First Report of Rhizoctonia solani AG-1-IB Infecting Potato with Stem Canker in Heilongjiang Province, China

Rhizoctonia solani is one of the most important soil-borne pathogenic fungi and causes disease of several important crops worldwide. The various symptoms of R. solani infection are damping off, root rot, stem cankers, crown rot, and blights (Ogoshi 1996). For potato (Solanum tuberosum L.), R. solani has been associated with black scurf and stem canker, causing both qualitative and quantitative damage (Djébali and Belhassen 2010). Isolates of R. solani anastomosis group 3 (AG3) are recognized as the predominant cause of these diseases on potato globally (Carling 1996) and in the northeast of China (Yang et al. 2017). In July 2017, two potato plants (cultivars Favorita and Youjin-885) displaying visible brown, slightly sunken, and sharped-edged lesions on subterranean stem and/or stolon typically associated with stem canker were collected from Harbin City (45°50′55″N, 126°50′27″E) and Kesan County (48°3′6″N, 125°51′46″E), located in the potato production regions of Heilongjiang Province, in China. Stem pieces (5 mm long) were taken from the margins of the healthy and diseased tissues and were surface disinfected with 70% ethanol for 30 s and 0.5% NaClO for 1 min, rinsed three times with sterile distilled water, and after drying placed on 2% water agar amended with 0.125 g/liter of streptomycin sulfate. After incubation for 48 to 72 h at 24°C, hyphal tips resembling Rhizoctonia were examined for morphological characters microscopically, transferred to potato dextrose agar (PDA), and incubated at 24°C in the dark until use. Characteristics of two fungal isolates were typical of R. solani Kühn, exhibiting hyphal branching at right angles, a septum near the branching point, and a slight constriction at the branch base, with 4.38 to 13.3 μm diameter. Hyphal cells were also determined to be multinucleate by staining with 1% safranin O and 3% KOH solution (Bandoni 1979). Meanwhile, the total DNA was extracted using the Plant Genomic DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions, and polymerase chain reaction amplification and sequencing of the internal transcribed spacer region of ribosomal DNA (rDNA) were conducted by the universal primers ITS1 and ITS4 (White et al. 1990). The resulting sequences (GenBank accession nos. MK288031 and MK288032) showed 99% identity to AG-1-IB isolates (e.g., MH172667 and GU585667). On the basis of morphological characteristics and nucleotide homology, two isolates were confirmed as R. solani AG-1-IB. Koch’s postulates were confirmed for each isolate by carrying out pathogenicity tests with 10 replicates. Symptomless mini-tubers (Favorita) of approximately the same size (10 to 20 g) were washed, visually inspected for black scurf, and allowed to sprout at room temperature for 10 days. Each mini-tuber was planted into autoclaved soil in a plastic pot (4-liter capacity) and placed in a greenhouse at 18/27°C (night/day) with 50% relative humidity and watered as required. The pots were inoculated by placing a 5-mm-diameter mycelial plug (from one PDA isolate) over the mini-tuber, which was then covered with potting mix. The control pots were inoculated with sterile PDA. The treatment of each isolate was performed on 30 plants, and the control was repeated. After 3 weeks, plants were removed and assessed for disease. No disease symptoms were noted on the control plants. Typical stem canker was observed on inoculated plants from each isolate. R. solani AG-1-IB was consistently reisolated from symptomatic stems and confirmed by the morphological and molecular characteristics mentioned above. To our knowledge, this is the first report of AG-1-IB causing disease on potatoes in Heilongjiang Province. Potato stem canker caused by R. solani AG-1-IB has been reported in Inner Mongolia Autonomous Region (Tian et al. 2011) and Shaanxi Province (Yang et al. 2015). Currently, it is not a major disease of potato, but it is considered an emerging problem owing to the high risk of soil-borne infection in northern China, which is the main seed potato producing area with limited crop rotation. Because seed potatoes are produced and transported across China, it will be important to monitor R. solani AG groups. This information will assist in monitoring pathogen spread and will be critical to developing disease management strategies.