First Report of Leaf Necrosis on Microstegium vimineum Caused by Bipolaris microstegii in Maryland.

Abstract

Japanese stiltgrass (JSG, Microstegium vimineum) is an invasive weed causing significant ecological changes in the United States. Severely diseased plants in a shaded location 2 × 4 m in size were discovered in August 2012 at a residence on Indian Springs Rd., Frederick, MD (39.46747° N, 77.46106° W). JSG in larger monoculture stands at sunny locations within 6 to 10 m of diseased plants had a few, small, necrotic spots. Diseased plants had leaves with brown, often large, elliptical, necrotic spots up to 0.5 × 1.5 cm. Lesions were surrounded by a diffuse chlorotic margin, and larger lesions had tan centers. Diseased plants were smaller in stature than neighboring, non-symptomatic plants. Symptoms were similar to those on JSG reported by Kleczewski and Flory (2). Field samples of diseased leaves in moist chambers at room temperature and lighting produced dematiaceous conidiophores and conidia typical of Bipolaris within 2 days. Subcultures of the isolate (FDWSRU 12-049) were made from the conidia. Cultures growing on modified potato carrot agar (broth from 140 g each of potatoes and carrots and 20 g agar in 1 L water), were gray, velutinous or tomentose, and had clumps of short aerial hyphae on the upper surface. Healthy plants were grown in potting soil from seeds collected at the disease site. A minimum of five 4-week-old plants were spray-inoculated in each of three replications by conidia from detached leaves in a suspension of 105 spores/ml, given a 16-h dew period at 25°C, and placed in a 25°C greenhouse for observation. Non-inoculated plants were included for comparison. Brown or dark tan necrotic, irregular, often linear, spots with entire margins developed on all inoculated individual plants and not on control plants. The Bipolaris species was recovered from inoculated plant samples incubated in moist chambers, thus fulfilling Koch's postulates. Conidia were produced sympodially on dematiaceous conidiophores, often in clusters of two to three spores at the terminus, were medium to dark brown, straight or slightly curved, nearly fusiform with obtuse apices, and typically had 8 to 10 distoseptate cells that were 74.8 ± 2.3 × 16.4 ± 0.3 μm, and Q = 4.6 ± 0.1 (mean ± ci, P = 0.05; n = 100). A sequence of the ITS1-5.8S-ITS2 region of DNA, extracted using a DNeasy Plant Mini Kit (QIAGEN), was 100% identical to that of the type specimen of B. microstegii from M. vimineum (BPI 883727; GenBank Accession No. JX089579), using BLAST. On the basis of fungal morphology and molecular characteristics (1), along with symptomatology and published information (2), the causal agent of this disease has been determined to be B. microstegii. Dried specimens of both the isolate and diseased leaves were deposited in the U.S. National Fungus Collections (BPI 892680) and sequence information was submitted to GenBank (KF150215). Bipolaris leaf spot was also found on JSG in Howard and Prince Georges counties, MD, in 2009, but the causal agents were not formally characterized (K. Rane, Univ. Maryland; personal communication). This is the first confirmed identification of B. microstegii on JSG in Maryland, a plant that occurs as extensive monocultures in natural areas. These results provide a basis for characterization of this disease in the mid-Atlantic region.