Old cultivars of roses (Rosa spp.) established on Rosa canina as rootstock are grown in field collections in the repository at Gaillard in eastern France, close to the Swiss border. During June 2005 and July 2006, several rose plants in full vegetation were severely wilted, leading ultimately to plant death. Necrotic lesions were present on the roots, and marginal tissue was excised and incubated in the dark at 20°C on PARP V8 agar plates (1). Similar isolates were obtained in both years, which were inoculated within 4 days on V8 agar medium and incubated at 25°C. The pathogen colonies grew approximately 30 mm daily on V8 agar. Colonies on corn meal agar (CMA) (1) were submerged, with or without a vague radiate pattern on potato carrot agar. Main hyphae were as much as 7 μm wide. Sporangia and zoospores were not produced. Hyphal swellings as much as 25 μm in diameter were abundant, terminal, and sometimes intercalary, often forming regular, dense chains in basipetal succession at hyphal tips and readily liberated. The morphological characters matched those described for Pythium intermedium (2). DNA sequencing of the ITS rDNA (ITS1, ITS2, and 5.8S rDNA) was generated using primers ITS5 and ITS4 in primary PCR (35 cycles: 96°C for 1 min, 55°C for 1 min, and 72°C for 2 min) and sequencing reactions (1). All isolates yielded identical ITS sequences, which were identical to the ex-type strain of P. intermedium, CBS 266.38 (GenBank Accession No. AY598647). The ITS sequence was deposited as GenBank Accession No. EF078693 (National Center for Biotechnology Information, NCBI, Bethesda, MD). Pathogenicity testing was performed on 3-year-old Rosa canina rootstocks from clonal propagation in 25 cm in diameter and 40 cm deep pots of compost 297 (Ricoter, Aarberg, Switzerland). Five-day-old mycelial plugs of our isolates grown on CMA (1) were inoculated in two pots, and roots were mechanically wounded. Sterile plugs were used on two control plants set aside. All plants were kept for 2 weeks at 20°C in a glasshouse with sufficient watering. Extensive necrotic lesions developed on inoculated roots, and severe wilting and leaves drying out were also observed. The controls showed no symptoms. P. intermedium was reisolated from margins of root lesions, thus fulfilling the postulates of Koch. P. intermedium was originally isolated from dead plant material (2) but is known as a typical soil inhabitant. It also has been isolated from water and living material from numerous plant species, but never from the genus Rosa until now. To our knowledge, this is the first record of P. intermedium on Rosa spp. grafted on Rosa canina in France. References: (1) L. Belbahri et al. FEMS Microbiol Lett 255:209, 2006. (2) A. J. van der Plaats-Niterink. Stud. Mycol. 21:1, 1981.
A new addition to the alien flora of Tunisia, Amaranthus spinosus L. (Amaranthaceae s.l.), with notes on A. diacanthus Raf.
