LAUSR

Stiltgrass [Microstegium vimineum (Trin.) A. Camus], is an annual C4 grass of Asiatic origin whose native range includes India, Pakistan, Nepal, China, Korea, and Japan (Cole et al 2004). In China, it is mainly distributed south of the Yangtze River, and is one of the most important weeds in autumn-maturing dryland crops, orchards, tea gardens, and plantations. With its high shade tolerance, M. vimineum also invades forest understories and crowds out the local vegetation (Warren et al. 2011). From June to August 2019, a leaf disease was observed causing severe defoliation of stiltgrass on the roadside of Sun Yat-sen Mausoleum in Nanjing City, Jiangsu Province, China (32.045964°N, 118.840064°E). Yellow or yellow-brown necrotic spots were observed on leaf tips and margins of the lower canopy, which later expanded to the entire leaf and progressed up the plant. Disease incidence was approximately 75-85% in August. Thirty symptomatic leaves were collected, and tissue samples (5 × 5 mm) were surface disinfected with 75% ethanol for 30 s, 0.02% NaClO for 30 s, 75% ethanol for 30 s, and washed twice with sterile water. Disinfected tissues were placed on potato dextrose agar (PDA) and incubated at 28°C for 5 days. Twenty-seven morphologically similar isolates were obtained from the leaves and purified by single-spore culturing for further study. Colonies on PDA were 70 to 85 mm in diameter after 4 to 5 days, initially white becoming gray-green with flocculent aerial mycelia. Conidiophores were solitary or clustered, 85 to 139 µm long × 5 to 8 µm wide (n = 50), and conidia were obclavate to ellipsoid or spindle shaped, brown, and measured 28 to 37 µm long × 13 to 18 µm wide (n = 50) with three false dissepiments. All characteristics were consistent with the morphology of Curvularia intermedia Boedijn (Sivanesan 1987). The rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and translation elongation factor (TEF) of a representative isolate (JSNJ-2019) were amplified using primers ITS1/ITS4, GPD1/GPD2 and EF1-983F/EF1-2218R (Manamgoda et al. 2014). The ITS sequence of JSNJ-2019 (GenBank: MZ613310) showed 99.83% (582/583bp) identity with C. intermedia (GenBank: MF370184 and GU073102); the GPDH sequence (GenBank: MZ701795) showed 99.66% (581/583bp) identity with C. intermedia (GenBank: LT715828) and the TEF sequence (Genbank: OM282974) showed 99.77% (864/866bp) identity with C. intermedia (GenBank: MF370186). Phylogenetic analysis based on the TEF sequences using Maximum-Likelihood and Bayesian methods placed JSNJ-2019 in the same clade with reference strain C. intermedia B19. The isolate was deposited in China Centre for Type Culture Collection (CCTCC) (Isolate code: CCTCC AF 2022041). For the pathogenicity assay, ten healthy M. vimineum plants grown in plastic pots (five to six leaf stage) were sprayed with 20ml conidial suspension (5×104 spores /ml); another ten healthy plants sprayed with sterile water served as controls. All inoculated and control plants were covered with transparent polyethylene bags immediately and were maintained in a greenhouse at 28±1℃. The transparent polyethylene bags were removed after 24 hours. The pathogenicity test was repeated three times. Five days post-inoculation, inoculated plants showed leaf blight symptoms as observed in the field, whereas no disease symptoms was observed on control plants. Reisolations were performed from inoculated plants, and the reisolated pathogen was confirmed as C. intermedia inter based on morphological and PCR assay (Konstantinova et al. 2002). No pathogens were isolated from control plants. Host range tests showed, C. intermedia JSNJ-2019 was pathogenic on corn, wheat, sorghum, barnyardgrass, crabgrass, green foxtail, Chinese sprangletop, cynodon, cogongrass, goosegrass, purslane and bedstraw and non-pathogenic on barley, rice, oat, cotton, bean, peanuts, rapeseed, tobacco and tea. These findings suggest C. intermedia could be used as a biocontrol agent against invasive M. vimineum and farmland weeds. However, application of C. intermedia as a bioherbicide should be limited to insensitive crop growing areas.