First Report of Foxtail Millet Seedling Damping-Off Caused by Binucleate Rhizoctonia AG-A in China.

Abstract

Foxtail millet (Setaria italica) is an important food and fodder grain crop in China. In June 2013, foxtail millet seedlings of cultivar Jigu19 showing symptoms of damping-off were observed with about 30% incidence in Handan, Hebei Province. Infected plants showed pale brown lesions on the stems and brown discoloration of the roots, which later turned into root rot. As a consequence, infected seedlings wilted and died prematurely. Segments of the diseased root tissue (5 mm long) were washed with sterile water, disinfected with 0.5% NaOCl for 2 min, rinsed with sterile water, placed on potato dextrose agar (PDA), and incubated at 25°C in the dark. Three isolates were obtained and designated HD-1, HD-3, and HD-5. Morphological characteristics of the three isolates were similar with white colonies bearing large amounts of floccose aerial hyphae and no production of sclerotia after 14 days. Hyphal cells were stained with DAPI and all the three isolates were binucleate. Total genomic DNA was extracted from the mycelial mat using CTAB (4). The rDNA internal transcribed spacer (ITS) region was amplified using the universal primer pair ITS1 and ITS4 (1). PCR amplicons were purified and sequenced. A BLASTn search revealed that the resulting sequences (GenBank Accession Nos. KM017960, KM017961, and KM017962 for HD-1, HD-3, and HD-5, respectively) shared 99% identity with other Ceratobasidium sp. AG-A isolates (JX913824, FJ440197). In addition, sequence identity of HD-1, HD-3, and HD-5 with each other was 99.8%, 99.7%, and 99.8%, respectively. Thus, the isolates were identified as Ceratobasidium sp. AG-A, i.e., binucleate Rhizoctonia (BNR) AG-A. Pathogenicity tests were conducted by placing autoclaved wheat seeds, each colonized with Rhizoctonia isolates at inoculum density of 25 propagules per gram (ppg), on the surface of a sterilized mix of soil, sand, and nutrient soil (1:1:1, v/v/v) in pots (3). Each pot contained five healthy seedlings of the foxtail millet cultivar Yugu 1, and every seedling was inoculated by placing three colonized wheat seeds under the sheath of foxtail millet at the 2- or 3-leaf stage; non-inoculated sterilized wheat seeds were used as a control. Plants were incubated at 25°C with 14 h light and 10 h dark in a growth room for 10 days, and then assessed for disease. Damping-off symptoms similar to those in the field appeared on inoculated plants; control plants were asymptomatic. BNR Rhizoctonia were re-isolated from diseased plants and confirmed to be AG-A based on morphological characteristics and rDNA-ITS sequence. BNR AG-A has been reported in China as a pathogen of Chinese mustard, Chinese cabbage, potato, and sugar beet, but there are no previous reports of its presence on foxtail millet. R. solani AG-1 and AG-4 are usually regarded as pathogenic to foxtail millet (2). To our knowledge, this is the first report of BNR AG-A causing foxtail millet damping-off in China. We found binucleate Rhizoctonia AG-A, the anamorph of Ceratobasidium sp., can infect foxtail millet at seeding stage, which may cause serious losses of the crop. Therefore, we should pay particular attention to it when considering seeding disease control and breeding disease resistance varieties of foxtail millet in the future.