Sugar beet (Beta vulgaris L.) is grown worldwide as the second largest sugar crop. Sugar beet crown and root rot is an economically serious disease mainly caused by Rhizoctonia solani (teleomorph Thanatephorus cucumeris) AG 2-2 and AG 4 (1). In July 2010, at the 25- to 27-leaf stage, symptoms typically associated with crown and root rot, including dark brown to black lesions at the base of the petioles or circular to oval dark lesions (up to 10.0 mm in diameter) at the taproot, were observed on 15% of sugar beet plants collected from three sites in Shanxi Province, northern China. Pieces of internal root tissues cut from the margins between symptomatic and healthy-appearing tissue were disinfected with 0.5% NaOCl for 2 min, rinsed three times with sterile water, then placed on water ager (WA) for incubation at 25°C in the dark. After 2 days, single hyphal tips of three Rhizoctonia-like isolates (designated SX-RSD1, SX-RSD2, and SX-RSD3) were transferred to potato dextrose ager (PDA). Colonies of all isolates were brown and developed dark brown sclerotia (0.5 to 1.0 mm diameter) on the media surface after 4 and 7 days, respectively. Mycelia were branched at right angles with septa near the branches and slight constrictions at the bases of the branches were present. Average hyphal diameters of the three isolates were 8.1, 7.3, and 7.6 μm, respectively. Hyphal cells were determined to be multinucleate (4 to 9 nuclei per cell) by staining with 4′-6-diamidino-2-phenylindole (DAPI) (2). Anastomosis groups were determined by pairing with reference strains (kindly provided by N. Kondo, Hokkaido University, Japan) (2), and all three isolates anastomosed with R. solani AG-2-2IIIB. All three isolates grew well on PDA at 35°C, which separates AG-2-2IIIB from AG-2-2 IV. The internal transcribed spacer (ITS) region of rDNA was amplified from genomic DNA of these isolates with primers ITS1 (5′-TCCGATGGTGAACCTGCGG-3′)/ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). Sequences (GenBank Accession Nos. KC413984, KC413985, and KC413986) were over 99% identical to those of 19 R. solani AG-2-2 IIIB isolates (e.g., FJ492146.3; strain F510). Therefore, based on the molecular characteristics and the anastomosis assay, these three isolates were identified as R. solani AG-2-2IIIB. To determine the pathogenicity of the isolates, wheat seeds were autoclaved twice for 60 min at 121°C on consecutive days and inoculated with each isolate (3). Subsequently, wheat seeds (three seeds per plant) were placed around 8-week-old sugar beet (cv. HI0305) plants at 2 cm intervals to each root and 10 mm deep in soil. Plants were grown at 25 to 27°C for 7 days in a glasshouse. All inoculated plants developed symptoms of root rot, whereas control plants inoculated with sterilized wheat seeds remained healthy. R. solani AG-2-2IIIB was consistently re-isolated from the symptomatic root tissue and was confirmed by both morphological and molecular characteristics described above, fulfilling Koch's postulates. To our knowledge, this is the first report of R. solani AG-2-2IIIB on sugar beet in Shanxi Province of China. R. solani AG2-2IIIB has been reported to be pathogenic on wheat in China (4), which is often grown in rotation with sugar beet. This rotation could increase the risk of soilborne infection to either crop by R. solani AG2-2IIIB. References: (1) R. M. Harveson et al. Compendium of Beet Diseases and Pests, American Phytopathological Society. St. Paul, MN. 2009. (2) W. C. Kronland and M. E. Stanghellini. Phytopathology. 78:820, 1988. (3) M. J. Lehtonen et al. Plant Pathol. 57:141, 2008. (4) D. Z. Yu et al., Hubei Agric. Sci. 3:39, 2000.
Identification of Rhizoctonia solani isolates from sugar beet roots by analyzing the ITS region of ribosomal DNA
