First report of Neophysopella vitis causing leaf rust on Parthenocissus semicordata in Guangdong Province, China.

Abstract

In April 2022, leaf rust disease of Parthenocissus semicordata was discovered in the urban greenbelt in Guangzhou city, China (23.06°N, 113.16°E). The disease incidence was 70% and disease severity was 75%. Chlorotic spots and red-brown necrotic flecks were present on the upper surface of infected leaves, and orange uredinia were distributed on the lower surface (Fig. 1 a-c). Two representative disease plants were collected as voucher specimens and dried, then deposited in Mycological Herbarium of the Zhongkai University of Agriculture and Engineering (MHZU GR0413, MHZU GR0414). Microscopic examination of the pustules of the samples revealed the presence of uredial paraphyses and urediniospores (Fig.1 d-f). Uredial paraphyses were hyaline, incurved, length 20-25 μm and dorsal wall 4.5-8.5 μm thick. Urediniospores were subglobose to ovoid, 16-24.5 × 10.5-17 μm. The wall of the urediniospore was hyaline or pale yellow, echinulate, and 1.0-2.0 μm thick. Telial structures were not observed. The morphological characteristics of uredial paraphyses and urediniospores were consistent with Yoshitaka's description of Neophysopella vitis (Yoshitaka 2000). Ten uredinia were picked using a sterile tweezer and the FlaPure Plant DNA Extraction Kit (Genesand Biotech Co., Ltd) was used to extract genomic DNA. Primers ITS4-rust (5'-CAGATTACAAATTTGGGCT-3') /ITS 5u (5'-CAAGGTTTCTGTAGGTG-3') were used to amplify internal transcribed spacer (ITS) and Rust2inv (5'-GATGAAGAACACAGTGAAA-3') /LR6 (5'-CGCCAGTTCTGCTTACC-3') to amplify large subunit (LSU) rDNA regions (Zhao et al. 2021). The sequences were submitted to NCBI (ITS: OQ991182 OQ991183, LSU: OQ979612 OQ979613), and blastn analyses of ITS and LSU showed 99.81% and 99.84% identity with the sequences from N. vitis (ITS: OQ304336, LSU: OM420266), respectively, found in GenBank. ITS combined with LSU sequences from the current study and reference sequences from Zhao et al. (2021) were used to construct Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees using CIPRES website. ML and BI phylogenetic trees showed that the sequences from the current study grouped with N. vitis, with bootstrap support and posterior probability of 98% and 1.0, respectively (Fig. 2). Morphological and molecular analyses confirmed that the rust fungus was N. vitis. In the pathogenicity test, urediniospores were picked from fresh diseased leaves with sterile tweezers, prepared into spore suspensions (1.0 × 106 urediniospores/ml), and sprayed on a one-week-old leaf from three healthy of potted plants of P. semicordata, while three other healthy leaves were sprayed with sterile water as control. Each treated leaf was wrapped in a plastic bag and incubated in the dark at 25℃ for 48 h after which plastic bags were removed and the plant were moved to a greenhouse at 25℃. At 15 days after inoculation, symptoms and the morphology of urediniospores were confirmed to be the same as those observed in the field collection and no symptoms or uredinia were observed in the control group (Fig.1 g, h). Yoshitaka first discovered leaf rust on P. semicordata caused by N. vitis in Nepal(Yoshitaka 2000). To our knowledge, this is the first report of N. vitis causing leaf rust on P. semicordata in China. P. semicordata is often used as an ornamental plant for exterior wall decoration in urban landscaping and court. The occurrence of this disease can lead to the decline of the P. semicordata leaves and the impairment of the plants aesthetically.