu Academy of Agricultural Sciences and used for further studies. To confirm the identity of the causal fungus, the internal transcribed spacer (ITS), 28S large subunit of nuclear ribosomal RNA (LSU) and partial translation elongation factor 1-alpha (TEF1-α) gene of the representative isolate CS-1 were amplified and sequenced using primers ITS1/ITS4 (White et al., 1990), LROR/LR7 (Chethan et al., 2017) and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively, and deposited on GenBank with accession numbers OP824764 (ITS), OP824767 (LSU)/span>and OP903926 (TEF1-α). Blastn analysis of all sequences resulted in E-value of 0.0 (ITS and LSU) and nearly 0.0 (TEF1-α), with Query cover values of 90% to 99% identity with C. fragariae, confirming the hypothesis based on morphological features examination. To conduct a pathogenicity test, three root segments of healthy plants were wounded using sterilized needles and inoculated by pipetting 10 μL of conidial suspension (1×107 conidia/mL) onto each wound, and controls were inoculated with 10 μL sterile distilled water. These root segments were kept in a moist chamber at 25°C in the dark. The experiment was repeated three times. After 14 days, root rot symptoms were observed on all of the inoculated root segments and identical to those observed in the field, whereas control root segments did not develop symptoms. The pathogen was re-isolated from the lesions of inoculated root segments, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of C. fragariae causing root rot on P. radix Rubra in China. This identification can aid in the selection of appropriate management measures for this disease.