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Chalara fraxinea Isolated from Diseased Ash in Norway

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.

Species variation in susceptibility to the fungus Neonectria neomacrospora in the genus Abies

ABSTRACT The fungus Neonectria neomacrospora has recently caused an epidemic outbreak in conifer species within the genus Abies in Denmark and Norway. Christmas tree producers in Europe and North America rely, to a large extent, on Abies species. The damage caused by N. neomacrospora, including dead shoot tips, red flagging of branches and potentially dead trees, have therefore caused concern about reduced quality and loss of trees, and thereby of revenue. Field observations of natural infection of 39 taxa, from 32 species, within the genus Abies in the Hørsholm Arboretum, Denmark, were evaluated; significant differences were seen between taxa, that is, species, and between some species and their subspecies. The Greek fir, Abies cephalonica, was the only species without damage. An inoculation experiment on detached twigs with mycelium plugs from a N. neomacrospora culture showed that all species could be infected. The damage observed in the inoculation experiment could explain 30% of the variation in the field observations based on species mean values. The epidemic outbreak and the high number of species susceptible to this fungus indicate that N. neomacrospora requires attention in the cultivation and conservation of Abies species.

First Report of Root Rot and Stem Canker Caused by Phytophthora cambivora on Noble Fir (Abies procera) for Bough Production in Norway

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 Kochs 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.

Overwintering Diseased Plant Parts and Newly Infected Flowers and Fruit as Sources of Inoculum for Colletotrichum acutatum in Sour Cherry

Production of inoculum of Colletotrichum acutatum from both previously infected and overwintered tissue, as well as newly developed plant tissue of sour cherry (Prunus cerasus), was studied in southern Norway. Plant parts were sampled from commercial, private, or research orchards, and incubated for 2 to 14 days (time depended on tissue type) in saturated air at 20°C. In early spring, abundant sporulation was found on scales of overwintered buds and shoots. A mean of 35% infected buds in four cultivars was observed, with a maximum of 72% of the buds infected in one of the samples. Over 3 years, the seasonal production of overwintered fruit and peduncles of cv. Fanal infected the previous year was investigated. In all three years, the infected plant material was placed in the trees throughout the winter and the following growing season; in two of the years, fruit and peduncles were also placed on the ground in the autumn or the following spring. Old fruit and peduncles formed conidia throughout the season, with a peak in May and June. Spore numbers declined over the season, but the decline was more rapid for plant material on the ground than in the trees. On average over 2 years, 68.7, 24.0, or 7.3% of the inoculum came from fruit placed in the trees, placed on the ground in spring, or placed on the ground the preceding autumn, respectively. The number of fruit and peduncles attached to the trees in a planting of cv. Hardangerkirsebær was followed from February to July one year, and although there was a decline over time, fruit and/or their peduncles were still attached in substantial numbers in July, thus illustrating their potential as sources of inoculum. In observations over 2 years in a heavily infected orchard of cv. Stevnsbær, 75 and 47% of flowers and newly emerged fruit, respectively, were infected. Artificially inoculated flowers and fruit produced conidia until harvest, with a peak in mid-July. It may be concluded that previously infected and overwintered, as well as newly emerged tissue of sour cherry, may serve as sources of inoculum of C. acutatum throughout the growing season.

Apple rootstocks may become infected by Neonectria ditissima during propagation

ABSTRACT Introduction and purpose: The ability of apple rootstocks to become infected by Neonectria ditissima, the cause of European canker, was studied over two years. Materials and methods: Rootstocks B9 and M9 with a size suitable for grafting (6-10 mm stem diameter, termed rootstocks), and smaller sized rootstocks (<5 mm stem diameter, termed transplants) of B9, M9, M26, MM106 and Antonovka were inoculated with N. ditissima at different times, either with contaminated map pins or with spore suspensions. In addition, the rootstocks were either defeathered (side shoots removed), topped (top shoot headed) or both, to create wounds that would normally occur during propagation, while wounds on transplants were made by removing leaves. Results and discussion: One month after inoculation, slightly sunken canker lesions had developed around the inoculation points of the map pins or wounds. No lesions developed on the non-inoculated controls. Map pin inoculation resulted in 30% to 89% infection and spore suspension sprayed on wounds from 5% to 45% infection. When the cankered areas were split open, brown lesions with necrotic tissue due to infection by N. ditissima appeared. The transplants of M9, M26 and MM106 inoculated with contaminated map pins in 2014 developed necrosis on 40% to 67% of the plants, but there were no differences in the incidence or severity among the different types. On the transplants of B9, Antonovka and M9 inoculated in 2015, there was more necrosis on B9 (42%) than on Antonovka (11%) and more sporulating lesions on B9 (29%) than on M9 (9%) or on Antonovka (4%). Conclusion: It can be concluded that rootstocks used for apple trees may become infected by N. ditissima, and wounds should thus be protected during propagation.

Delphinella shoot blight and Grovesiella canker on Abies lasiocarpa in western USA

ABSTRACT The impact of Delphinella shoot blight (Delphinella abietis) and Grovesiella canker (Grovesiella abieticola) on subalpine (Abies lasiocarpa) and corkbark fir (A. lasiocarpa var. arizonica) in a provenance trial in Idaho (ID) was evaluated in 2013. Both pathogens were previously reported from North America on fir species. D. abietis had been found on subalpine fir in USA, but not in ID, and G. abieticola on grand fir (Abies grandis) in ID, but not on subalpine or corkbark fir. D. abietis kills current-year needles and in severe cases buds and shoots, and G. abieticola results in dead shoots and branches and can eventually kill whole trees. Significant differences between provenances in susceptibility to D. abietis and G. abieticola were observed in the provenance trial in ID. In general, subalpine fir was more susceptible to both diseases than corkbark fir. In 2013, D. abietis was also found on subalpine fir in the Puget Sound area of Washington State and G. abieticola was seen on white fir (Abies concolor), but neither disease was detected in native stands of subalpine fir in Washington State. Morphological features of both fungi were described from samples collected in the provenance trial in ID in May 2016.