RESUMO
European ash (Fraxinus excelsior), also known as common ash, occurs naturally inland in lower areas of southeastern Norway and along the southern coast of the country. It is important both as a forest and ornamental tree. During the last decade, dieback has become a disastrous disease on F. excelsior in many European countries. The anamorphic fungus Chalara fraxinea T. Kowalski (1), described for the first time from dying ash trees in Poland, is now considered the cause of ash dieback (2). In May of 2008, C. fraxinea was isolated from 1.5 m high diseased F. excelsior in a nursery in Østfold County in southeastern Norway. Symptoms included wilting, necrotic lesions around leaf scars and side branches, and discoloration of the wood. From symptomatic branches, small pieces (approximately 1 cm3) were excised in the transition area between healthy and discolored wood. After surface sterilization (10 s in 70% ethanol + 90 s in NaOCl), the pieces were air dried for 1 min in a safety cabinet, cut into smaller pieces, and placed on media. The fungus was isolated on potato dextrose agar (PDA) and water agar (WA). On PDA, the cultures were tomentose, light orange, and grew slowly (21 mm mean colony diameter after 2 weeks at room temperature). Typical morphological features of C. fraxinea developed in culture. Brownish phialides (14.8 to 30.0 [19.5] × 2.5 to 5.0 [4.1] µm, n = 50) first appeared in the center of the colonies on the agar plugs that had been transferred. The agar plugs were 21 days old when phialides were observed. Abundant sporulation occurred 3 days later. Conidia (phialospores) extruded apically from the phialides and formed droplets. Conidia measured 2.1 to 4.0 (3.0) × 1.4 to 1.9 (1.7) µm (n = 50). The first-formed conidia from each phialide were different in size and shape from the rest by being longer (6 µm, n = 10) and more narrow in the end that first appeared at the opening of the phialide. Internal transcribed spacer sequencing confirmed that the morphological identification was correct (Accession No. EU848544 in GenBank). A pathogenicity test was carried out in June of 2008 by carefully removing one leaf per plant on 10 to 25 cm high F. excelsior trees (18 trees) and placing agar plugs from a 31-day-old C. fraxinea culture (isolate number 10636) on the leaf scars and covering with Parafilm. After 46 days, isolations were carried out as described above from discolored wood that had developed underneath necrotic lesions in the bark and subsequently caused wilting of leaves. All the inoculated plants showed symptoms, and C. fraxinea was successfully reisolated. No symptoms were seen on uninoculated control plants (eight trees) that had received the same treatment except that sterile PDA agar plugs had been used. References: (1) T. Kowalski. For. Pathol. 36:264, 2006. (2) T. Kowalski and O. Holdenrieder, For. Pathol. Online publication, doi: 10.1111/j.1439-0329.2008.00565.x, 2008.
RESUMO
In 2004, damages resembling those caused by Phytophthora spp. were observed in a 15-year-old bough plantation of noble fir (Abies procera). When removing bark upward from the roots and base of a diseased tree, a reddish brown discoloration with distinct borders to surrounding wood appeared. The discoloration extended approximately 1.5 m above ground, but only on one side of the stem. This resulted in dead basal branches (flagging) on the cankered side of the tree. Other dying trees in the same field did not show flagging symptoms but turned chlorotic to brown after being girdled by the expanding stem canker. Approximately 25% of the trees were dead or dying. Isolations were carried out from the area between healthy and diseased tissue both from roots and base of the stem of the tree with flagging symptoms. Samples were rinsed in running tap water and plated on the Phytophthora selective medium PARP (17 g of cornmeal agar, 10 mg of pimaricin, 250 mg of ampicillin, 10 mg of rifampicin, and 100 mg of pentachloronitrobenzene (PCNB) in 1 liter of water), with and without hymexazol added (50 mg/l). Morphological characters of the isolated Phytophthora sp. included nonpapillate sporangia (37 to 64 µm), internal proliferation, and characteristic hyphal swellings. The isolate was heterothallic and produced amphigynous antheridia when crossed with tester strains of P. cryptogea. The mating type was A2. The internal transcribed spacer (ITS) rDNA sequences were identical to P. cambivora (GenBank Accession No. AY880985). Thus, both morphological characters and DNA analysis supported the species identification. A pathogenesis test to fulfill Koch's postulate was carried out during 2005. Inoculation was done by placing agar with culture in the growth medium close to the roots of noble fir seedlings. Eleven weeks after inoculation, clearly visible stem canker symptoms were observed. The ITS sequences of the reisolated Phytophthora sp. were determined and found identical to P. cambivora. P. cambivora was reported to cause root rot and stem canker in a noble fir Christmas tree plantation in the United States (1). P. citricola and P. citrophthora are known to cause problems on Lawson Falsecypress/Port-Orford-cedar (Chamaecyparis lawsoniana) in Norway, but damages by Phytophthora spp. have never been reported in Abies spp. plantations or forest stands in Norway. Currently, we are also working on Phytophthora problems discovered in two different Christmas tree plantations (A. lasiocarpa and A. nordmanniana). Reference: (1) G. A. Chastagner et al. Plant Dis. 79:290, 1995.
