First Report of a Rust Disease on Bletilla striata Caused by Coleosporium bletiae in Anhui province of China.

Abstract

Bletilla striata is a perennial Orchidaceae which is widely distributed in Anhui, Hubei, Guizhou, Guangxi, Sichuan, Yunnan and other provinces in China (He et al. 2017). It has the effects of convergence, haemostasis, clearing away heat and detoxification, reducing swelling and generating muscle (He et al. 2017; Xu et al. 2019). With the reduction in wild resources of B. striata, cultivation can promote the effective conservation of B. striata germplasm resources. Fusarium root rot, leaf spot disease, and other diseases have been reported on this plant in China (Wang et al. 2022, Xu et al. 2022, Zhou et al. 2022). Coleosporium bletiae on B. striata has been reported in Guizhou, Sichuan, and Hubei provinces in China, with frequent occurrence and serious disease incidences in Guizhou province (Xu et al. 2022). In April to November 2021, rust was found in Anhui Bletilla planting base (30°09' to 30°46'N; 115°45' to 116°30'E). Approximately 17% of the B. striata plants showed disease symptoms (Figure S1). Uredinia are hypophyllous, scattered, round, powdery, and are associated with chlortic spots 1-4 mm wide on the upper side of the leaves. After mid-October, the temperature gradually decreased, and reddish-brown telia appeared around uredinia in circular shape. A total of 60 samples with typical symptoms were collected. Urediospore and teliospores were obtained by sterile needles, and their sizes were observed and measured under a microscope with sterile water. Urediospores were orange‒yellow, mainly oval in shape, 27.2-36.8 µm× 17.6-22.8 µm (n=60), and uniformly covered by verrucae on the surface (Figure S2 a-b). Teliospores were clavate, with a smooth surface and golden yellow contents, and gradually narrowed from top to base, ranging in size from 85.33-109.66 μm× 15.27-20.35 μm (n=60) (Figures S2 c-e). Three samples with typical symptoms were selected; approximately 200 μg of Urediospore of the rust pathogen were colleced per sampleand placed in 1.5 mL sterile centrifuge tubes for genomic DNA extraction by the CTAB method (Xu et al. 2022). The internal transcribed spacer (ITS) region of rDNA and large subunit (LSU) RNA genes were amplified using primer pairs ITS1/ITS4 (Leclerc et al., 2000) and NL1/NL4 (Zhang et al. 2012), respectively. The obtained sequences were deposited in GenBank (OP363678 for ITS and OP363683 for LSU), which were 100% identical with 100% coverage to the ITS sequence (MN108161, locality: Hubei) and the LSU sequence (KX386038) of Coleosporium bletiae. Based on morphological characteristics and DNA sequences, the isolates were identified as C. bletiae (Figures S2 and Figure S3). To confirm pathogenicity, 2-year-old healthy plants of B. striata were sprayed with 5 mL of urediospore suspension (2.6×105/mL). Plants sprayed with sterile distilled water were used as control. The test was conducted in an artificial climate incubator at 25 ± 1°C with a 12 h light/12 h dark photoperiod (relative humidity = 90%). The experiment was repeated three times. Two weeks after inoculation, typical yellowish chlorosis spots appeared on the leaf surface and a few yellow spore piles appeared on the leaves, which was consistent with the field symptoms (Figure S4). Control plants remained healthy. To our knowledge, this is the first report of C. bletiae causing rust on B. striata in Anhui province of China. Correct identification of this disease is of great importance to develop management strategies to control the disease. There may be seedling transport and spore transmission between Hubei and Anhui.