White clover (Trifolium repens), also known as Dutch clover (family Fabaceae), is an herbaceous, perennial plant, widely planted as pasture crop and occasionally used as lawn plant. From June to September in 2012 and 2013, approximately 5 to 8% of the plants as garden lawn were infected in the areas surveyed in Tonghua County, Jinlin Province. Ash green and water-soaked lesions appeared initially on the petiole and leaves. Subsequently, petioles collapsed with soft watery rot followed by collapse of leaves and eventually the entire plant. Aerial hyphae appeared on all the infected parts, followed by production of light brown to brown sclerotia. Seven isolates with the morphological characteristics of Rhizoctonia solani Kühn were isolated from symptomatic petioles and leaves which were surface disinfested in 70% alcohol for 30 s and 0.5% sodium hypochlorite for 1 min and plated on potato dextrose agar (PDA). Hyphal tips were transferred to a fresh plate of PDA and the cultures were examined for morphological characters microscopically. Mycelia of all isolates were branched at right angles with a septum near the branch and a slight constriction at the branch base. Hyphal cells were determined to be multinucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at 400× magnification with a microscope. The internal transcribed spacer (ITS) region of the nuclear rDNA was amplified by using the primers ITS4 and ITS5 (2). The ITS sequences of isolates BSYJ14 (GenBank Accession No. HF678123), BSYJ31 (HF571130), BSYY21 (HF678126), and BSY22 (KC572140) exhibited 100% identity with that of R. solani AG 1-1B (AB122138 and HQ185364). ITS sequences of another three isolates, BSYJ11 (HF678122), BSYJ32 (HF678125), and BSYJ12 (HF678121) exhibited 99% identity with the ITS sequence of R. solani AG 1-1B. Pathogenicity tests were performed on healthy, potted T. repens. Five potted plants were inoculated at the base of the petiole with a 0.6-cm diameter mycelial plug from 3-day-old PDA cultures for each isolate, and the inoculation sites were covered with moistened sterile absorbent cotton. Another five potted plants were inoculated with sterile PDA plugs as controls. All plants in the experiments were covered with plastic bags and kept in a greenhouse at 20 to 25°C for 72 h, then the plastic bags were removed. After 5 to 7 days, the symptoms of watery rot were observed on petioles and leaves of all plants inoculated with these isolates, while control plants remained healthy. R. solani AG 1-IB was re-isolated from all plants inoculated with the isolates. The isolates were confirmed by morphological characteristics of the hyphae and hyphal fusions with the original isolates. The pathogenicity test was carried out twice with similar results. R. solani has been reported to cause root rot on T. pratense in northwestern China (4) and summer blight on T. pratense in Japan (3). To our knowledge, this is the first report of R. solani AG 1-IB causing summer blight on T. repens in China.
References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. E. L. Cooke et al. Mycol. Res. 101:667, 1997. (3) T. Tsukiboshi et al. Bull. Natl. Grassl. Res. Inst. 39:50, 1988. (4) W.-Y. Zhuang (ed.). Fungi of Northwestern China. Mycotaxon Ltd. Ithaca, NY, 2005.