first report of ophiostoma karelicum from birch stands - Poznań

University of Agriculture, Kraków, Poland
FIRST REPORT OF OPHIOSTOMA KARELICUM
FROM BIRCH STANDS IN POLAND
R. Jankowiak
Key words: Betula verrucosa, ophiostomatoid fungi, pathogenicity, Scolytus ratzeburgi
The ascomycete genus Ophiostoma with both Sporothrix and/or Pesotum anamorphs
includes species causing blue-stain in timber as well as species that are serious
tree pathogens, e.g. O. ulmi and O. novo-ulmi. These fungi are associated with
different species of bark beetles (Coleoptera, Scolytinae; Kirisits 2004). Information
about hardwoods beetle-associated Ophiostoma species in Poland is very limited.
Ophiostoma dentifundum, O. grandicarpum, O. introcitrinum, O. moniliforme, O.
proliferum, O. quercus and O. stenoceras were isolated from Quercus wood (Kowalski
and Butin 1989) while O. ulmi and O. novo-ulmi were found in association with
Scolytus spp. and Dutch elm disease (Siemaszko 1939, Mańka 1953, Przybył 2002).
Ophiostoma karelicum was isolated in September 2010 from the galleries of
Scolytus ratzeburgi. The samples were collected from two sites in Poland (Babimost
Forest District – 52°10'32''N, 15°48'35''E; Myszyniec Forest District – 53°22'27''N,
21°22'57''E). Twenty eight galleries of S. ratzeburgi were collected from six dying
trunks of birches (Betula verrucosa). Six fragments of each gallery were placed on a
2% malt extract agar (MEA) medium containing 0.05% cycloheximide. A total of
114 isolates of O. karelicum were obtained from 79% galleries. Of these, 54 and 70
were isolated from galleries collected in Babimost and Myszyniec, respectively. In
cultures, Pesotum-like structures only developed. To obtain ascomata, eight isolates
from each site were crossed. Isolates of O. karelicum formed fast growing,
hyaline to white colony on MEA medium (Phot. 1 h). Bases of teleomorph dark,
90–205 m in diameter, ornamented with dark hairs (Phot. 1 a). Ascomatal necks
dark, smooth, straight or curved, 390–964 m long excluding ostiolar hyphae,
22.5–45.0 m wide at base, 8.8–22.5 m wide at apex (Phot. 1 b). In culture necks
often two-three per ascoma. Ostiolar hyphae hyaline, septate, 12.6–75.0 m long.
Asci not observed. Ascospores hyaline, one-celled, cylindrical in frontal view,
allontoid in side view, 2.3–4.4 × 1.1–1.7 m without sheath (Phot. 1 c).
Anamorphs: 1) Pesotum: synnemata simple or branched, with pigmented stipe,
204–616 m long, 20–91 m wide at base, conidia hyaline, one-celled, obovoid,
with truncated base, 3.0–4.9 × 1.4–2.6 m (Phot. 1 d, e), 2) Sporothrix:
conidiogenous cells arising directly from hyphae, 6.4–43.3 m long; conidia
hyaline, obovoid with truncated base, 2.8–5.8 × 0.9–2.8 m (Phot. 1 f, g).
Phytopathologia 59: 55–58
© The Polish Phytopathological Society, Poznań 2011
ISSN 2081-1756

56 Short communications
Phot. 1. Ophiostoma karelicum on 2% MEA: a – ascoma with neck, b – apex of the ascomatal neck,
c – ascospores, d – Pesotum anamorph, synnemata, e – Pesotum anamorph, conidia, f – Sporothrix
anamorph, conidiogenous cells, g – Sporothrix anamorph, conidia, h – 14-day-old colony.
Phots. c, d–f have been done using contrast phase (photo by R. Jankowiak)

Short communications 57
PCR amplicons of two isolates (009/O.k, 022/O.k) generated using primers
ITS 1F and ITS 4 were sequenced (White et al. 1990) and found identical to those
of O. karelicum from the GenBank database (Acc. No. EF486693, EF032478).
A preliminary inoculation test was conducted with two randomly chosen isolates
of O. karelicum obtained and identified in this study (009/O.k, 022/O.k). Inoculations
followed the procedure described by Krokene and Solheim (1998). On the
3 th of January 2011, the stems of two-year-old B. verrucosa seedlings were superficially
wounded by removing a bark flap (4 × 7 mm). For inoculation, a disc (3 mm
diameter) was cut from a margin of a 12-day-old culture of the test isolates, placed
on each wound and then covered by the bark flap and a Parafilm ® strip. Fifty seedlings
(25 per each isolate) were inoculated with the O. karelicum, whereas 25 plants
were inoculated with sterile MEA as control treatment. After 11 weeks of incubation,
inoculations with O. karelicum resulted in long necrotic lesions in the phloem
and sapwood of the test seedlings (Phot. 2 a, b). The average lesion length for O.
karelicum was 57.8 mm (18–120 mm) and for control 3.03 (0–7 mm). Both isolates
of O. karelicum caused blue-stain of the sapwood in the two-year-old seedlings. The
average depth of sapwood penetration was 1.06 mm (0.2–1.8 mm; Phot. 2 c, d).
None of control plants or plants inoculated with O. karelicum died during the
11-week-long observation period.
Phot. 2. Pathogenicity test results. Lesions on the surface of stem caused by Ophiostoma karelicum
(a) and control (b). The lesions were observed 11 weeks after inoculation.
Cross section through a birch stem in the inoculations points: c – O. karelicum, d – control.
The arrow indicates blue-stain in the sapwood (photo by R. Jankowiak)

58 Short communications
This is the first finding of O. karelicum in Poland. It has also been only reported
from the same host (Linnakoski et al. 2008), and from Pinus sylvestris and Picea abies
in Finland and Russia (Linnakoski et al. 2010). In the present study, O. karelicum
was unable to cause mortality of any of the test seedlings. However, this fungus
generated serious necrotic lesions in the phloem and sapwood of two-year-old B.
verrucosa seedlings. This suggests that O. karelicum is a pathogen with medium level
of virulence to B. verrucosa.
Acknowledgements
The author gratefully acknowledges Miroslav Kola ik (Institute of Microbiology,
Prague, Czech Republic) for identification of O. karelicum using molecular
techniques.
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Author’s address:
Dr. Robert Jankowiak, Department of Forest Pathology, University of
Agriculture, Al. 29 Listopada 46, 31-425 Kraków, Poland, e-mail:
rljankow@cyf-kr.edu.pl
Accepted for publication: 12.03.2011