Typhula cf. subvariabilis, new snow mould in Antarctica

ABSTRACT We collected snow blight of moss, Polytrichum juniperinum on King George Island, maritime Antarctica. Host died in a circle of about 10–30 cm after snow melts. Clamp connected hyphae and no sclerotia were observed on tip of host leaves. DNA sequence of ITS region from moss symptoms were perfectly matched with fruit bodies of Typhula sp. on Macquarie Island in the maritime Antarctica and high homology with Typhula cf. subvariabilis from Iran. Therefore, we suggested that T. cf. subvariabilis caused snow blight on moss in Antarctica, and this is first record of Typhula snow blight in Southern Hemisphere. These results also suggested that fungi in same genera gained similar ecological niche in both Polar Regions.


Introduction
Snow moulds are cryophilic (cold-adapted) fungi (Hoshino andMatsumoto 2012, 2013) and fungallike microbes, and they attack dormant plants such as forage crops, winter cereals and conifer seedlings under snow cover (Matsumoto and Hsiang 2016). Therefore, snow moulds are a generic name including diverse fungi and fungal-like microbes belonging to various taxa (oomycetes, ascomycetes and basidiomycetes). Important pathogens of agricultural crops from temperate to frigid zones in the Northern Hemisphere are Pythium spp. (e.g. Pythium iwayamai) in oomycetes, Microdochium nivale and Sclerotinia spp. (e.g. Spirorbis borealis) in ascomycetes and Typhula spp. (e.g. T. incarnata and Typhula ishikariensis complex) in basidiomycetes .
The snow moulds Pythium polare and related species occur frequently as pathogens of moss and vascular plants species in the Arctic and Antarctic, chiefly in maritime area (Petrov 1983;Tojo and Newsham 2012;. Two psychrophilic snow moulds Spirorbis borealis and Typhula ishikariensis are also widely distributed not only in the cool temperate zone and frigid zone but also in Arctic regions such as Alaska and the Yukon (Lebeau and Longston 1958;McBeath 2002), Greenland (Hoshino et al. 2006), Finnmark (northern Norway: Årsvoll 1975Matsumoto and Tronsmo 1995;Matsumoto et al. 1996), Iceland (S. borealis not found: Kristinsson and Guðleifsson 1976;Hoshino et al. 2004), Lapland (northern Finland and Sweden: Jamalainen 1949, 1957Ekstrand 1955), Svalbard (Hoshino et al. 2003), Russian Arctic . Their distribution pattern suggests that S. borealis and T. ishikariensis are highly adapted to the Arctic environment. S. borealis and T. ishikariensis were not found from Antarctica. However, Sclerotinia antarctica on Antarctic hair grass Deschampsia antarctica was found in Antarctic peninsula. The fungus is morphologically similar to S. borealis (Gamundi and Spideni 1987) and needs comparisons with S. borealis. However, there is no Typhula species of snow moulds recorded from Antarctica.
Basidiomycetous hyphae were observed in dead moss leaves from both Polar regions (Wilson 1951;Tojo and Newsham 2012), and these hyphae have less taxonomical features such as conidial and other sexual stages. We collected basidiomycetous hyphae in dead moss leaves on King George Island, South Shetland Islands, maritime Antarctica, and DNA sequences of these pathogens had high homology with Typhula species. In this paper, we described first record of snow mould Typhula species and their position in Antarctica.

Collection
The specimens of hyphae from snow blight of Polytrichum juniperinum were collected from coastal area in Geographer cove, Fildes Peninsula, King George Island (62°02′, 58°21′W), South Shetland Islands, Antarctica. The specimens of basidiocarps of Typhula sp. were also collected from Handspike Corner Herb. Fields, Macquarie Island (54°30′, 158°5 7′E), Australia. Dates of collection are shown below. These specimens were kept in the mycological herbarium of National Museum of Nature and Science (TNS), Tokyo, Japan.

Morphological observations
Colours of hyphae and basidiocarps were described according to the colour identification chart of the Royal Botanic Garden Edinburgh (Flora of British Fungi) (Royal Botanic Garden Edinburgh 1969).
For light microscopic observations of hyphae with moss shoots, dried or fresh specimens were mounted in water. For observation of hyphae in moss shoots using a transmission electron microscope (H-800, Hitachi Ltd., Tokyo, Japan), samples were prepared according to Malajczuk et al. (1977).

Phylogenetic analyses
DNA was extracted from the specimens by the protocol of DNeasy Plant Mini (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS) region of genomic rDNA was amplified with the primer pairs ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), as described by Hsiang and Wu (2000). The polymerase chain reaction (PCR) product was purified using a QIAquick PCR Purification Kit (Qiagen) and sequenced on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using the primer ITS1.
Multiple alignments of the ITS sequences were performed, and the nucleotide substitution rate was calculated. A phylogenetic tree was constructed by the neighbour-joining method (Kimura 1980;Saitou and Nei 1987) using the programme CLUSTAL W (Thompson et al. 1994) with bootstrap values based on 1000 replications (Felsenstein 1985).

Symptoms of snow blight of Antarctic moss
The moss colonies of P. juniperinum were spread over wet or submerged areas near Great Wall Station (Chen et al. 1993). Many moribund moss colonies were found in P. juniperinum after the snow bed had melted (Figure 1). Host died in a circle of about 10-30 cm after snow melts. In our observations, many fungal infections in moss were found in moss vegetation near the seashore in the research area. However, we could not find any obvious disease in moss along the coasts of Svalbard (Hoshino et al. 1999). Fungal infections were seen in circular patterns or irregular patches, and sclerotina were not observed in these symptoms. Mycelia on moss shoots were visible to the naked eye immediately after thawing (Figures 1(b) and 2 (a)). Similar observation was reported from both Polar regions (Tojo and Newsham 2012). Figures 2 and 3 show microscopic images of a moribund leaf of P. juniperinum. Clamp connected hyphae mycelia grew well on the moss leaf and were intertwisted at the growth point of the moss (Figure 2). Several numbers of hypha-like structures observed in host moss cells (Figure 3). These hyphalike structures were almost similar size of those of T. ishikariensis infected in bentgrass (Oshiman et al. 1995). We also obtained DNA from fungal infected moss shoots and only one kind of fungal ITS sequence from these specimens (detail was described in next section). Therefore, we regarded that one pathogenic fungus caused snow mould disease with P. juniperinum in Antarctica. Unfortunately, we did not success to isolate fungi in moribund moss leaves. In Arctic, Wilson (1951) studied infections of Rhacomitrium carpet on Jan Mayen Island and reported that the moss disease was caused by an unidentified basidomycete.
Phylogenic analysis and morphological characteristics of Typhula sp. from Macquarie Island ITS sequences were obtained from two moss symptoms (LC005082 and LC005083, Table 1). Phylogenetic comparisons of our specimens with previous data on ITS sequences of Typhula spp. and related genera are shown in Figure 4. Fungi in moss symptoms were perfectly matched with the basidiocarp of Typhula sp. GAL006938 collected on Macquarie Island, Subantarctica, Australia, and was highly homology with Typhula sp. Wh-1 that caused snow mould disease to winter wheat in Iran (Hoshino et al. 2007). DNA sequence data also indicated that this fungus in moss cells is in the genus Typhula, and this is first record of Typhula snow blight in Antarctica.
These morphological characteristics of basidiocarps of Typhula sp. GAL006938 and sclerotia of Typhula sp. Wh-1 were similar with Typhula subvariabilis (Berthier 1974;Berthier 1976). Several saprophytic species of Typhula and related genera of Macrotyphula and Pistillaria are formed basidiocarps from sclerotia and substrates. Those species are psychrotrophic and if their mycelium can survive and     grow at environmental condition, they form the basidiocarps directory from substrates (Hoshino et al. unpublished results). On subantarctic islands such as King George Island (62°02′, 58°21′W) and Macquarie Island (54°30′, 158°57′E), annual ground temperature is enough low for mycelial growth of Typhula sp. Therefore, this fungus formed basidiocarps directory from substrates and we did not find sclerotium on these symptoms. We regarded that Typhula sp. caused snow mould disease on moss colonies in Antarctic Islands and this pathogen was similar with T. subvariabils (T. cf. subvariabiris).

Evolution of snow moulds in Antarctica
We found fungal disease on the moss, Polytrichum juniperium on King George Island, Antarctica. Sclerotia were not found on diseased plants; however, molecular data suggested that Typhula cf. subvariabilis was present there. These findings suggest that three genera of representative snow moulds Pythium, Sclerotinia and Typhula could share the same niches in both Polar regions.
Other fungal species are also recognised as snow moulds in Antarctica (Tojo and Newsham 2012). Zygomycetous snow moulds are not found in the Northern Hemisphere, but Rhizopus sp. was pathogen on the moss Bryum anatrcticum at Cape Bird on Ross Island (Greenfield 1983). Unfortunately, the specimens are not available (Greenfield, personal communication). Several ascomycetes such as Thyronectria antarctica var. hyperantarctica, Coleroa turfosorum, Bryosphaeria megaspora, Epibryon chorisodontii and an unidentified Plectomycete were recorded from ring infection and macroscopic section of mosses (Hawksworth 1973;Longton 1973;Fenton 1983). Cryophilic fungi are less in the number of species than that of fungi in temperate region , and flora in Polar regions are different from other regions of cryosphere in temperate and frigid zones. Therefore, there must be new niches for cryophilic fungal phytopathogens in Polar regions, and novel cryophilic fungi are likely to have evolved as phytopathogens to adapted in polar climate and vegetation.
Pathogenicity and cold-adaptation are essential factors for fungi to become cryophilic fungal phytopathogens, and these factors co-evolved in Typhula (Hoshino 2005). T. ishikariensis complex well adapted Arctic environment. However, their basidiospores did not adapt in long-distance dispersionhave by airborne (Cunfer and Bruehl 1973); therefore, this fungus was not found from low latitude snowy regions (Hoshino et al. 2007). Antarctica is geographically isolated from other continents. T. cf. variabilis was distributed in low latitude snowy regions such as Middle East, and their basidiospores probably succeeded to survive and adapt in the Antarctic environments. We should survey the geographical distribution of T. cf. subvariabilis around Antarctica.