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CryoLetters 23, 151-156 (2002)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK
CHILLING RESISTANCES OF ISOLATES OF Pythium ultimum var.
ultimum FROM THE ARCTIC AND TEMPERATE ZONES
T. Hoshino1*, M. Tojo2, H. Kanda3, M.L. Herrero4, A.M. Tronsmo4, M. Kiriaki1,
Y. Yokota1 and I. Yumoto1
1 Research Institute of Biological Resources, National Institute of Advanced Industrial Science
and Technology (AIST), 2-17-2-1, Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido
062-8517, Japan
2 Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1,
Gakuen-chou, Sakai, Osaka 599-8531, Japan
3 National Institute of Polar Research, 1-9-10, Kaga, Itabashi-ku, Tokyo 173-8515 Japan
4 The Norwegian Crop Research Institute, Fellesbygget, N-1432 Ås, Norway
Abstract
Chilling resistances in moss pathogenic fungi, Pythium ultimum var. ultimum, from
Longyearbyen, Svalbard (78°N, 15°E), located in the Arctic Zone and in the same isolates from
Temperate Zone, were determined. Both strains had similar optimum growth temperatures.
However, the strains from Svalbard could grow and survive at 0 - 5°C. In addition, chilling
treatment induced irregular mycelial morphology in the Arctic isolates. On the other hand, the
isolates from Japan did not grow at temperatures below 5°C and were destroyed after chilling
stress (0°C for 3 days or at 4°C for 1 week). The results suggested that isolates from Svalbard
highly adapted to the severe spring condition in Polar environments.
Key words: chilling resistance, fungi, Pythium ultimum var. ultimum, Svalbard
INTRODUCTION
Moss vegetation plays an important role as a producer in early stages of primary
succession in arctic and antarctic regions. Moss colonies also offer habitats for terrestrial
invertebrates. Therefore, moss communities are a key part of the terrestrial ecosystems in Polar
regions. On the other hand, microorganisms, especially fungi in Polar regions, are well known
for their formation of mycorrhizae, basidiolichens and decomposer associations (1). In addition,
some fungi have been reported to actively attack mosses growing in the Arctic, including
Northern Norway (Alta in Finnmark: 2), Svalbard in the Barents Sea (3-5), Jan Mayen in the
Greenland Sea (6) and Ellesmere Island in northern Canada (7); and in the Antarctica, including
South Orkney Island (7), King George Island (8) and on Cape Bird, Victoria Land (9).
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Wilson (6) studied infections of Rhacomitrium carpet on Jan Mayen Island and reported
that the moss disease was caused by an unidentified basidiomycete. We also reported that
Pythium ultimum var. ultimum and Pythium sp. HS group caused parasitic disease in Sanionia
uncinata in northern Norway (2), Svalbard (4, 5), western Greenland (Hoshino unpublished
results) and maritime Antarctica (8). Thyronectria antarctica var. hyperantarctica (7, 10), an
undetermined plectomycete (7), Coleroa turfosorum, Bryosphaeria megaspora, Epibryon
chorisodontii (11) and Rhyzopus sp. (9), which may cause parasitic disease in mosses, have
been isolated from various regions of the Antarctic Zone. Therefore, fungi also play another
ecological role in Polar regions, since some fungi may cause not only disintegration of dead
moss shoots but also infection in moss colonies.
Pythium ultimum var. ultimum is cosmopolitan and grows in cool to moderately warm
climates (12). There are only two reports of isolation of P. ultimum var. ultimum in the arctic
region, in Iceland (13) and Svalbard (4). However, oomycota, including the genus Pythium spp.,
has less resistance to freezing than do other fungi (14, 15). Strains from the Arctic showed moss
pathogenic activity in the snow melt period (4, 5). Thus, Arctic strains must adapt the severe
climate of early spring.
There have been no physiological studies on moss pathogenic fungi in the arctic and
antarctic regions. The aim of this study was to elucidate the physiological characteristics of
moss pathogenic isolates of P. ultimum var. ultimum from the Arctic.
MATERIALS AND METHODS
Fungal strains
Two strains of P. ultimum var. ultimum (LY-1 and LY-2) were used in this study. These strains
were collected from dead moss leaves in Longyearbyen (78°N, 15°E), Svalbard in 1997 (4).
Two strains of the Temperate Zone (OPU407 and OPU447), which were obtained from the
culture collection in Osaka Prefecture University (Japan), were used for comparison in this
study. All strains were maintained on corn meal agar (CMA, Difco, MD, USA) slants at 20°C.
Growth temperature
Mycelial discs of 5 mm in diameter were cut from the margin of an active growing colony,
transferred to the centers of CMA plates (9 cm in diameter), and cultured at 9 different
temperatures from 0 to 40°C, in duplicate. After 1, 2 and 3 days of cultivatio n, the colony
diameters were determined. The linear mycelial growth rate per day was calculated after an
initial lag period.
Determination of the mycelial chilling resistance of Pythium ultimum var. ultimum
Chilling resistance of mycelium was determined by the regrowth of mycelia after chilling stress.
Each strain was grown on potato dextrose agar (PDA, Difco, MD, USA) plates at 20°C and did
not produce sexual (oospore) or non-sexual organs (hyphal s welling) in PDA. Mycelial discs of
5 mm in diameter, cut from the margin of an active growing colony in PDA, were placed on
PDA plats 2.5cm in diameter and chilled to 4 or 0°C in a program freezer (MPF-1000, EYELA,
Tokyo, Japan) at a cooling rate of 10°C/h. After chilling, the mycelial discs were transferred to
fresh PDA plates and incubated at 20°C. Mycelial growth was observed daily for up to 3 days.
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RESULTS AND DISCUSSION
Chilling resistance of strains from the Arctic
The strains from the Arctic and Temperate Zone reacted differently to chilling stress. The
effects of chilling stress on selected strains (OPU407 for Temperate strain and LY-1 for
Svalbard strain) are shown in Fig. 2A. OPU407 strain was destroyed after chilling stress (0°C
Growth temperature
The taxonomic characteristics (morphologies of oogoniums, an
theridiums, oospores and
hyphal swellings) of strains (LY-1 and LY-
2) from Svalbard corresponded to those of strains
from the Temperate Zone (4). The effects of culture temperature on mycelial growth of strains
from Svalbard and Japan are shown in Fig. 1.
Mycelial growth of strains from Svalbard
occurred at temperatures between 0 and 30
°C, the optimum temperature being 25°C. These
strains were psychrotolerant and could grow at temperatures of less than 5
°C, whereas strains
from Japan (OPU407 and OPU447) did not grow at temperatures less than 5
°C. The mycelial
growth rates of strains from Japan were much lower than those of strains from Svalbard at 10
°C,
but the optimal growth temperature of strains from Svalbard was the same as that of strains
from Japan. Similar results were also obtained from Pythium
sp. HS group from the Arctic (2,
5) and Antarctica (8).
Tojo et al. isolated P. ultimum var. ultimum
from carrot seedlings growing in a greenhouse
in Barentsburg (78
°N, 14°E), Svalbard (16). This strain showed the same thermal dependence
of mycelial growth as did isolates from the Temperat
e Zone. These results suggested that the
strains isolated from dead moss leaves in Svalbard originally developed in the Temperate Zone
and adapted to the arctic conditions.
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for 3 days or 4°C at 1 week). The same results were obtained from strain OPU447 collected
from Hokkaido, Japan (data not shown). Strains from the Temperate Zone had lost their ability
to grow under snow cover and in early spring in the Arctic. On the other hand, strains LY-1 and
LY-2 from Svalbard survived after chilling stress.
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Irregular morphology by chilling stress
Figure 2B shows the hyphal morphology of the LY-1 strain during chilling stress. Chilling
stress did not cause temporary inhibition of mycelial growth, however, induced irregular
growth of hyphal tips. About 32% of the hyphal tips showed irregular morphology, a "knot like"
structure. It is known that Pythium spp. can form hyphal swellings (non-sexual organs) in
culture medium. However, these knot-like hyphal tips did not related to hyphal swellings. Some
studies have shown that heat (17, 18) and freezing (19) stresses induced changes in the hyphal
morphology of fungi. However, this is the first of chilling stress inducing the formation of
irregular morphology in fungi.
These knot-like hyphae were stained by Congo Red (data not shown). Dead hyphal cells of
Pythium was stained by Congo Red (20). Therefore, chilling stress induced partial necrosis in
the hyphal tips of the Arctic isolates. On the other hand, OPU407 and 447 strains did not show
such irregular morphology because strains from the Temperate Zone were destroyed after
chilling stress. However, OPU 407 and 447 strains were kept at 10°C (minimum growth
temperature) for 1 day, hyphae of these strains formed knot-like structure. Therefore, irregular
growth of P. ultimum var. ultimum is one of the responses against chilling stress.
Acknowledgment: This research project was financially supported by grants from Ministry of
Economy, Trade and Industry (Japan). T. Hoshino also thanks The Research Council of Norway
for financial support of this research.
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Accepted for publication 18/3/02
