Taxonomic revision of the Typhula ishikariensis complex

Abstract

Typhula ishikariensis and the related fungi were separated into three biological species by morphological and physiological characteristics, as well as DNA sequences and mating reactions. We propose that the T. ishikariensis complex should be divided into three species (T. ishikariensis, T. canadensis and T. hyperborea) and two varieties (T. ishikariensis var. ishikariensis and var. idahoensis). Typhula hyperborea was reappraised to be recognized also as a separate species of the T. ishikariensis complex.

1. Introduction

Snow mold is incited by many fungi that attack dormant plants such as forage crops, winter cereals and conifer seedlings under snow cover (Matsumoto & Hsiang, 2016). Although some of their taxonomic and ecological features have only recently been elucidated (e.g., Saito, 1998; Hoshino, Tkachenko, Kiriaki, Yumoto, & Matsumoto 2004b; Ikeda, Hoshino, Matsumoto, & Kondo, 2015), the taxonomic confusion of the most important fungus, Typhula ishikariensis S. Imai still remains unsolved, and requires thorough comparison. McDonald (1961), after literature review, concluded that T. ishikariensis had priority among related fungi. Since then, different fungi related to T. ishikariensis were reported from different regions by different authors such as Bruehl and Cunfer (1975), Årsvoll and Smith (1978), Matsumoto, Sato and Araki (1982), and Matsumoto, Tronsmo and Shimanuki (1996).

Imai (1930) first described this fungus on dead leaves and stems of winter wheat and red clover in Hokkaido, Japan. A similar fungus, T. idahoensis Remsberg was described as a snow mold pathogen on winter wheat in the USA (Remsberg, 1940a, 1940b). In Scandinavia, Ekstrand (1937, 1939, 1955) also found similar fungi and named T. borealis H. Ekstr. 1955 and T. hyperborea H. Ekstr. 1955. His first diagnoses of T. borealis at 1937, 1939 and T. hyperborea at 1937 were invalid (no Latin diagnosis or description; ICN Shenzhen Code Art. 39.1). Legitimate nomenclatures accompanied with Latin diagnoses of both species were published 1955 (Ekstrand).

Tomiyama (1955) recognized the significance of T. ishikariensis as an important snow mold pathogen on winter wheat in Hokkaido, Japan. Gulaev (1948) described T. graminearum Gulaev Nom. Inval. Art. 39.1 (ICN Shenzhen Code) on grasses and conifer seedlings, and Kuznetzova (1953) also described T. humulina Kuznezowa on hop roots in Russia. Potatosova (1960a, 1960b) on the basis morphological features considered these species to be synonyms of T. idahoensis, and Hoshino et al. (2004b) suggested that T. graminearum and T. humulina should be regarded as T. ishikariensis by mating experiments.

Later in the USA, Bruehl and Cunfer (1975) claimed that T. ishikariensis and T. idahoensis should be regarded as separate species based on morphology, host range, and distribution. They also revealed genetic isolation of both species by mating experiments (Bruehl, Machtmes, & Kiyomoto, 1975). However, T. ishikariensis and T. idahoensis mated rarely (Bruehl et al., 1975), but Christen and Bruehl (1979) confirmed that their hybrids were virulent and capable of survival in nature. Bruehl and Machtmes (1980) ultimately concluded that these two species were indistinguishable based on culture morphology.

Årsvoll and Smith (1978), on genetic evidence, regarded isolates from Europe and North America as a single species and established three varieties in the T. ishikariensis complex, i.e., var. ishikariensis S. Imai, var. idahoensis (Remsberg) Årsvoll & J.D. Sm., and var. canadensis J.D. Sm. & Årsvoll based on sclerotial rind cell pattern.

Matsumoto et al. (1982) found two biological species (biotypes A and B) in Japan that were sexually incompatible with each other. They also differed in morphology, distribution, host range, and pathogenicity (Matsumoto, 1989) - different enough to be regarded as separate species. However, both biotypes were genetically related through the medium of North American fungi (Matsumoto, Sato, Araki, & Tajimi, 1983). Matsumoto et al. (1996) divided T. ishikariensis strains from Norway into three groups based on mating reactions of tester monokaryons of biotypes A and B. Isolates of group I, sharing common culture morphology (Matsumoto & Tronsmo, 1995), were basically compatible with biotypes A but showed various mating reactions. Group II was compatible with biotype B, and group III was not compatible with either biotype.

These results imply that populations of T. ishikariensis in each locality are genetically separate to various extent but that they have potential to exchange genetic traits especially under laboratory conditions. Hsiang and Wu (2000) examined phylogenic relationships of T. ishikariensis in North America to detect differences at an infraspecific level, using internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP). Thus, the situation of the T. ishikariensis complex is comparable to that of Armillaria mellea (Vahl) Kumm. (Cha, Sung, & Igarashi, 1994) and Heterobasidion annosum (Fr.) Bref. (Garbelotto & Gonthier, 2013).

In this study, we aimed to elucidate the extent of variation within the T. ishikariensis complex, using 480 strains from various localities in Northern Hemisphere based on di-mon mating reactions, phylogenic analyses, morphological characteristics, host range and their distribution. The results obtained here indicate biological species differentiation sufficient to propose a new universal nomenclature system of the T. ishikariensis complex.

2. Material and methods

2.1. Fungal strains from overwintered plants

Fungal sclerotia were collected from decayed leaves and stems of various plants during our surveys (Table 1). Collected sclerotium samples were packed in paper envelopes and dried at room temperature during transportation. Fungal sclerotia were surface-sterilized first in 70% (v/v) ethanol and in 0.5% (as active chlorine) sodium hypochlorite solution and, then, thoroughly rinsed in sterilized distilled water. They were then cut with sterilized steel blades, placed on potato dextrose agar (PDA: Difco, Sparks, MD, USA) and incubated at 4°C. Mycelia from growing colony margins were transferred and maintained on PDA slants at 0°C.

Table 1 - Biological species separation within the strains belonging to the Typhula ishikariensis complex based on mating reaction of testers.

Localities a	Number of strains designated as b
	Biological species I		II	III
	A+ B+	A+ B-	A-B+	A-B-
West Greenland (7)	0	0	0	48
East Greenland (3)	1	0	0	13
Iceland (3)	0	4	2	0
Svalbard (4)	0	0	0	3
Tromsø og Finmark, Norway (3)	0	0	2	58
Switzerland (5)	0	72	0	1
Liechtenstein (2)	0	5	0	0
Austria (2)	0	5	0	0
Russia
European (8)	0	31	0	7
Siberia (7)	0	41	21	8
Far East (12)	0	25	8	18
Hokkaido, Japan (3)	0	38	45	0
Alaska, USA (3)	0	0	2	0
Quebec, Canada (2)	3	13	2	4

^a Figures in parentheses indicate the number of sampling site.

^b Figures indicate the number of strains. Isolates belonging to different vegetative compatible groups (VCGs) were regarded as separate strains. VCGs between localities were not determined. A: biotype A; B: biotype B; +: compatible; -: incompatible.

2.2. Production of basidiomata

Sclerotia formed on oatmeal agar (Difco) plates were placed on the surface of a mixture of humid unsterile commercial artificial soil (Hokusan Co. Ltd., Kita-Hiroshima, Japan) and sea sand (14−20 mesh, Wako Pure Chemical Industries, Ltd., Osaka, Japan) in glass dishes (55 mm diam, 45 mm in height) and incubated under the fructification regime of 12 h light at 10°C and 12 h dark at 4°C up to two mo (Kawakami, Matsumoto, & Naito, 2004).

2.3. Morphological observations

Colors of basidiomata and sclerotia were described according to the color identification chart of the Royal Botanic Garden Edinburgh (Flora of British Fungi) (Anonymous, 1969). Basidiospores from fresh specimens were mounted in water for light microscopic examination. About 30 basidiospores were randomly chosen for determination of length and width excluding the apiculus. Surface features of basidiomata and sclerotia were observed by phase-contrast microscopy and scanning electron microscopy (SEM). For SEM, basidiomata and sclerotia were cut on a piece of double-sided adhesive tape attached to a specimen holder and then coated with platinum-palladium using a JFC-1100 Ion Sputter (JEOL, Tokyo, Japan). They were examined using a JSM-T330A SEM (JEOL) operating at 10 kV.

2.4. Mating experiments

Basidiomata were soaked separately in 500 μL autoclaved water in test tubes and kept overnight in a refrigerator (4°C). Test tubes were shaken to remove basidiospores from basidiomata. Basidiospore suspension was appropriately thinned, spread on PDA plates containing lactic acid, and incubated at 4°C for two weeks. Colonies with smooth hyphae (monokaryons) were subcultured on PDA slants at 4°C. Monokaryons of biotypes A (strains PR7-6-7 and PR9-4-3 from Japan) and B (strains 35-8 and 8-2 from Japan) were designated as testers and paired with dikaryons of collected strains (di-mon mating; Bruehl et al., 1975) on PDA plates and incubated at 4°C for one month. A small agar block was cut from monokaryon colonies near the colony junction and transplanted to another PDA plate. Growth from the block was then examined for the presence of clamp connections on hyphae after incubation for 5 to 7 days at 4°C. The presence of clamp connections on hyphae was the criterion of mating compatibility.

2.5. Phylogenic analyses

Sclerotia were harvested from one-month-old PDA cultures grown at 4°C, and DNA was extracted by the protocol of DNeasy Plant MiniPrep (QIAGEN GmbH, Hilden, Germany). ITS1-5.8S-ITS2 (ITS) region of genomic rDNA were amplified using the primer pairs ITS1 and ITS4 (White, Bruns, Lee, & Taylor, 1990), and mitochondrial large ribosomal RNA (the partial mitochondrial LrRNA) with ML5 and ML6 (Bruns et al., 1998). PCR products were purified using QIAquick PCR Purification Kit (QIAGEN GmbH, Germany) and sequenced in one direction on ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using each primer set. A total of 186 sequences newly generated from the present study were deposited in DDBJ/EMBL/GenBank (Table 2).

Table 2 - Sequenced specimens used this study.

Fungal name	Voucher no.	Strain no.	Localities	Accession numbers
				ITS a	ML b
Pistillaria petasitis		PP1, HIT	Hokkaido, Japan	LC005088	LC192706
Typhula incarnata		IS11in, HIT	Akureyri, Iceland		LC192600
		IS12in, HIT	Glaumbæ, Iceland		LC192599
		IS13in, HIT	Reykholt, Iceland	LC192841	LC192598
		S1in, HIT	Skælingur, Faroe Is.		LC192602
		V1in, HIT	Vestmanna, Faroe Is.		LC192601
		Rin, HIT	Błonie, Poland	LC192842	LC192603
		Komi1in, HIT	Komi, Russia		LC192606
		Y1in, HIT	Mari El, Russia	LC192843	LC192605
	IRAN 11961F	H-I2, HIT	East Azerbaijian, Iran	AB267391	LC192604
		IK1in, HIT	Irkutsk, Russia	LC192844	LC192607
		SD1in, HIT	Sakhalin, Russia	LC192845
		SD11in, HIT	Sakhalin, Russia		LC192609
		YS1in, HIT	Sakhalin, Russia	LC192608	LC192608
		QBC1in, HIT	Quebec, Canada		LC192610
T. intermedia		TI1, HIT	Hokkaido, Japan		LC192704
T. ishikariensis var. ishikaiensis	TNS-F-24965	IS-1, HIT	Akureyri, Iceland	LC192584	LC192611
	TNS-F-24696	IS-3, HIT	Akureyri, Iceland	LC192585	LC192612
	TNS-F-24697	IS-4, HIT	Akureyri, Iceland	LC192846	LC192613
		OPU1809	Buskerud, Norway	LC192577	LC192615
		2-2-5, NIBIOd	Oppland, Norway	LC192578	LC192616
		5-5-11, NIBIO	Troms og Finnmark, Norway		LC192618
		KP-10, MBGe	Murmansk, Russia	LC192569	LC192628
	TNS-F-24681	KP-13, MBG	Murmansk, Russia	LC192851	LC192629
	TNS-F-24682	KP-16, MBG	Murmansk, Russia	LC192570	LC192630
	TNS-F-24683	SPb-2, HIT	St Petersburg, Russia	LC192571	LC192625
		SPb-4, HIT	St Petersburg, Russia		LC192626
	TNS-F-24684	SPb-6, HIT	St Petersburg, Russia	LC192572	LC192627
	TNS-F-24679	92-Tr-13, MBG	Moscow, Russia	LC192567	LC192631
	TNS-F-24680	MSC-1, HIT	Moscow, Russia	LC192568	LC192632
	TNS-F-24676	C-2, HIT	Chuvashia, Russia	LC192565
	TNS-F-24677	C-4, HIT	Chuvashia, Russia	LC192566	LC192633
	TNS-F-24678	C-6, HIT	Chuvashia, Russia	LC192852	LC192634
	TNS-F-24675	T. humulina, HIT	Mari El, Russia	LC192564	LC192638
	TNS-F-2673	Y-2, HIT	Mari El, Russia,	LC192562	LC192635
		Y-6, HIT	Mari El, Russia,		LC192637
		E-2, HIT	Sverdlovsk, Russia	LC192558	LC192641
	TNS-F-24670	E-5, HIT	Sverdlovsk, Russia	LC192854	LC192643
	TNS-F-24671	T. graminearum, HIT	Sverdlovsk, Russia	LC192560	LC192644
	TNS-F-24663	N3A3, HIT	Novosibirsk, Russia	LC1928554	LC192645
	TNS-F-24664	N4A1, HIT	Novosibirsk, Russia	LC192555	LC192646
		N4A3, HIT	Novosibirsk, Russia	LC192855	LC192647
		N4B2, HIT	Novosibirsk, Russia	LC192856	LC192648
		N5-1, HIT	Novosibirsk, Russia		LC192649
		IK1-1, HIT	Irkutsk, Russia		LC192651
		IK2-1, HIT	Irkutsk, Russia		LC192652
		IK3-1, HIT	Irkutsk, Russia		LC192653
	TNS-F-24661	YS1-1, HIT	Sakhalin, Russia	AB194769
		YS3-3, HIT	Sakhalin, Russia	AB127951	AB187583
		YS5-3, HIT	Sakhalin, Russia	AB194770
		YS8-2, HIT	Sakhalin, Russia	AB194771
		Y10-1, HIT	Sakhalin, Russia	AB194772
		Al-222, HIT	Hokkaido, Japan	LC192859	LC192673
	TNS-F-24716	MAFF306136	Hokkaido, Japan	AB127949	LC192671
		MF1-5, HIT	Hokkaido, Japan	LC192858	LC192672
		Rebun, HIT	Hokkaido, Japan	AB127950
		PR7-6, HIT	Hokkaido, Japan	AB127952	LC192670
		Noheji, HIT	Aomori, Japan	LC192860
	TNS-F-24704	QBC4-1, HIT	Quebec, Canada	LC192591	LC192677
		QBC7-1, HIT	Quebec, Canada		LC192680
T. ishikariensis var. idahoensis	CUP25153		Montana, USA	LC613246
	CUP27221		Idaho, USA	LC192550
	TNS-F-24709	ida1-72, HIT	North America	LC192595	LC192675
	TNS-F-24710	ida2-101, HIT	North America	LC192596	LC192676
		idal-3, HIT	North America		LC192674
T. canadensis		KUS2-1, HIT	Kulusk, Greenland		LC192701
		IS5, HIT	Akureyri, Iceland		LC192614
	TNS-F-24692	5-2-21, NIBIO	Troms og Finnmark, Norway	LC192581	LC192622
	TNS-F-24693	5-4-13, NIBIO	Troms og Finnmark, Norway	LC192582	LC192623
		IK4-3, HIT	Irkutsk, Russia		LC192654
		KPK2-1	Kamchatka, Russia		LC192661
		KPK3-1, HIT	Kamchatka, Russia		LC192662
		KPK4-1, HIT	Kamchatka, Russia		LC192663
		KPK5-1, HIT	Kamchatka, Russia		LC192664
		KPK11-2, HIT	Kamchatka, Russia		LC192666
		KS1-1, HIT	Kamchatka, Russia		LC192659
	TNS-F-24659	KS1-2, HIT	Kamchatka, Russia	LC192574	LC192660
	TNS-F-24662	SD-1, HIT	Sakhalin, Russia	LC192553	LC192658
		YS2-1, HIT	Sakhalin, Russia		LC192655
		YS3-1, HIT	Sakhalin, Russia		LC192656
	DAOM 160550		Saskatchewan, Canada	LC192551
	TNS-F-24707	QBC7-4, HIT	Quebec, Canada	LC192594	LC192681
		QBC10-1, HIT	Quebec, Canada		LC192682
		QBC10-2, HIT	Quebec, Canada		LC192683
		Cam14, HIT	North America		LC192687
		Can II, HIT	North America		LC192686
	TNS-F-2411	DE7228, HIT	North America	LC192597	LC192685
T. hyperborea	TNS-F-24694	FERM P-18741	Barentsburg, Svalbard	LC192583	LC192624
		SSM1-1, HIT	Sisimiut, Greenland		LC192688
	TNS-F-24703	SSM2-1, HIT	Sisimiut, Greenland	LC192588	LC192689
		SSM3-1, HIT	Sisimiut, Greenland		LC192690
	TNS-F-24698	OPU1811	Nuuk, Greenland	LC192586	LC192691
	TNS-F-24699	OPU1812	Nuuk, Greenland	LC192587	LC192692
	TNS-F-24700	OPU1814	Nuuk, Greenland	LC192862	LC192693
		OPU1816	Nuuk, Greenland		LC192694
		AGM1-1, HIT	Tasiilaq, Greenland		LC192695
		AGM2-1, HIT	Tasiilaq, Greenland	LC613245	LC192696
		AGM4-1, HIT	Tasiilaq, Greenland		LC192697
	TNS-F-24701	AGM5-1, HIT	Tasiilaq, Greenland	LC192589	LC192698
		KUS1-1, HIT	Kulusk, Greenland		LC192699
	TNS-F-24703	KUS1-2, HIT	Kulusk, Greenland	LC192590	LC192700
	TNS-F-24687	OPU1810	Oppland, Norway	LC192579	LC663960
	TNS-F-24688	5-4-9, NIBIO	Troms og Finnmark, Norway	LC192847
	TNS-F-24689	6-1-1, NIBIO	Troms og Finnmark, Norway	LC192580	LC192619
	TNS-F-24690	6-1-9, NIBIO	Troms og Finnmark, Norway	LC192848	LC192620
	TNS-F-24691	Tana, NIBIO	Troms og Finnmark, Norway	LC192849	LC192621
	TNS-F-24674	Y4-2, HIT	Mari El, Russia	LC192563	LC192563
	TNS-F-24672	K-1, HIT	Tatarstan, Russia	LC192561	LC192639
	TNS-F-24666	E-1, HIT	Sverdlovsk, Russia	LC192557	LC192640
	TNS-F-24668	E-3, HIT	Sverdlovsk, Russia	LC192853	LC192642
	TNS-F-24669	E-4, HIT	Sverdlovsk, Russia	LC192559
	TNS-F-24665	N6A-1, HIT	Novosibirsk, Russia	LC192556	LC192650
	TNS-F-24658	KPK8-2, HIT	Kamchatka, Russia	LC192573	LC192665
		KK2-1, HIT	Kamchatka, Russia		LC192668
	TNS-F-24660	KK8-1, HIT	Kamchatka, Russia	LC192575	LC192669
		KE1-1, HIT	Kamchatka, Russia		LC192667
	TNS-F-24657	AND1-1, HIT	Chukotka, Russia	LC192576
	TNS-F-24705	QBC5, HIT	Quebec, Canada	LC192592	LC192678
	TNS-F-24706	QBC6, HIT	Quebec, Canada	LC192593	LC192679
	TNS-F-24708	QBC13, HIT	Quebec, Canada	LC192861	LC192684
T. intermedia	SAPA 100038	MAFF 244400	Hokkaido, Japan	AB267394	LC192704
T. maritima	TNS-F-17093	NBRC 104266	Hokkaido, Japan		LC192703

^a 18S rRNA, ITS1, 5.8S rRNA, ITS2, LSU rRNA, partial and complete sequence.

^b mitochondrial gene for large subunit ribosomal RNA, partial sequence.

^c Hachinohe Institute of Technology, Aomori, Japan.

^d Norwegian Institute of Bioeconomy Research, Ås, Norway.

^e N.V. Tsitsin Main Botanical Garden, Russian Academy of Sciences

Representative sequences of Typhula spp. and related fungi were included in the dataset of the present phylogenetic analyses based on the recent phylogenetic study on typhuloid fungi reported by Olariaga, Huhtinen, Læssøe, Petersen, & Hansen (2020). Each ITS and the partial mitochondrial LrRNA dataset consisted of 129 or 109 taxa including two outgroups, Pleurotus ostreatus (Jacq.) P. Kumm. and P. eryngii (DC.) Quél., following Olariaga et al. (2020). Namely, each dataset was aligned with Muscle v.3.6 (Edgar, 2004a, b) and then, manually edited in BioEdit v.7.0.1 (Hall, 1999). Ambiguously aligned regions were excluded from the analyses. The final alignments were deposited in TreeBASE (https://treebase.org) under the accession number S27664. Phylogenetic analyses were performed for ITS and the partial mitochondrial LrRNA datasets under maximum likelihood (ML) and Bayesian inference (BI). Briefly, the analyses were performed using MEGA X (Kumar et al., 2018) after testing the best models. According to the lowest BIC (Bayesian Information Criterion) scores, K2 and T92+G were chosen as the optimal substitution models for the analyses of the ITS and the partial mitochondrial LrRNA datasets, respectively. BI analyses were conducted by the same method reported by Kasuya and Ono (2018), using MrBayes v.3.0b4 (Huelsenbeck & Ronquist, 2001). In BI analyses, the best-263 fit substitution models for the ITS and the partial mitochondrial LrRNA datasets were estimated using MrModeltest v.3.7 (Nylander, 2004) based on the hierarchical likelihood-ratio test (hLRT). The GTR+G+I model was selected as the best evolutionary model for each dataset.

2.6. Characterization of Typhula hyperborea

2.6.1. Growth temperature relations

Mycelial discs of 5 mm diam were cut from the margins of actively growing colonies, transferred to the center of PDA plates, and incubated at 5 different temperatures from 0 to 25°C, in triplicates. After 1, 2 and 3 wk of incubation, colony diameters were measured. The linear mycelial growth rate per week was calculated after initial lag period.

2.6.2. Pathogenicity

Five each of Engmo timothy seedings were transplanted to 9 cm plastic pots filled with steamed, fertilized peat/soil mixture, and grown for 6 wk in a greenhouse at 12−18°C, supplemented with 8000 lux form cool white and warm fluorescent light for 12 h, and hardened at 1°C for 2 wk under the same light conditions, according to Tronsmo (1984, 1985).

A total of 4 pots were used for inoculation experiments for each strain. Inoculated plants were incubated under the simulated snow cover conditions at 1°C for 8 wk, as described by Årsvoll (1977). Plant damage was evaluated three weeks after recovery in the greenhouse. Inoculation experiments were all made in Ås, Norway.

3. Results and discussion

3.1. Biological species designation

Strains of the T. ishikariensis complex collected from snowy regions of Northern Hemisphere were divided into three biological species based on mating reactions of testers of biotypes A and B: biological species I strains were mostly compatible only with biotype A and included those compatible with both biotypes; biological species II strains were all compatible exclusively with biotype B; and both biotypes did not react when paired with biological species III (Table 1).

3.2. Phylogenic analyses

The ITS dataset consisted of 127 ingroups and two outgroup taxa. It had an aligned in length of 763 characters including gaps, of which 37 characters were constant, 402 variable and phylogenically uninformative, and 324 phylogenically informative. The partial mitochondrial LrRNA dataset consisted of 107 ingroup and two outgroup taxa. It had an aligned length of 1088 characters including gaps, of which 19 characters were constant, 700 variable and phylogenetically uninformative, and 318 phylogenetically informative.

The ML and BI analyses resulted in trees that were almost identical in topology for both of ITS and the partial mitochondrial LrRNA sequence datasets. Hence, only the ML trees with the highest log likelihood (-1006.12 and -832.25) were shown in Figs. 1 and 2 for ITS and the partial mitochondrial LrRNA, respectively. Phylogenetic analyses revealed the monophyly of Typhula from outgroups (Pleurotus spp.) and also that of the T. ishikariensis complex for Typhula of ITS (Fig. 1: 82/100, 82/0.61) and the partial mitochondrial LrRNA (Fig. 2: 100/1.00, 89/0.74).

Fig. 1 − A phylogenetic tree of partial ITS sequences of selected typhuloid fungi including Typhula ishikariensis complex constructed by the ML method, inferred by using K2 model. Bootstrap values (BS) of maximum likelihood greater than 50% and Bayesian posterior probabilities (PP) above 0.5 are shown along the nodes in the topology (BS/PP). Scale bar indicates the number of substitutions per site.

Circles: localities from Asia including Russian Far East and Siberia; triangle: from Europe; squares: from North America and Greenland. Diamond: specimens or strains annotated in T. idahoensis from North America. White: biological group I (A+ B-); grey: II (A- B+); black: III (A- B-).

Fig. 2 − A phylogenetic tree of the partial mitochondrial LrRNA sequences of selected typhuloid fungi including Typhula ishikariensis complex constructed by the ML method, inferred by using T92+G model. A discrete Gamma distribution was used to model evolutionary rate differences among site [5 categories (+G, parameter = 0.4547)]. Bootstrap values (BS) of maximum likelihood greater than 50% and Bayesian posterior probabilities (PP) above 0.5 are shown along the nodes in the topology (BS/PP). Scale bar indicates the number of substitutions per site. Symbols were same as figure 1.

We here referred to biological species I, II, and III as T. ishikariensis, T. canadensis stat. nov. (syn. T. ishikariensis var. canadensis), and T. hyperborea, respectively. The partial mitochondrial LrRNA supported biological species separation (T. ishikariensis including both varieties: 97/0.97, T. canadensis: 97/0.92, T. hyperboea: 99/0.97). However, mating reactions and phylogenic clades were discrepant in some strains due to their anomalities in mating reactions as follows: i) T. ishikariensis var. idahoensis strains, belonging to biological species II, constituted a distinct subclade within the clade of T. ishikariensis var. ishikariensis (Figs. 1, 2). The phylogenic evidence as well as morphological similarities of both fungi (Bruehl & Machtems, 1980) led us regard T. ishikariensis var. idahoensis as a valid taxon within the T. ishikariensis complex. ii) A group of T. ishikariensis strains from North America was compatible with both testers of biotypes A and B and considered as hybrids between var. ishikariensis and var. idahoensis (Årsvoll & Smith, 1979; Christen & Bruehl, 1979), and some strains in Norway showed the same feature (Matsumoto et al., 1996). We failed to determine sequences of both ITS and the partial mitochondrial LrRNA and were, consequently, unable to conduct their phylogenic analyses. These strains were identified as T. ishikariensis var. ishikariensis on morphological evidence; iii) there were three strains of T. canadensis from North America that mated with biotype A testers (Table 1; Fig. 1); and iv) the T. hyperborea clade included a strain from Wisconsin, USA that mated with biotype B and biological species III (Fig. 1) and showed normal growth on PDA at 10°C (Millett & Maxwell, 1997; Millet, 1999). T. hyperborea usually failed to show normal growth on PDA at 10°C (Matsumoto et al., 1996; Hoshino, Tronsmo, Matsumoto, Ohgiya, & Ishizaki, 1997).

3.3. Morphological characteristics

Cultural morphology and physiological characteristics of the T. ishikariensis complex were well described by many authors (e.g., Remsberg, 1940a; Ekstrand, 1955; Potatosova, 1960a; Årsvoll & Smith, 1978; Bruehl & Machtmes, 1980, Smith, 1987; Smith, Jackson, & Woolhouse, 1989; Matsumoto, 1989; Matsumoto & Tajimi, 1991; Matsumoto et al., 1996; Hoshino, Kiriaki, Yumoto, & Kawakami, 2004a; Hoshino et al., 2004b). We summarized taxonomically important characteristics of the T. ishikariensis complex including our findings in Table 3.

Table 3 - Summary of taxonomic characteristics of the Typhula ishikariensis complex.

Taxonomic species	Mating reaction a	Mycelia growth on PDA		Sclerotia			Basidiomata		Host	Distribution
		at 0 c	at 10˚C	Formed on host tissues	Color	Size (mm)	Caulocystidia (µm)	Size of spore (µm)
T. ishikariensis var. ishikariensis	A+, B-	less aerial mycelia	normal	easily dislodged	light to dark brown	0.5−2	short (>50)	2.5−5.0×7.5−12.3	monocots, dicots, Gymnosperm	Northern Hemisphere
T. ishikariensis var. idahoensis	A-, B+	less aerial mycelia b	normal	frequently subepidermal	light brown to almost black	0.5−2	mixed short (>50, main) and long (50-120, rare)	2.5−8.0×6.5−13.0	almost monocots	North America
T. canadensis	A-, B+	aerial mycelia abundant in some strains	normal	frequently subepidermal	light brown to almost black	0.2−2	long (50−120)	2.7−4.5×7.8−12.6	almost monocots	Northern Hemisphere absent from central Asia to middle Europe
T. hyperborea	A-, B-	aerial mycelia abundant	irregular (feather-like) without strains from Greenland c	easily dislodged	light to dark brown	0.5−2	short (>50)	3.7−7.1×7.6−14.8	almost monocots	Arctic and Boreal Zone of North Europe and Russia, Alps, North America

^a see Table 1 for detail. Each taxon but var. idahoensis includes strains that showed anomalous mating reaction. For detail, see the test.

^b Remsberg (1940) and Ǻrsvoll & Smith (1978) suggested abundant aerial mycelia in this species. However, Bruehl & Machtmes (1980) reported that T. ishikariensis and T. idahoensis were similar in cultural morphology among ca. 400 isolates they tested.

^c Strains from Nuuk, West Greenland showed normal growth on PDA at 10˚C.

Aerial mycelia of T. ishikariensis are more or less appressed to PDA plates and less fluffy as compared to T. canadensis and T. hyperborea. Remsberg (1940a) and Årsvoll and Smith (1978) described the abundant production of aerial mycelia by T. ishikariensis var. idahoensis. However, Bruehl and Machtmes (1980), after examination of ca. 400 isolates of T. ishikariensis and T. idahoensis (= T. ishikariensis var. idahoensis), concluded that both taxa were indistinguishable in culture morphology. Smith (1987) and Smith et al. (1989) validated Bruehl's observations. Typhula canadensis from Alaska (Anchorage), Canada, Iceland, Norway and Russian Far East (Magadan) has fluffy aerial mycelia on PDA (e.g., Årsvoll & Smith, 1978; Matsumoto et al., 1996; Matsumoto 1997; Hoshino et al., 2004a; Tkachenko, 2013). Especially, strains with small sclerotia of T. canadensis produce aerial mycelia abundantly (Årsvoll & Smith,1978). Since, T. hyperborea sensu H. Ekstr. also has abundant aerial mycelia under cultural conditions (Ekstrand, 1955). Some strains of T. hyperborea from abundant aerial mycelia on PDA even at 0°C (Matsumoto et al., 1996), especially after freezing at -40°C (Hoshino et al., 1998).

Four specimens of T. ishikariensis sensu stricto collected by Imai were available from The Tottori Mycological Institute in Japan (Fig. 3A−D). The host plant and locality of the specimen suggested that Imai collected T. ishikariensis var. ishikariensis. Their sclerotia had turned dark brown (19 bay) due presumably to desiccation (Fig. 3B−D). Typhula ishikariensis var. ishikariensis strain, MAFF306136 from alfalfa (Medicago sativa L.) was similar to Imai's specimen (Fig. 3E). Typhula ishikariensis var. ishikariensis (represented by biotype A) has brown sclerotia on host surface, while T. ishikariensis var idahoensis frequently beneath the epidermis (Bruehl & Machtmes, 1980). Typhula canadensis (represented by biotype B) has brown to black (12 Fulvous to 36 Fulvous black) sclerotia often buried in plant tissues as does T. ishikariensis var. idahonsis. Mature sclerotia of T. canadensis are almost black (36 Fulvous black) and easily distinguish from brown (15 Brick to 19 Bay) sclerotia of T. ishikariensis. T. canadensis is highly variable in sclerotium size, strains from Alaska (Anchorage), Canada, Iceland, Norway and Russian Far East (Magadan) produce small sclerotia (0.2−0.5 mm) (e.g., Årsvoll & Smith, 1978; Matsumoto et al., 1996; Matsumoto 1997; Hoshino et al., 2004a; Tkachenko, 2013), and in Japan, populations from localities with deep, persistent snow cover tend to produce large sclerotia more than 2.0 mm in (Matsumoto & Tajimi, 1990). Similar fungus was also collected in Russian Far East (Kamchatka and Sakhalin) and Siberia (Tkachenko, 2013). Sclerotia of T. hyperborea formed on PDA are brown (15 Brick to 19 Bay) and indistinguishable from those of T. borealis (= T. ishikariensis var. ishikariensis) in size, shape, and color (Ekstrand, 1955). Our strains of biological species III, T. hyperborea from Northern Norway, the Arctic, Alps, European part of Russia (Volga-Ural), Siberia and Russian Far East (Chukotka and Kamchatka) had similar characteristics of the original description of T. hyperborea by Ekstrand (1955) (Matsumoto & Tronsmo, 1995; Matsumoto et al., 1996; Hoshino et al., 1997, 1998, 2001; Hoshino, Tronmso, & Saito, 2003; Hoshino, Saito, & Yumoto, 2006). Sclerotial rind cell pattern was considered as important to distinguish T. ishikariensis varieties (Årsvoll & Smith, 1978). However, rind cell pattern of strains from Japan and Norway was highly variable and unable to use as a taxonomic criterion (Matsumoto et al., 1996). Our findings agreed with the results of Matsumoto et al. (1996), and regard that rind cell pattern is of limited taxonomic significance (data not shown).

Fig. 3 − Morphological characteristics of Typhula ishikariensis.

A−D: lectotype (TNS-F-40457). E: epitype (TNS-F-24716). A: specimen label. B: dried basidiomata. C, D: dried sclerotium on rotted red clover stem. E: fresh basidiomata of T. ishikariensis var. ishikariensis from alfalfa.

Members of the T. ishikariensis complex normally produced clavate basidiomata which consist of fertile heads and stipes, and each basidisium has four sterigmata (Imai, 1930; Remsberg, 1940a; Corner, 1950; Kuznetzova, 1953; Potatosova, 1960c; Parmasto, 1965; Berthier, 1976; Årsvoll & Smith, 1978; Matsumoto & Tajimi, 1991). However, there has been few descriptions of baidioma morphology of T. hyperborea sensu H. Ekstr. Some of our strains of T. hyperborea developed basidomata with short stems and pointed heads (Fig. 4A). These features are unique to T. hyperborea. Typhula ishikariensis and T. canadensis are indistinguishable in basidoma except the length of caulocystidia; basidiomata of T. canadensis have long caulocystidia (ca. 50−120 μm; Fig. 4C and E). The caulocystidia of T. ishikariensis var. idahoensis are mostly short but rarely long (Fig. 4F and G). Those of T. ishikariensis var. ishikariensis and T. hyperborea are exclusively short (Fig. 4B, D, H and I). Berthier (1976) observed stems of T. idahoensis, however, he did not describe the above trait. Other previous reports on basidioma formation by T. canadensis (Årsvoll & Smith,1978; Matsumoto & Tajimi, 1991; Kawakami et al., 2004: Hoshino et al., 2004a, 2004b) and other species (Imai, 1930; Remsberg, 1940a; Corner, 1950; Kuznetzova, 1953; Potatosova, 1960c; Parmasto, 1965) did not refer to caulocystidia, either. Further studies, using a number of strains under different environmental conditions, are necessary to evaluate the significance of caulocystidia as a taxonomic criterion.

Fig. 4 − Morphological characteristics of Typhula hyperborea and their relatives.

A, D, H, I: T. hyperborea TNS-F-24687 (epitype). B: T. ishikariensis var. ishikariensis (epitype TNS-F-24716). C, E: T. canadensis MAFF 306142. F, G: T. ishikariensis var. idahoensis CUP27223 (syntype) A: fresh basidiomata. B−D: stem surface of fresh basidiomata. E−I: stem surface of dried basidiomata.

Basidiospores of T. hyperborea sensu H. Ekstr. from northern Scandinavia were short oviform and distinct from those of T. borealis (Ekstrand, 1955; Fig. 5A). Typhula hyperborea also had similar basidiospores (Fig. 5B). Ekstrand (1955) distinguished between T. borealis and T. hyperborea sensu H. Ekstr. in Scandinavia based on basidiospore dimension, and we subsequently compared basidiospores drawn in his literature in terms of Q-value (ratio of length and width) to reveal the difference (Fig. 5A). Our strains from various localities in northern Hemisphere were, on the contrary, indistinguishable by basidiospore dimension (Fig. 5B). Spore dimensions of T. ishikariensis and T. canadensis overlapped each other, and basidiospores of T. hyperborea tended to exceed the range of T. ishikariensis and T. canadensis. If Ekstrand (1955) distinguished T. hyperborea from T. borealis solely by basidiospore dimension, the separation of both fungi may be arbitrary and is considered to ignore differences in features reflecting genetic background.

Fig. 5 − Basidiospore dimensions of the Typhula ishikariensis complex.

A: White circles: T. borealis and black circles: T. hyperborea sensu H. Ekstr. in Ekstrand (1955). B: our strains. White circles: T. ishikariensis, grey circles: T. canadensis, black circles: T. hyperborea.

3.4. Host range

The members of the T. ishikariensis complex differ in host range; T. ishikariensis var. ishikariensis attacks both mono- and dicots and rarely Gymnosperm (Gulaev, 1948; Potatosova, 1960a, 1960b, 1960c; Hoshino et al., 2004b). Imai by himself or Imai and Tanaka collected three specimens from rotted stalks of red clover in a snowy locality, Sapporo (former Kotoni village), Hokkaido, Japan (Fig. 3A). The original description of T. borealis is similar to that of T. ishikariensis (Ekstrand, 1955), and the wide host range of T. borealis, including winter cereals and grasses, clover, winter rape and beets, overlaps with that of T. ishikariensis (Imai, 1930; Tomiyama, 1961; Ylimäki, 1969; Årsvoll & Smith, 1978; Smith, 1989). These results suggest that T. borealis (strains from southern Norway) should be regard as T. ishikariensis var. ishikariensis, and our mating and phylogenic analyses agreed with these results (Table 1, Figs. 1 and 2). The host range of T. canadensis and T. hyperborea, as well as T. ishikariensis var. idahoensis, is restricted to monocots (Table 3) except Stellaria spp. whose tops get moribund and liable to the attack by fungus before persistent snow cover (Matsumoto & Hsiang, 2016).

3.5. Physiological characteristics of Typhula hyperborea

Typhula hyperborea (= T. ishikariensis group III) showed irregular growth as the extension of hyphae was inhibited and formed feather-like colony at 10˚C on PDA. Its optimal growth temperature was below 10˚C (ca. 0−4˚C) (Matsumoto et al., 1996; Hoshino et al., 1997). However, when strains were cultured on corn meal agar (CMA; Difco), mycelial growth was improved in most strains (Fig. 6). Since PDA seemed to contain little radical scavengers and since incubation at high temperatures promoted oxygen uptake in the psychrophilic species of the genus Typhula (Dejardin & Ward, 1971), PDA was amended with 100 µg/mL β-carotene or 5 mM sodium ascorbate. Consequently, mycelial growth was improved to various extent (Fig. 6), indicating that the injurious effects of active oxygen produced by excess respiration may be alleviated by radical scavengers in most strains of T. hyperborea. Its irregular growth at higher temperatures was ascribed to the loss of vital properties of intracellular proteins (Hoshino et al., 1997).

Fig. 6 − Effects of free radical scavengers on mycelial growth of Typhula ishikariensis and T. hyperborea.

White bars: PDA, shaded bars: PDA amended with 5 mM sodium ascorbate, dotted bars: PDA with 100 μg/mL β-carotene, black bars: corn meal agar. Cultures were incubated at 10˚C.

Pathogenisity of T. hyperborea was determined, using timothy (Table 4). Strains that showed irregular growth on PDA at 10˚C were more virulent than those with normal growth and T. ishikariensis. These results may imply that pathogenic strains of T. hyperborea are higher in metabolic turnover to exploit host nutrients, which works well exclusively under exposure to stress conditions due to freezing temperatures.

Table 4 - Inoculation test of Typhula ishikariensis on timothy.

Strains, localities	Biological species	Regrowth after inoculation/ un-inoculated control (g dry weight/plant)	Irregular growth on PDA at 10˚C
OPU1809, Norway	T. ishikariensis	0.85	-
OPU1810, Norway	T. hyperborea	0.61	+
FERM P-18741, Svalbard	T. hyperborea	0.17	+
OPU1811, Greenland	T. hyperborea	0.98	-
OPU1812, Greenland	T. hyperborea	0.00	+
OPU1813, Greenland	T. hyperborea	0.88	-
OPU1814, Greenland	T. hyperborea	0.72	-
OPU1817, Greenland	T. hyperborea	0.73	-
OPU1816, Greenland	T. hyperborea	0.00	+

3.6. Taxonomy

Typhula ishikariensis var. ishikariensis S. Imai, Trans. Sapporo Nat. Hist. Soc. 11:75, 1930 Figs 3B-E, 4B

Mycobank no. MB 427232.

Basionym: Typhula ishikariensis S. Imai, MB 819232

Synonyms: Typhula humulina Kusnezowa, Botanicheskie Materialy 9:145 (1953), MB 3027256

Typhula borealis H. Ekstr., Meddn. Växtskyddsant. Stockh. 67:52 (1955), MB 532444

Diagnosis: Sclerotium color of T. ishikariensis var. ishikariensis, var. idahoensis and T. hyperborea were light to dark brown. Those of T. canadensis were dark brown almost black. Basidiomata of T. ishikariensis var. ishikariensis and T. hyperborea had short, and T. canadensis had long caulocystidia. Less aerial mycelia and normal growth on PDA at 10˚C. T. canadensis had abundant aerial mycelia, and T. hyperborea showed irregular growth on PDA at 10˚C. Mycelia of T. ishikariensis var. ishikariensis mainly mated within biological species I.

Type: JAPAN, Hokkaido, Kotoni, leaves of Trifolium pretense L., 6 Nov 1929, leg. S. Imai (lectotype, TNS-F-40457; syntype, TNS-F-40455, JAPAN, Hokkaido, Kotoni, leaves of T. pretense, 30 Oct 1929, leg. S. Imai & I. Tanaka; epitype, TNS-F-24716, JAPAN, Hokkaido, Sapporo, Hitsujigaoka, leaves of Medicago sativa L., May 1976, leg. N. Matsumoto; ex-epitype strain, MAFF306136).

Gene sequences ex-epitype: AB127949 (ITS), LC197671 (mitochondrial gene for large subunit ribosomal RNA).

Description: Sclerotia readily detached from host, globose to subglobose, light to dark brown (15 brick to 19 bay), 0.5−2 mm diam, surface smooth and rough when dry. One or more basidiomata emerging from a sclerotium, clavate or cylindrical, 4−20 mm tall, white to pale yellow (2 B to 6 E). Basidia having four sterigmata (Imai, 1930; Kuznetzova, 1953; Hoshino et al., 2004b). Basidiospores ovoid to ellipsoidal, 7.5−12.3×2.5−5 μm. Caulocystidia short, >50 μm.

Host: monocots (grasses, winter cereals, tulip), dicots (beets, forage legumes, hop, rapeseed), and Gymnosperm (pine).

Distribution: widely distributed from Europe through Asian Far East (Matsumoto, 1989; Tkachenko, 2013) to North America (Bruehl & Cunfer, 1975; Årsvoll & Smith, 1978; Millett & Maxwell, 1997; Millett, 1999). This fungus dominates in snowy regions in the Temperate to Frigid Zones of Europe such as Alps (Schmidt, 1976; Årsvoll & Smith, 1978), Scandinavia (Kristinsson & Guðleifsson, 1976; Årsvoll, 1977; Matsumoto & Tronsmo, 1995; Matsumoto et al., 1996; Hoshino et al. 2004a), Baltic countries to European part of Russia and Northern Ukraine (Tkachenko et al., 1997). Similar morphological fungus was recorded from other countries of central Europe such as Germany (Andres, Hindorf, Fehrmann, & Trägner-Born, 1987), Poland (Dynowska, 1983) and former Czechoslovakia (Benada, 1976).

Mating group: T. ishikariensis var. ishikariensis from North America and Europe, Norwegian group I and Japanese biotype A.

Additional specimens examined: TNS-F-40456, 40458 (Imai also observed these specimens), 17091, 24661, 24663, 14664, 24667, 24670, 24671, 24673, 24675, 24676, 24677, 24678, 24679, 24680, 24681, 24682, 24683, 24684, 24685, 24686, 24695, 24696, 24697, 24704, UPS:BOT:F-707367, PACMA 5487 (T. borealis), JHP-549 2643 (T. borealis), Herbarium of Norwegian Institute of Bioeconomy Research (NIBIO): 2 packets of T. ishikariensis, 19 packets of T. borealis, 18 packets of Typhula “borealis”, 29 packets of T. cf. borealis.

Note: The lectotype is in rather poor condition and includes only two basidiomata (Fig. 3B). We did not determine DNA sequences from the lectotype. Thus, we selected an epitype from a prevalent mycelial compatibility group in Hokkaido, Japan in this species. Japanese name: Ishikari-gamanoho-take (Ishikari Province-bulrush-mushroom, Imai, 1930).

Typhula ishikariensis var. idahoensis (Remsberg) Årsvoll & J.D. Sm., Can. J. Bot. 56:361 (1978) Figs 4F, G

Mycobank no. MB 348940.

Basionym: Typhula idahoensis Remsberg, Mycologia 32:89 (1940), MB 291704

Diagnosis: Sclerotia of var. idahoensis and T. canadensis were frequently formed subepidermal in host tissues. Those of var. ishikariensis and T. hyperborea were easily dislodged from host tissues. Morphological characteristics similar to var. ishikariensis: however, rarely mates with var. ishikariensis (biological specie I) and often with T. canadensis (biological species II).

Type: USA, Idaho, Hill City, wheat leaves and stems, May 1939, leg. C.W. Hungerford (lectotype: CUP 27221; syntype, CUP 25153, USA, Montana, Bozeman, 11−24 Mar 1936, Triticum vulgare, leg. P.A. Young, CUP 25316, USA, Idaho, Soda Spring, 5 Oct 1936, Triticum vulgare, leg. Finch, CUP 27220, USA, Idaho, Felt, May 1930, wheat leaves and stems, leg. C.W. Hungerford, CUP 27222, USA, Idaho, Felt, Apr 1931, wheat leaves and stems, leg. C.W. Hungerford, CUP 27223, USA, Idaho, Tetonia, May 1931, on Agropyron cristatum, leg. C.W. Hungerford).

Gene sequences lectotype: LC192550 (ITS); syntype CUP 25153: LC613246 (ITS).

Description: Sclerotia are frequently subepidermal and subspherical in host, globose to subglobose, light to dark brown (15 brick to 19 bay, original description: chestnut-brown to bone-brown: Remsberg, 1940a), 0.5−2 mm diam, surface smooth and rough when dry. One or more basidiomata emerging from a sclerotium, clavate or cylindrical, 4−20 mm tall, white to pale yellow (2 B to 6 E). Basidiospores ovoid to ellipsoidal, 7.5−12.3×2.5−5 μm. Caulocystidia mainly short (>50 μm) and rarely enlonged (<50 μm).

Host: monocots (grasses, winter cereals).

Distribution: North America (Remsberg, 1940a, 1940b; Bruehl et al., 1975; Årsvoll & Smith, 1978)

Mating group: T. ishikariensis var. idahoensis in North America and T. canadensis in North America and Europe, Norwegian group II and biotype B in Japan.

Additional specimens examined: CUP 17507, 17510, 19191, 25316, 27220, 27221a, 37857, UPS:BOT:F-683949 (isosyntype), WSP 12015, 12016, 12017, 12018, 13554, 15993, 16235, 16236, 23669, 35987, 37317, 37322, 46544, 46679, 46711, 46832, 46833, 46834, 46835, 46888, 46890,51550, 71681, TNS-F-24709, 24710, Herbarium of NIBIO: 9 packets of T. idahoensis, 55 packets of Typhula cf. idahoensis

Note: Basidia having 4, 6 or 8 sterigmata (Remsberg 1940a). Cunfer (1974) reported that this fungus had tetrapolar incompatibility with multiple alleles. Japanese name: Hokubei-Ishikari-gamanoho-take (North American-Ishikari Province-bulrush-mushroom, newly named).

Typhula canadensis (J.D. Sm. & Årsvoll) Tam. Hoshino, T. Kasuya, & N. Matsumoto, stat. nov. Figs 4C, E

Mycobank no. MB 819233.

Basionym: Typhula ishikariensis var. canadensis J.D. Sm. & Årsvoll, Can. J. Bot. 56: 362 (1978), MB 348939

Diagnosis: Sclerotium color of T. canadensis was dark brown to almost black. Those of other T. ishikariensis complex were light to dark brown. Some strains of Basidiomata of T. canadensis and var. idahoensis had long caulocystidia, however those of other T. ishikariensis complex were short. T. canadensis had abundant aerial mycelia and normal growth on PDA at 10˚C. Mycelia of T. canadensis mainly mated within biological species II.

Type: CANADA, Saskatchewan, Price Albert National Park, Poa annus L. and Poa pratensis L., 9 May 1974. leg. J. Drew Smith (holotype, DAOM160550).

Gene sequence holotype: LC192551 (ITS).

Description: Sclerotia highly variable in size: small sclerotia 0.2−0.5 mm often suspended in aerial mycelium, large sclerotia up to 2 mm diam, often produced involving host tissues, globose to subglobose, dark brown (16 cigar brown) almost black (36 fuscous black or 37 olivaceous black), surface smooth and rough when dry. One or more basidiomata emerging from a sclerotium, clavate or cylindrical, 5−25 mm tall, white to pale brown (2 B to 6 F). Basidia with four sterigmata. Basidiospores ellipsoidal, 7.6−12.5×2.5−4 μm. Caulocystidia enlonged, 50−120 μm. Morphological characteristics similar to T. ishikariensis; however, rarely mates with var. ishikariensis and often mates with var. idahoensis.

Host: mainly monocots (grasses, winter cereals, Cyperaceae, tubers of Chinese yam) and rarely on a few dicots such as Stellaria spp.

Distribution; Far East (Matsumoto, 1989; Matsumoto & Tajimi, 1991; Tkachenko, 2013), Siberia (Tkachenko, 2013), Norway (Matsumoto & Tronsmo, 1995; Matsumoto et al., 1996), Iceland (Kristinsson & Guðleifsson, 1976; Hoshino et al., 2004a), North America (Årsvoll & Smith, 1978; Millett & Maxwell, 1997; Millett, 1999).

Mating group: T. ishikariensis var. canadensis in North America and Europe, Norwegian group II and biotype B in Japan. T. ishikariensis var. idahoensis in North America.

Additional specimens examined: TNS-F-24662, 24692, 24693, 24707, 24711

Note: Japanese name: Kuro-tsubu-Ishikari-gamanoho-take (black-specked-Ishikari Province-bulrush-mushroom, newly named).

Typhula hyperborea H. Ekstr., Meddn. Växtskyddsant. Stockh. 67:55 (1955) Figs 4A, D, H, I

Mycobank no. MB 89234.

Diagnosis: T. hyperborea had abundant aerial mycelia on PDA at 0˚C and showed irregular growth (feather-like colonies) on PDA at 10˚C. Mycelia of T. hyperborea mainly mated within biological species III.

Type: SWEDEN, Norrbotten, Luleå, 1939, (lectotype, strain 7575, Fig. 14 in Meddn. Växtskyddsant. Stockh. 67:49 (1955); epitype, TNS-F-24687, NORWAY, Oppland, Fron, Kvarvet, leaves of Deschampsia cespitosa (L.) P. Beauv., May, 1992, leg. N. Matsumoto; ex-epitype: OPU1810).

Gene sequences ex-epitype: LC192579 (ITS), LC663960 (mitochondrial gene for large subunit ribosomal RNA).

Description: Sclerotia readily detached from host, globose to subglobose, light to dark brown (15 brick to 19 bay), 0.5−2 mm diam, surface smooth and rough when dry. One or more basidiomata emerging from a sclerotium, clavate or cylindrical, 5−25 mm tall (under artificial condition), head often tapered, white to pale brown (2 B to 6 F, 30 clay pink). Basidia with four sterigmata. Basidiospores ellipsoidal, 7.6−14.8×3.7−7.1 μm. Caulocystidia short, >50 μm. Mycelium abundant at 0˚C, but growth irregular at 10˚C on PDA (Matsumoto et al., 1996; Hoshino et al., 1997).

Host: monocots (grasses, winter cereals)

Distribution; the Arctic (Hoshino et al., 2003; Hoshino et al., 2006), Kamchatka, Siberia (Hoshino et al., 2001), Ural-Volga in Russia, Alps, Northern Scandinavia (Ekstrand, 1939, 1955; Røed, 1956; Matsumoto & Tronsmo, 1995; Matsumoto et al., 1996), North America (Pouleur, 1988; Pouleur & Couture, 1988). Similar fungus was also recorded in Northern Scandinavia (Jamalainen, 1957; Årsvoll, 1977), Russian Arctic (Petrov, 1983; Shiryaev, 2004, 2006, 2009, 2013a, 2013b) and Yukon in Canada (Lebeau & Longsdon, 1958).

Additional specimens examined: TNS-F-24672, 24674, 24688, 24657, 24658, 24659, 24660, 24665, 24666, 24668, 24669, 24689, 24690, 24691, 24694, 24698, 24699, 246700, 24701, 24702, 24703, 24705, 24706, 24708, 24712, 24713, Herbarium of NIBIO: 18 packets of Typhula “borealis”, 29 packets of T. cf. borealis.

Note: Årsvoll and Smith (1978) suggested that T. hyperborea might correspond to T. ishikariensis var. canadensis or to some intermediate forms but obtained neither exsiccata nor cultures of T. hyperborea. We found several specimens labeled T. “borealis” or T. cf. borealis at Herbarium in Norwegian Institute of Bioeconomy Research, Norway (Ekstrand used these names or or T. cfr. borealis in his publications; Jamalainen, 1957, 1974); however, we were unable to find type specimens of both fungi.

We designated the illustration of basidiospores by Ekstrand (1955) as the lectotype. No other morphological characteristics were shown by Ekstrand (1955). Thus, we selected an epitype from a strain collected from a representative locality in the vicinity of the original description of Ekstrand (1955).

Japanese name: saihate-Ishikari-gamanoho-take (northmost-Ishikari Province-bulrush-mushroom, newly named).

Disclosure

The Authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of the countries where they were performed.

Acknowledgments

We thanks to Dr. Eiji Nagasawa, Tottori Mycological Institute, Japan, Prof. Dr. Leif Sundheim, Norwegian Institute of Bioeconomy Research, Prof. Dr. Timothy D. Murray, Washington State University, USA and Prof. Dr. Henning Knudsen, University of Copenhagen, Denmark for their support to examine the specimens. We also thank the following persons for technical support in the collection of fungal sclerotia: Drs. M.R. Asef (Iran), P.E. Aspholm (Norway), late Dr. A.N. Berkutenko, Mr. I.A. Borzdyko, Drs. G.B. Borovskii, Yu.A. Chikin (Russia), C. Cripps (USA), L. Couture (Canada), E. V. Deineko, K.A. Funtov (Russia), M. Gaard (Faroe Is.), T. Hsiang (Canada), A. Kawakami (Japan), H. Kristinsson (Iceland), G.A. Lazarev (Russia), F. Mascher-Frutshi (Switzerland), J.H. McBeath (USA), L.G. Mihaleva, Ms. N.N. Molodkina, late Dr. V.A. Nedodoluzhko, Drs. Yu.A. Neofitov, E.D. Nikitina (Russia), O. Nissinen (Finland), T.A. Penzina (Russia), M. Pronczuk (Poland), R.I. Safin (Russia), I. Saito (Japan), D. Schmidt (Switzerland), Yu.V. Sidorchuk, Ms. L.V. Sukhareva, Drs. A.A. Taran, K.G. Tkachenko, A.Ya. Yakovlev (Russia), R. Zare (Iran), V.F. Zhirov (Russia). This research was financially supported in part by a Grant-in-Aid for Scientific Research (KAKENHI) (no. 19570100) from the Japanese Society for the Promotion of Science (JSPS), New Energy and Industrial Technology Development Organization (NEDO) in Japan, Institute for Fermentation, Osaka (IFO) and the Research Council of Norway.

References

• Andres, H., Hindorf, H., Fehrmann, H., & Trägner-Born, J. (1987). Untersuchungen zum Auftreten und zur Verbreitung von Typhula-Arten an Wintergetreide im öslichen Franken und Bayerischen Wald. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 94, 491-499.
• Anonymous (1969) Royal Botanic Garden Edinburgh, Flora of British fungi, colour identification chart. Her Majesty's Stationary Office, Edinburgh.
• Årsvoll, K. (1977). Effects of hardening, plant age, and development in Phleum pratense and Festuca pratensis on resistance to snow mould fungi. Meldinger fra Norges Landbrukshøgskole 56(28), 1-14.
• Årsvoll, K., & Smith, J. D. (1978). Typhula ishikariensis and its varieties, var. idahoensis comb.nov. and var. canadensis comb. nov. Canadian Journal of Botany 56, 348-364. http://doi.org/10.1139/b78-042
• Benada, J. (1976). The occurrence of Typhula ishikariensis Lasch ex Fr. on winter wheat in Czechoslovakia. Sborník ÚVTIZ - Ochrana Rostlin 12, 315-317.
• Berthier, J. (1976). Monographie des Typhula Fr., Pistillaria Fr. et genres voisins. Numero Special du Bulletin de la Societe Linneenne de Lyon 45, 1-195.
• Bruehl, G. W. (1988). Typhula spp., the snow moulds. Advances in Plant Pathology 6, 553-559. http://doi.org/10.1016/B9978-0-12-033706-4-4.50041-4
• Bruehl, G. W., & Cunfer, B. M. (1975). Typhula species pathogenic to wheat in the Pacific Northwest. Phytopathology 65, 757-60. http://doi.org/10.1094/Phyto-65-755
• Bruehl, G. W., & Machtmes, R. (1980). Cultural variation within Typhula idahoensis and T. ishikariensis and the species concept. Phytopathology 70, 867-871. http://doi.org/10.1094/Phyto-70-867
• Bruehl, G. W., Machtmes, R., & Kiyomoto, R. (1975). Taxonomic relationships among Typhula species as revealed by mating experiments. Phytopathology 65, 1108-1114. http://doi.org/10.1094/Phyto-65-1108
• Bruns, T. D., Szaro, T. M., Gardes, M., Cullings, K. W., Pan, J. J., Taylor, D. L., Horton, T. R., Kretzer, A., Garelotto, M., & Li, Y. (1998). A sequence database for the identification of ectomycorrhizal basidiomycetes by phylogenetic analysis. Molecular Ecology 7, 257-272. http://doi.org/10.1046/j.1365-294X.1998.00337.x
• Cha, J. Y., Sung, J. M., & Igarashi, T. (1994). Biological species and morphological characteristics of Armillaria mellea complex in Hokkaido: A. sinapina and two new species, A. jezoensis and A. singular. Mycoscience 35, 39-47. http://doi.org/10.1007/BF02268526
• Christen, A. A., & Bruehl, G. W. (1979). Hybridization of Typhula ishikariensis and Typhula idahoensis. Phytopathology 69, 263-266. http://doi.org/10.1094/Phyto-69-263
• Corner, E. J. H. (1950). A monograph of Clavaria and allied genera. Oxford University Press.
• Cunfer, B. M. (1974). Sexual incompatibility and aspects of the mono- and dikaryotic phases of Typhula idahoensis. Phytopathology 64, 123-127. http://doi.org/10.1094/Phyto-64-123
• Dejardin, R. A., & Ward, E. W. B. (1971). Growth and respiration of psychrophilic species of the genus Typhula. Canadian Journal of Botany 49, 339-347. http://doi.org/10.1139/b71-057
• Dynowska, M. (1983). Badania nad grzybami z rodzaju Typhula Fr. emend Karst. pochodzącymi z terenu województwa olsztynskiego I. Acta Mycologica 19, 283-293.
• Edgar, R. C. (2004a). Muscle: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792-1797. http://doi.org/10.1093/nar/gkh340
• Edgar, R. C. (2004b). Muscle: A multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics, 5, 113. http://doi.org/10.1186/1471-2105-5-113
• Ekstrand, H. (1937). Trådklubbra på vintersäd. Växtskyddsnotiser 1, 3-4.
• Ekstrand, H. (1939). Höstsädens och vallgräsens övervintring. Statens Växtskyddsanstalt 1, 16-19.
• Ekstrand, H. (1955). Höstsädens och vallgräsens övervintring. Statens Växtskyddsanstalt Meddelande 67, 1-125.
• Garbelotto, M., & Gonthier, P. (2013). Biology, epidemiology, and control of Heterobasidion species worldwide. Annual Review of Phytopathology 51, 39-59. http://doi.org/10.1146/annurev-phyto-082712-102225
• Gulaev, V. V. (1948). Rotting of pine tree seedlings in forest nurseries (in Russian). Tatarskii respublikanskii otdel VNITOLES i Tatarskaya lesnaya opytnaya stantsiya VNIIKH. 9, 37-50.
• Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98.
• Hoshino, T., Kiriaki, M., Yumoto, T., & Kawakami, A. (2004a). Genetic and biological characteristics of Typhula ishikariensis from Northern Iceland. Acta Botanica Islandica 14, 59-70.
• Hoshino, T., Saito, I., & Tronsmo, A. M. (2003). Two snow mold fungi from Svalbard. Lidia 6, 30-32.
• Hoshino, T., Saito, I., & Yumoto, I. (2006). New findings of snow mold fungi from Greenland. Monographs on Greenland, Bioscience 56, 89-94.
• Hoshino, T., Tkachenko, O. B., Kiriaki, M., Yumoto, I., & Matsumoto, N. (2004b). Winter damage caused by Typhula ishikariensis biological species I on conifer seedlings and hop roots collected in the Volga-Ural regions of Russia. Canadian Journal of Plant Pathology 26, 391-396. http://doi.org/10.1080/07060660409507158
• Hoshino, T., Tkachenoko, O. B., Tronsmo, A. M., Kawakami, A., Morita, N., Ohgiya, S., Ishizaki, K., & Matsumoto, N. (2001). Temperature sensitivity and freezing resistance among isolates of Typhula ishikariensis from Russia. Búvísindi 14, 61-65.
• Hoshino, T., Tronsmo, A. M., Matsumoto, N., Araki, T., Georges, F., Goda, T., Ohgiya, S., & Ishizaki, K. (1998). Freezing resistance among isolates of a psychrophilic fungus, Typhula ishikariensis, from Norway. Proceedings of NIPR Symposium on Polar Biology 11, 112-118.
• Hoshino, T., Tronsmo, A. M., Matsumoto, N., Ohgiya, S., & Ishizaki, K. (1997). Effects of temperature on growth and intracellular proteins of Norwegian Typhula ishikariensis isolates. Acta Agriculturæ Scandinavia Section B. Soil and Plant Science 47, 185-189. http://doi.org/10.1080/09064719709362459
• Huelsenbeck, J. P., & Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogeny. Bioinformatics, 17, 754-755. http://doi.org/10.1093/bioinformatics/17.8.754
• Ikeda, S., Hoshino, T., Matsumoto, N., & Kondo, N. (2015). Taxonomic reappraisal of Typhula variabilis, Typhula laschii, Typhula intermedia, and Typhula japonica. Mycoscience 56, 549-559. http://doi.org/10.1016/j.myc.2015.05.002
• Imai, S. (1930). On the Clavariaceæ of Japan. II. Transactions of the Sapporo Natural History Society 11, 70-77.
• Jamalainen, E. A. (1957). Overwintering of Gramineae-plants and parasitic fungi II. On the Typhula sp-.fungi in Finland. The Journal of Scientific Agricultural Society of Finland 29, 75-81. http://doi.org/10.23986/afsci.71435
• Jamalainen, E. A. (1974). Resistance in winter cereals and grasses to low-temperature parasitic fungi. Annual Review of Phytopathology 12, 281-302. http://doi.org/10.1146/annurev.py.12.090174.001433
• Kawakami, A., Matsumoto, N., & Naito, S. (2004). Environmental factors influencing sporocarp formation in Typhula ishikariensis. Journal of General Plant Pathology 70, 1-6. http://doi.org/10.1007/s10327-003-0086-3
• Kasuya, T., & Ono, Y. (2018). Herpobasidium filicinum (Eocronartiaceae, Platygloeales) occurs on Dennstaedtia wilfordii (Dennstaedtiaceae) in Japan. Mycoscience 59, 443-448. http://doi.org/10.1016/j.myc.2018.03.001
• Kristinsson, H., & Guðleifsson, B. I. (1976). The activity of low-temperature fungi under the snow cover in Iceland. Acta Botanica Islandica 4, 44-57.
• Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35, 1547-1549. http://doi.org/10.1093/molbev/msy096
• Kuznetzova, A. (1953). New species of the fungus Typhula humulina A. Kuzn. On subterranean stems of hop (in Russian and Latin). Botanicheskie Materialy Otdela Sporovykh Rastenii 9, 142-145.
• Lebeau, J. B., & Longsdon, C. E. (1958) Snow mold of forage crops in Alaska and Yukon. Phytopathology 48, 148-150.
• Matsumoto, N. (1989). Autecology of the pathogenic species of Typhula. Research Bullutin of the Hokkaido National Agricultural Experiment Station 152, 91-162.
• Matsumoto, N. (1997). Evolution and adaptation in snow mold fungi (in Japanese). Soil Microorganisms 50, 13-19. https://doi.org/10.18946/jssm.50.0_13
• Matsumoto, N., & Hsiang, T. (2016). Snow mold. The battle under snow between fungal pathogens and their plant hosts. Springer.
• Matsumoto, N., & Tajimi, A. (1990). Continuous variation within isolates of Typhula ishikariensis biotypes B and C associated with habitat differences. Canadian Journal of Botany 68, 1768-1773.
• Matsumoto, N., & Tajimi, A. (1991). Typhula ishikariensis biotype B and C, a single biological species. Transactions of the Mycological Society of Japan 32, 273-281.
• Matsumoto, N., & Tronsmo, A. M. (1995). Population structure of Typhula ishikariensis in meadows and pastures in Norway. Acta Agriculturæ Scandinavia Section B. Soil and Plant Science 45, 197-201.
• Matsumoto, N., Sato, T., & Araki, T. (1982). Biotype differentiation in the Typhula ishikariensis complex and their allopatry. Annuals of the Phytopathological Society of Japan 48, 275-280. http://doi.org/10.3186/jjphytopath.48.275
• Matsumoto, N., Sato, T., Araki, T., & Tajimi, A. (1983). Genetic relationships within the Typhula ishikariensis complex. Transactions of the Mycological Society of Japan 24, 313-318.
• Matsumoto, N., Tronsmo, A. M., & Shimanuki, T. (1996). Genetic and biological characteristics of Typhula ishikariensis isolates from Norway. European Journal of Plant Pathology 102, 431-439. http://doi.org/10.1007/BF01877137
• McDonald, W. C. (1961). A review of the taxonomy and nomenclature of some low-temperature forage pathogens. Canadian Plant Disease Survey 41, 256-260.
• Millett, S. M. (1999). Distribution, Biological and Molecular Characterization, and Aggressiveness of Typhula Snow molds of Wisconsin Golf Courses. University of Wisconsin-Madison.
• Millett, S. M., & Maxwell, D. (1997). Typhula snow molds of Wisconsin golf courses. In: A. Nishimune & N. Iriki (Eds.), Plant-Microbe Interactions at Low Temperature under Snow (pp. 119-124). Hokkaido National Experimental Station.
• Nylander, J. A. A. (2004). MrModeltest, ver. 3.7. Distributed by the author. Uppsala: Evolutionary Biology Centre, Uppsala University.
• Olariaga, I., Huhtinen, S., Læssøe, T., Petersen, J. H., & Hansen, K. (2020). Phylogenetic origins and family classification of typhuloid fungi, with emphasis on Ceratellopsis, Macrotyphula and Typhula (Basidiomycota). Studies in Mycology 96, 155-184. http://doi.org/10.1016/j.simyco.2020.05.003
• Parmasto, E. H. (1965). Key to the Clavariaceae of USSR (in Russian). Akademiya Nauka.
• Petrov, V. F. (1983). Pathogenic microflora of root growth in perennial grasses in Khibiny (in Russian). The Bulletin of Applied Botany, Genetics and Plant Breeding 82, 38-45.
• Potatosova, E. G. (1960a). Critical review of species of Typhula genus on cultivated plants of the USSR (in Russian). Thesis of candidate of agricultural sciences, Academy of Sciences USSR.
• Potatosova, E. G. (1960b). Fungi of Typhula genus in the USSR (in Russian). Botanicheskii Zhurnal 45, 567-572.
• Potatosova, E. G. (1960c). Typhulosis on winter crop (in Russian). Trudy Vsesoyuznogo-Nauchnoissledovatelscogo Instituta Zaschity. Rastenii 7, 135-142.
• Pouleur, S. (1988). Inventaire des moisissure nivéales au Québecat culture in vitro du Typhula ishikariensis. Memorire de maîtrise, Université Laval.
• Pouleur, S., & Couture, L. (1988). Présence et distribution des moisissures nivéales des céréales d'automne au Québec. Canadian Journal of Plant Pathology 10, 183-187. http://doi.org/10.1080/07060668809501752
• Remsberg, R. E. (1940a) Studies in the genus Typhula. Mycologia 32, 52-96. http://doi.org/10.2307/3754544
• Remsberg, R. E. (1940b) The snow molds of grains and grasses caused by Typhula itoana and Typhula idahoensis. Phytopathology 3, 178-180.
• Røed, H (1956). Parasittære vinterskader på engvekster og høstæd I Norge. Nordisk Jordbrugsforskning 38, 428-432.
• Saito, I. (1998). Non-gramineous hosts of Myriosclerotinia borealis. Mycoscience 39, 145-153. http://doi.org/10.1007/BF02464052
• Schmidt, D. (1976). Observations on snow moulds affecting grasses. Revue Suises d'Agriculture 8, 8-15.
• Shiryaev, A. G. (2004). Clavarioid fungi of Urals. I. Boreal forest zone. Mikologiya i Fitopatologiya 38, 59-72.
• Shiryaev, A. G. (2006). Clavarioid fungi of Urals. III. Arctic Zone. Mikologiya i Fitopatologiya 40, 294-306.
• Shiryaev, A. G. (2009). Clavarioid fungi of the tundra and forest-tundra zones of Kola Peninsula (Murmansk region) (in Russian). Novitates Systematicae Plantarum Non Vascularium 43, 134-149. http://doi.org/10.31111/nsnr/2009.43.134
• Shiryaev, A. G. (2013a) Geographical specificy of the tundra and boreal biota of clavarioid fungi in Chukotka (in Russian). Byulleten' Moskovskogo Obshchestva Ispytatelei Prirody Otdel Biologicheskiii 118, 67-79.
• Shiryaev, A. G. (2013b). Spatial heterogeneity of the species composition of Clavarioid fungi's complex in the Eurasian Arctic. Contemporary Problems of Ecology 6, 381-389.
• Smith, J. D. (1987) Winter-hardiness and overwintering diseases of amenity turfgrasses with special reference to the Canadian Prairies. Technical Bulletin 1987-12E.
• Smith, J. D., Jackson, N., & Woolhouse, A. R. (1989) Fungal diseases of amenity turf grasses. E. & F. N. Spon.
• Tkachenko, O. B. (2013). Snow mold fungi in Russia. In: R. Imai, M. Yoshida, & N. Matsumoto (Eds.), Plant and microbe adaptations to cold in a changing world. Proceedings from plants and microbe adaptations to cold 2012 (pp. 293-303). Springer.
• Tkachenko, O. B., Matsumoto, N., & Shimanuki, T. (1997). Mating patterns of east-Europe isolates of Typhula ishikariensis S. Imai with isolates from distant region. Mikologiya i Fitopatologiya 31, 68-72.
• Tomiyama, K. (1955). Studies in the snow blight disease of winter cereals (in Japanese). Hokkaido National Agriculture Experimental Station Report 47, 1-234.
• Tomiyama, K (1961) Snow blight of winter cereals in Japan. In Recent advances in botany, from lectures & symposia presented to the IX International Botanical Congress, Montreal, 1959, vol. 1 (pp. 549-552). University of Toronto Press.
• Tronsmo, A. M. (1984). Predisposing effect of low temperature on resistance to winter damage in grasses. Acta Agriculturæ Scandinavia 34, 210-220. http://doi.org/10.1080/00015128409435390
• Tronsmo, A. M. (1985). Effects of dehardening on resistance to freezing and to infection by Typhula ishikariensis. Acta Agriculturæ Scandinavia 35, 113-116. http://doi.org/10.1080/00015128509435764
• White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M. A. Innis, D. H. Gelfand, J. J. Sninsky, & White T. J. (Eds.), PCR Protocols: a guide to methods and applications (pp. 315-322). Academic Press.
• Ylimäki, A. (1969). Typhula blight of clover. Annales Agriculturae Fenniae 8, 30-37.