First Report of Leaf Spot Disease On Microstegium vimineum Caused by Bipolaris setariae in China

Microstegium vimineum, a Poaceae annual C4 plant, occurred widely in crop fields, tea gardens, orchards, under forests and roadsides in most provinces and regions south of the Yellow River, China. It was introduced into the eastern USA causing ecological and environmental damage (Stricker, 2016). In October 2015, M. vimineum plants with leaf spots were observed on the roadside of Mingling Road (32.04521°E, 118.84323°N), Nanjing, China. In an early stage of disease development, light brown or brown, round or oval shaped lesions appeared on the upper surface of leaves. In a middle stage, the lesions gradually expanded and the edges of the diseased leaves were lightly curled. In a late stage, leaves were withered or curled and the entire plant died. Initial disease incidence was up to 85% among natural populations of the weed. Diseased leaves collected from field were surface disinfected (75% ethanol for 30s; 1% sodium hypochlorite solution for 30s; 75% ethanol for 30s; sterile deionized water for 1min) and placed on water agar (20g agar per liter) (Kleczewski et al., 2010). Plates were incubated in the dark at 28℃ for 3 days. Following incubation, leaves, spores and conidiophores were examined using light microscopy. Single spores were obtained by using the single-spore procedure, plating out a loopful of spores onto water agar, and then carving individual spores out with associated agar under a microscope. Single spores were isolated, plated onto MV-agar (30g M. vimineum leaves, 20g agar per liter), and placed under 365 nm wavelength black light. Fungal colonies were transferred onto PDA medium, after 4 days colonies measured between 83 to 86 mm in diameter, appeared flat and dark brown, with short, light gray aerial hyphae. Conidiophores were solitary or clustered, light brown to medium brown, with pale apical color and multiple septa. The upper part was usually geniculated, 5.5-9.5 μm wide. Conidia were light yellowish brown to medium yellowish brown, mostly fusiform, straight or curved, fusoid or navicular, often slightly curved, rarely straight, smooth, 5-9 (mostly 7) septa, 48-70×10-14.5 μm (average 57×12.5 μm); hilium slightly prominent, and truncated at the base. Through morphological observation, the fungus was preliminarily identified as Bipolaris sp.. Four to five seeds of M. vimineum were planted in pots (10 cm in diameter) filled with nutrient soil, placed in the greenhouse and watered regularly. Four pots were inoculated with a conidia suspension of 1×105 sp/mL, at 4-5 true stage. Inoculated seedlings were maintained under 80% humidity and 28℃ for 24h in the dark, and then transferred to a greenhouse. Three pots of uninoculated seedlings were used as controls. Two days after the inoculation, buff-colored, irregular-shaped spots appeared centered on leaf veins. Within a week, diseased leaves became crinkled and their edges were yellow to brown due to proliferation of the spots. By 15 days, large areas of brown spots appeared on the leaves, some leaves turned yellow-brown and severely curled, and 80% of the plants had died. The diseased symptoms were similar to that of the field sample. The fungus re-isolated resulted morphologically identical to the original isolate grown on PDA medium and used for inoculation, thus fulfilled Koch’s postulates. The CTAB method was used to extract DNA from isolates of diseased leaves taken directly from the field, and the internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase gene (GPDH) were amplified using primer pairs of ITS1/ITS4 and GPD/GPD2 (Manamgoda et al., 2014) respectively. The ITS amplified sequence (Genbank accession MW446193) shared 100% identity with the reference sequence of Bipolaris setariae (MN215638.1) and the GPDH amplified sequence (MW464364) shared 99.83% identity with the reference sequence of B. setariae (MK144540.1). Field experiments were conducted in Laboratory Base of Nanjing Agricultural University, where M. vimineum plants were planted. Spore suspensions with concentrations of 105, 104, 103, 102, and 101 sp/mL were prepared, distilled water was used for control, and there were four replicates of each treatment. Twenty four plots were randomly arranged, the experimental unit consisted of 50 to 60 plants in an area of 0.5m×0.6m. The interval distance between plots was about 20 cm so as to prevent the mutual influence among treatments. M. vimineum plants were inoculated at 3-4 true leaf stage. Inoculation was done at sunset, and 60 mL spore suspension was sprayed onto each plot. After spraying, the waterproof-breathable black cloth was used to cover the plots, and removed 36 hours later. The outdoor temperature was 20~28℃. After 10 days, the symptoms of M. vimineum were observed and the disease index was recorded. SPSS 20 software (SPSS Inc., Chicago, IL, USA) was used for variance analysis, and Origin 9.0 (OriginLab, Hampton, MA, USA) was used to calculate the half lethal concentration (ED50) and 90% lethal concentration (ED90) of the strain MLL-1-5 on M. vimineum. Symptoms appeared on inoculated M. vimineum seedlings immediately after dark treatment. Within a week, all seedlings inoculated with the highest spore concentration were dead. Plants sprayed with water remained healthy. ED50 and ED90 of the strain MLL-1-5 was 1.9×101 and 1.4×103 sp/mL respectively, which indicated aggressiveness of the strain MLL-1-5 B. setariae. After 28 days, infected M. vimineum plants did not recover. This is the first report of leaf spot disease on M. vimineum caused by B. setariae in China. M. vimineum is a widely distributed and extremely harmful weed in China and United States. No biocontrol agents against M. vimineum are currently available. B. setariae may have potential as a biocontrol agent against M. vimineum both in China and the United States.