First Report of Sclerotinia Stem Blight Caused by Sclerotinia trifoliorum on Trifolium ambiguum in Poland.

Abstract

Kura clover (Trifolium ambiguum M. Bieb.), a rhizomatous, persistent legume native to the Caucasus region, has received recent attention in North America and New Zealand as a pasture and silage crop. It is reported to be resistant to most pathogens affecting other clovers, including Sclerotinia trifoliorum Eriks. (3,4), one of the most destructive pathogens of clovers in northern Europe. Kura clover (cv. KTA202) was established in May 2009 near Mochełek, Poland (53° 13' N, 17° 51' E) on a Luvisol soil. By May 2011, 70% of plants grown in an experimental field (350 m2) had died, and 20% of the remaining plants were yellow and wilted. At crowns and the lower stem regions, wet, brown lesions with delicate white mycelium were observed. Lesion development was followed by death of the entire plant in a few days. By early June, only a few asymptomatic plants per square meter remained in the field. Tissue fragments of 20 symptomatic plants were surface-sterilized with 1% NaOCl for 1 min and plated on potato dextrose agar (PDA). A fungus with morphological characteristics of S. trifoliorum was consistently isolated. DNA isolation from sclerotia was performed with the DNeasy Plant Mini Kit (Qiagen, USA). Amplification and sequencing of the ITS region of rDNA was performed with primers ITS1/ITS4. NCBI Blast analysis of the 542-bp segment showed a 99% homology with most of S. trifoliorum and S. sclerotiorum strains in GenBank (e.g., AY547267.1 and EU082466.1). Sequence of isolate St0211TA was deposited in GenBank (Accession No. JQ743329). To determine growth rate of hyphae, morphology, and dimensions of sclerotia, colonies were grown in three replications on PDA at 20 ± 1°C in the dark. S. trifoliorum (CBS 122377) and S. sclerotiorum from our local collection were used as controls. Mean growth rate of S. trifoliorum isolates (20.5 mm/day) was slower compared to S. sclerotiorum (32.3 mm/day). Sclerotia began to form on delicate and smooth mycelium of S. trifoliorum on the entire surface of the plate in 7 to 8 days. Sclerotial size on day 28 was 2.0 to 9.0 × 2.0 to 7.0 mm (average 4.2 × 3.6 mm). Ultimately, the identification of S. trifoliorum was confirmed on the basis of ascospore morphology. Apothecia grew from sclerotia in wet sand at 12°C after 12 weeks. Asci contained dimorphic ascospores: four larger 13.0 to 16.0 × 6.0 to 9.0 mm (average 14.1 × 7.4 μm) and four smaller 10.0 to 12.0 × 5.4 to 6.0 mm (average 10.6 × 6.0 μm), typical for this species (1). Isolate St0511TA, which most intensively produced apothecia, was deposited in CBS (No. 133234). Koch's postulates were fulfilled by pathogenicity tests carried out on 2-week-old T. ambiguum seedlings grown in pots (6 × 30 plants), sprayed with a mycelial fragment suspension, and incubated at 15°C (2). Brown, wet spots with delicate white mycelium were observed on cotyledons after 3 days. After 5 days, approximately 10% of cotyledons were killed and mycelium appeared on stems and leaves, and after 10 days, 73% of seedlings were dead. S. trifoliorum was reisolated from all symptomatic tissues. To our knowledge, this is the first report of S. trifoliorum stem blight on T. ambiguum in the field.