Windflowers (Pulsatilla spp.) are perennial medicinal plants in the family Ranunculaceae with high economic as well as medicinal value in China. It is a commonly used traditional Chinese medicine (1). In 2012, a stem rot disease was observed on windflower (P. koreana Nakai) at flowering stages in fields of Liaoning Province, China. Disease incidence ranged from 10 to 65% (average of 45%) and resulted in approximately 25 to 60% yield loss. The infected area of plants initially takes on a dark green or brown water-soaked appearance and then becomes paler. Soon after, plants turn brown or black and die. If moisture conditions remain conducive, a cottony mycelium cover was observed on the affected area. Later, the mycelium usually produces numerous black sclerotia that were up to 1 cm long in affected plant parts. Diseased tissue were surface sterilized for 1 min in 1% NaOCl (v/v), rinsed with sterilized distilled water, and plated on potato dextrose agar (PDA). Isolates were mesophilic, with an optimum growth temperature of around 20°C. Colonies on PDA were white, with abundant aerial mycelium. Some mycelium in the colony center, especially that submerged in the medium, was light brown. Sclerotia were spherical to subspherical, elongated or fused to form irregular shapes, 2.5 to 9.0 × 2.0 to 6.8 mm. They were tightly attached to the agar surface by their under surface, which could be seen through the bottom of the petri dishes. These characteristics were consistent with Sclerotinia nivalis (2,3). Two isolates were selected for molecular identification. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 (4) and sequenced. The obtained sequences (GenBank Accession Nos. JX206828 and JX424615) showed 99 and 100% homology with the sequences of S. nivalis in GenBank (Accession No. AB516670). To demonstrate pathogenicity, mycelial blocks of the isolates grown on PDA were placed on the base of the stems of ten 1-month-old Pulsatilla koreana plants. The same numbers of control plants were treated with PDA plugs as a control. The inoculated plants were incubated at 25°C with a 12-h photoperiod. After 4 days, the initiation of stem necrosis was observed, and 9 days after inoculation, the plants collapsed and died. Control plants had no symptoms. The same fungus was reisolated from all inoculated plants, satisfying Koch's postulates. To our knowledge, this is the first confirmed report of windflower as a natural host for S. nivalis in China.
References: (1) S. C. Bang et al. J. Nat. Prod. 68:268, 2005. (2) G. Q. Li et al. Mycol. Res. 104:232, 2000. (3) I. Saito. Mycoscience. 38:227, 1997. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.