RESUMO
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 Mochelek, 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. References: (1) E. N. Njambere et al. Plant Dis. 92:917, 2008. (2) L. H. Rhodes, Sclerotinia Crown and Stem Rot Resistance, http://www.naaic.org/stdtests/scleroti.htm , 1991. (3) A. K. Slesaravichyus et al., Selektsiya i Semenovodstvo Moskva 6:21, 1988. (4) N. L. Taylor and R. R. Smith, Adv. Agron. 63:153, 1998.
RESUMO
Numerous Fusarium species have been associated with Fusarium head blight of wheat. In Poland, Fusarium poae was reported as the dominant species isolated from wheat grain during seasons with low amounts of rainfall during anthesis (1). F. langsethiae was described as a new toxigenic Fusarium species (3) and causal agent of Fusarium head blight (2), which has been isolated from infected oats, wheat, and barley in northern and central Europe (Norway, Austria, Germany, Czech Republic, Denmark, and England) (2). On the basis of morphological similarities, F. langsethiae has long been identified as a "powdery" form of F. poae. However, F. langsethiae produces type A trichothecene toxins such as T-2, whereas F. poae produces nivalenol and other 8-keto trichothecenes, scirpentriol, and 15-acetoxyscirpenol. In 2006, we obtained several isolates of F. langsethiae from kernels collected from winter wheat ears with head blight symptoms. Isolates were collected in the central (Sobiejuchy 52°54'N, 17°43'E; Minikowo 53°29'N, 17°56'E) and northern (Radostowo 53°59'N, 18°45'E) regions of Poland. Strains were isolated on potato dextrose agar (PDA) medium (pH 5.5). Further analyses were conducted on single-spore isolates. Initial species identification of all isolates was conducted on the basis of morphological features. The strains were grown in darkness at 25°C on PDA in plastic petri dishes to diagnose colony color, odor, and growth rate. The cultures also were incubated on saltwater nutrient agar (SNA) at 25°C for 7 days in near-UV light (Philips TLD 36W/08) and darkness in a 12/12-h cycle to promote conidia formation. The calculated average mycelial growth rate per day was based on the difference in millimeters between the colony diameters after 4 and 7 days of incubation. Growth rates ranged from 5.4 to 10.3 mm/day for nine strains. Mycelium was whitish or pinkish white, sparse, and 1 to 3 mm high with no odor. All colonies showed a powdery mycelium surface. Microconidia was napiform or globose, nonseptate, sporadically 1-septate, with an average length of 6.4 µm (range 3.9 to 13.7 µm) and width of 5.6 µm (range 2.9 to 8.8 µm). Microconidia were formed in heads, borne on unbranched or branched monophialides that were 8.5 to 16.3 µm long. All strains had slim, bent monophialides, typical for F. langsethiae, and always a few, short, thick, and squat ones resembling F. poae. In young cultures, monophialides may be formed directly on hyphae. Formation of macroconidia, sclerotia, and chlamydospores were not observed after 3 weeks of incubation. Species identification was confirmed by PCR assay with the use of SCAR (sequence characterized amplified region) primers producing a 310-bp DNA fragment (4), which was deposited in GenBank (Accession No. EU088404). To our knowledge, this is the first report of F. langsethiae in Poland. References: (1) C. Sadowski et al. J. Appl. Genet. 43A:69, 2002. (2) M. Torp and A. Adler. Int. J. Food Microbiol. 95:241, 2004. (3) M. Torp and H. I. Nirenberg. Int. J. Food Microbiol. 95:247, 2004. (4) A. Wilson et al. FEMS Microbiol. Lett. 233:69, 2004.
