First Report of Potato Stem Canker Caused by Rhizoctonia solani AG-2-2IIIB in Heilongjiang Province, China

Abstract

Rhizoctonia solani is one of the most important soil borne pathogenic fungi, causing disease in several important crops worldwide. The symptoms of R. solani infection are attributed to various plant diseases, such as damping off, root rot, stem cankers, crown rot and blights (Ogoshi 1996). In potato, R. solani has been associated with black scurf and stem cankers causing qualitative and quantitative damage to infected crops (Djébali and Belhassen 2010). Isolates of R. solani AG3 have been reported as the predominant cause of stem canker on potato (Carling 1996) and the same results were also obtained in the cities of Harbin and Suihua in Heilongjiang Province, China (Yang et al. 2017). During the summer of 2019, diseased potato plants (cv. Youjin-885) with symptoms typically associated with stem canker (Supplementary Fig. 1A) were found in Jiamusi City of Heilongjiang in Northeast China, where potatoes are grown widely. Stem pieces (5 mm long) were taken from the margins of the healthy and diseased tissues and were surface sterilized with 70% ethanol for 30 s and 0.5% NaClO for 1 min, then rinsed 3 times with sterile distilled water and placed on potato dextrose agar (PDA) at 25°C in the dark. After incubation for 48–72 h, hyphae tips resembling Rhizoctonia were microscopically examined for morphological characteristics, and transferred to fresh plates of PDA. The characteristics of the observed isolate were typical of R. solani Kühn, which include hyphal branching at right angles, a septum near the branching point and a slight constriction at the branch base (Supplementary Fig. 1B), amongst other characteristics. Hyphal cells were also determined to be multinucleate by staining with 1% safranin O and 3% KOH solution (Bandoni 1979). Meanwhile, PCR amplification and DNA sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was performed by using the universal primers ITS1/ITS4 (White et al. 1990) and the AG2-2IIIB specific primers (see Supplementary Fig. 1C for PCR results and primer sequences) described by Carling et al. (2002). The resulting sequences that were amplified with the ITS1/ITS4 primers (GenBank accession no. MT258822) and the AG2-2IIIB specific primers (GenBank accession no. MT679722) showed more than 98% and 100% identity to those of more than 10 AG-2-2IIIB isolates present in GenBank, respectively. Based on the morphological characteristics and molecular analysis, the isolate was identified as R. solani AG-2-2IIIB. Koch's postulates were carried out to prove the pathogenicity of the isolate. First, disease-free minitubers (cv. Favorita) of approximately the same size (10-20 g) were allowed to sprout at room temperature for 10 days. The minitubers were then planted into autoclaved soil in a plastic pot (4 L capacity), placed in a greenhouse at 18 - 27°C (night-day) with 50% relative humidity and watered as required. The pots were inoculated by 7-mm-diameter mycelial plugs (from one PDA petri plate) over the minituber, which was then covered with potting mix. The control pots were inoculated with sterile PDA. Each treatment consisted of 10 plants, and the experiment was repeated three times. After 3 weeks, plants were removed and assessed for disease. Typical stem cankers were observed on plants grown in pots inoculated the mycelial plug, but the control plants remained disease free. Moreover, R. solani AG-2-2IIIB was reisolated consistently from symptomatic stems, and the identity of the reisolates were confirmed by the above mentioned morphological and molecular analyses. To our knowledge, this is the first report of AG-2-2IIIB causing disease on potatoes in Heilongjiang Province, the main potato-producing area of China. Potato black scurf caused by R. solani AG-2-2IIIB has been reported in Inner Mongolia Autonomous Region (Tian et al. 2011). Although it is not considered a major potato disease in Heilongjiang Province, with the recent rapid development of the potato industry, it can become a major problem due to the high risk of soil borne infection and disease dispersal with regard to seed potato transportation across China. The information presented in this note will assist in developing management strategies for this disease.