日 植 病 報 48: 275-280 (1982)

Ann. Phytopath. Soc. Japan 48: 275-280 (1982)

Biotype Differentiation in the ishikariensis Complex and Their Allopatry in Hokkaido

Naoyuki MATSUMOTO*, Toru SATO** and Takao ARAKI***

松 本 直 幸 ＊・佐藤 徹 ＊＊・荒木 隆 男＊＊＊:雪腐 黒 色 小 粒 菌 核 病 菌 の生 物 型 と そ れ らの 北 海 道 内 に お け る異 所 性

Abstract

Typhula ishikariensis isolates were collected from various parts of Hokkaido. There were two genetically different groups which did not mate with each other; one was termed biotype A, and the other was further divided into two by cultural characteristics, i.e., biotypes B and C. Biotype A was identical to T. ishikariensis and T. ishikariensis var. ishikariensis. Biotype B was not identical to T. idahoensis or T. ishikariensis var. idahoensis. Biotype C was similar to T. ishikariensis var. canadensis. Biotype A tended to occur inland where deep cover lasts for a long time. Biotype B was obtained mainly from the coastal regions where snow cover is thin and short in time. The distribution of biotype C was irregular. Taxonomic significance of the genetics and allopatry in the T. ishikari- ensis complex is discussed. (Received August 31, 1981)

Key Words: snow mold fungi, , distribution.

Introduction

Pathogenic species of Typhula had been confused in taxonomy until McDonald14) reviewed the literature on these fungi in 1961. Although he suggested the need for the comparison of many specimens from different sources by one investigator, he placed T. ishikariensis S. Imai and T. idahoensis Remsberg in synonymy. Bruehl et al.2,3) discriminated between both species on the bases of morphology and genetics. They mated to a limited extent, but most of the hybrid offsprings were presumed incapable of survival in nature. Arsvoll and Smith1), on the other hand, did not regard T. idahoensis as a separate species on genetical evidence and established three varieties in T. ishikariensis, i.e., var. ishikariensis, var. idahoensis, and var. canadensis. They could obtain inter- variety hybrids which formed normal basidiocarps. Genetical relationship between var. idahoensis and var. canadensis is closer than that between var. ishikariensis and var. idahoensis or var. canadensis. Christen and Bruehl8) and Bruehl and Machtmes5)

＊ Hokkaido National Agricultural Experiment Station , Hitsujigaoka 1, Toyohira-ku, Sapporo 061-01 北 海 道 農 業 試 験 場 ＊＊ Present address: National Research Institute , Nishinasuno, Tochigi 329-27 草 地 試 験 場 ＊＊＊ Present address: National Institute of Agricultural Sciences , Kannondai, Yatabe, Ibaraki 305

農 業 技 術 研 究 所 276 日本植 物病 理 学 会 報 第48巻 第3号 昭 和57年7月

thereafter reconsidered that interspecific hybrids of T. ishikariensis•~T. idahoensis could

exist in nature since they found putative interspecific hybrids in nature.

In this study, we examined whether such relationships existed within our T.

ishikariensis isolates and found similar differentiation.

Materials and Methods

Natural dikaryons of T. ishikariensis of different origins in Hokkaido were obtained from either basidiocarps or surface-sterilized sclerotia by placing them on potato-dextrose

agar (PDA) at 10C. Monokaryons were obtained as follows; a basidiocarp formed in nature or in an artificial condition (12-hr photoperiod, 10C) was secured with adhesive

tape to the inside of a Petri dish lid, and basidiopores were allowed to discharge onto

the dish containing sterile water. The spore suspension was spread on PDA plates.

Germlings were transferred to PDA slants several days later and examined on clamp

connections after a few weeks. The absence of clamp connections was used for the

criterion of monokaryon. The mono- or dikaryotic condition of the isolates was

confirmed by Giemsa staining.

Mating experiments between monokaryons (mon-mon mating) or between dikaryon

and monokaryon (di-mon mating) were conducted as described by Bruehl et al.3) with

slight modifications. Agar discs 5mm in diam with mycelia were cut from the margin of actively growing cultures and placed 1.5cm apart in pairs on PDA plates. After two

weeks a small block was cut from the junction of the colonies for mon-mon matings or

from the original monokaryon colony for di-mon matings and transferred to the

unoccupied portion of the same plate. Growth from the block was examined on clamp

connections after several days. The symbol "+" shows the compatible combination in

which mating occurred and the offspring grew vigorously, having normal hyphae with abundant clamp connections. When the growth was normal and vigorous but had no

clamp connections, the combination was regarded as incompatible. The symbol "-" is

given in this case. The symbol "•}" indicates the combination in which the growth was meager and abnormal, having a few clamp connections. All the experiments were

conducted at 10C.

Results

Grouping of T. ishikariensis isolates

More than one hundred isolates were used for the mating experiments, and a portion

of the results is illustrated in Tables 1 and 2. Di-mon mating experiments with Hokkaido

dikaryons revealed that there were two genetically different groups (Table 1). One

group of the isolates which mated with both the monokaryon A 7-6 and the American T. ishikariensis monokaryon 70-5-7* will be referred to as biotype A. The other group

consisting of the isolates which did not mate with these two monokaryons but mated

with the monokaryn Tu 6 and sometimes with the American T. idahoensis monokaryon

5999-5-3* could be further divided into two by cultural morphology, i.e., biotypes B and ＊ 輸入 許 可 番 号:農 林 水 産 省 指 令53横 植 第1339号(昭 和53年5月19日) ,同53横 植 第2156号(昭 和53年8月22 日),お よび 同54横 植 第2753号(昭 和54年12月12日). Ann. Phytopath. Soc. Japan 48 (3). July, 1982 277

Table 1. Grouping of Typhula ishikariensis isolates from Hokkaido by mating experiments

a) Mating offspring vigorous, having abundant clamp connections. b) Mating offspring vigorous, but clamp connections lacking. c) Mating offspring abnormal with poor growth. A few clamp connections present.

Table 2. Mating reaction of monokaryons of Typhula ishikariensis biotypes to T. ishikariensis and T. idahoensis dikaryons from USA and to T. ishikariensis var. canadensis dikaryons from Canada

a) Mating offspring vigorous, having abundant clamp connections. b) Mating offspring vigorous, but clamp connections lacking. c) Mating offspring abnormal with poor growth. A few clamp connections present.

C. Biotype C resembled T. ishikariensis var. canadensis. Crosses between biotype B dikaryons and the T. idahoensis monokaryon often failed to produce vigorous offsprings as did the isolates YOG-3, HCy-4, and HiTu-1.

Monokaryons were obtained from each biotype and used for di-mon mating experi- ments with the dikaryons of T. ishikariensis and T. idahoensis from the USA* and T. ishikariensis var. canadensis from Canada* (Table 2). Monokaryons of biotype A were compatible with T. ishikariensis but incompatible with T. idahoensis and T. ishikariensis var. canadensis. In most crosses between biotype B monokaryons and T. ishikariensis dikaryons, the products usually exhibited "•}" reaction. In crosses with T. idahoensis, however, biotype B monokaryons were seldom dikaryotized. T. ishikariensis var. canadensis 278 日本 植 物 病理 学 会 報 第48巻 第3号 昭 和57年7月 was compatible with biotype B. Biotype C monokaryons reacted the same way as did biotype B monokaryons.

Allopatry of the biotypes in Hokkaido The distribution patterns of biotypes A and B were allopatric in Hokkaido (Fig. 1). Biotype A tended to occur inland, where deeper snow cover lasts for a longer period of time (Fig. 2A) and sunshine in November is generally poor (Fig. 2B). T. incarnata

Fig. 1. Localities of Typhula ishikariensis biotypes in Hokkaido. •› : biotype A, •œ: biotype B, •£: biotype C.

A B Fig. 2. A: Mean annual number of days with snow cover equal to or more than 50cm. B: Mean hours of bright sunshine in November. Reproduced from Climatic Atlas of Japan vol.2 (1972), Japan Meteorological Agency, Chijinshokan, Tokyo. Ann. Phytopath. Soc. Japan 48 (3). July, 1982 279

Lasch ex Fr. was also prevalent there and was sometimes dominant over biotye A in some of the regions. Biotype B was obtained mainly from the remaining coastal regions along the Pacific Ocean and the Sea of Okhotsk. Biotype B overlapped with Bubak & Vleugel in eastern part of Hokkaido17). The distribution pattern of biotye C was irregular.

Discussion

The results show that there are three biotypes in T. ishikariensis isolates from

Hokkaido. Biotype A would be identical to T. ishikariensis in the USA and to T. ishikari- ensis var. ishikariensis in . Isolates of biotype A mated the same way as reported for these two taxa by other workers1,3), and moreover, common incompatibility factors were identified between Japanese and American isolates (Matsumoto, unpublished). Biotype

A and the American T. ishikariensis isolates also resembled in cultural characteristics on

P DA, i.e. scarce aerial mycelia, brown sclerotia, etc. When biotype B dikaryons were crossed with the T. idahoensis monokaryon, nearly half of the offsprings exhibited "•}" reaction, and biotype B monokaryons seldom mated with the T. idahoensis dikaryons.

These findings suggest that these two taxa are not closely related in genetics. Cultural characteristics on PDA were also different; biotype B had abundant aerial mycelia and black sclerotia, whereas the T. idahoensis isolates resembled the American T. ishikariensis

isolates. In fact, Bruehl and Machtmes5) proved it difficult to distinguish single isolates of T. ishikariensis and T. idahoensis in culture. Biotype C is almost identical to T. ishikariensis var. canadensis except that var. canadensis is highly compatible with var. idahoensis1). Putative intergrade isolates between biotypes B and C were occasionally obtained and were tentatively classified as biotype B because of their larger sclerotia.

We assume that the allopatry of biotypes A and B may be due to the difference in

climatic conditions before and during winter. Cloudy or rainy weather before winter

in the biotype A area is unfavorable for host plants to develop winter hardiness. In the biotype B area, however, plants can develop winter hardiness to a higer level by more

abundant sunshine. Light has influence on winter hardiness level11,12). In fact, winter

wheat plants grown in these two areas differ in some charcteristics important for winter

hardiness (Amano and Kunii, personal communications). Snow cover in the biotype B

area is not as deep as in the biotype A area and does not last so long. Biotype B is

more virulent than biotype A13). Hardier plants and less snow cover in the biotype B

area may have permitted the virulent biotype to thrive. Why biotype A is prevalent

instead of biotype B in the biotype A area, where plants attain a lower level of winter

hardiness and deeper snow cover lasts longer, remains to be explained. Biotype A could

be predominant over biotype B in its area by unknown factor(s), and biotype B might

have been expelled from there as a result of competition for the niche, i.e. the competi-

tive exclusion principle9). A similar interaction may exist between biotype A and T.

incarnata or between biotype B and S. borealis. The irregular distribution pattern of

biotype C can not be explained at present and needs further investigation.

As far as biotypes A and B are concerned, we believe that the process of speciation

has been completed and each biotype is different enough to be regarded as a separate

species. In the USA5), however, "unidentifiable isolates" which mate with both T. 280 日本植 物 病 理 学 会 報 第48巻 第3号 昭 和57年7月

ishikariensis and T. idahoensis exist in nature, and both species are indistinguishable in culture. Speciation there can be assumed to be still in the process. In Norway1), only T. ishikariensis var. ishikariensis exists, and it often mates with other varieties. Thus, the T. ishikariensis complex includes several populations differing in genetics and morphology. Biotypes A and B (and C) would occupy both ends of variation within the complex, and the American and Norwegian taxa lie between them. Gene exchange between the groups in the same species is known to be either improbable or restricted in many fungi by heterokaryon incompatibility6,10) or by the failure of hyphal anastomosis15,16). Caten and Jinks7) regarded these groups (or subspecies) as the unit of evolution rather than separate species. Comparative studies with various isolates of different origins are necessary to clarify the present complicated situation of the T. ishikariensis complex. Until then, we will use "biotype" for our own isolates of T. ishikariensis.

We wish to thank Dr. G.W. Bruehl, Washington State University, and to Dr. J.D. Smith, Agriculture Canada, for providing us with Typhula isolates. We are also grateful to Drs. F. Igarashi, T. Iwata, T. Kunii, M. Ozaki, I. Saito, K. Tsuboki, and M. Yamakawa, Hokkaido Prefectural Agricultural Experiment Stations, for helping us collect sclerotial materials.

Literature cited 1. Arsvoll, K. and Smith, J.D. (1978). Can. J. Bot. 56: 348-364. 2. Bruehl, G.W. and Cunfer, B.M. (1975). Phytopathology 65: 755-760. 3. Bruehl, G.W., Machtmes, R. and Kiyomoto, R. (1975). Ibid. 65: 1108-1114. 4. Bruehl, G.W. and Machtmes, R. (1979). Can. J. Bot. 57: 1252-1254. 5. Bruehl, G.W. and Machtmes, R. (1980). Phytopathology 70: 867-871. 6. Carr, A.J.H. and Olive, L.S. (1956). Amer. J. Bot. 46: 81-91, 7. Caten, C.E. and Jinks, J.L. (1966). Trans. Br. mycol. Soc. 49: 263-266. 8. Christen, A.A. and Bruehl, G.W. (1979). Phytopathology 69: 263-266. 9. Hardin, G. (1960). Science 131: 1292-1297. 10. Jinks, J.L. and Grindle, M. (1963). Heredity 18: 407-411. 11. Lawrence, T., Cooper, J.P. and Breese, E.L. (1973). J. agric. Res., Camb. 80: 341-348. 12. Lorenzetti, F., Tyler, B.F., Cooper, J.P. and Breese, E.L. (1971). Ibid. 76: 199-209. 13. Matsumoto, N. and Sato, T. (1982). Ann. Phytopath. Soc. Japan 48: In Press. 14. McDonald, W.C. (1961). Can. Plant Dis. Surv. 41: 256-260. 15. Ogoshi, A. (1976). Bull. Natl. Inst. Agric. Sci., Ser. C, No.30. 63 pp. 16. Puhalla, J.E. (1978). Phytopathology 69: 1186-1189. 17. Tomiyama, K. (1955). Hokkaido Natl. Agric. Exp. Stn. Rep. No.47. 324 pp.

和 文 摘 要

雪腐黒色小粒菌核病菌の生物型 とそれ らの北海道内における異所性

松本直幸 ・佐藤 徹 ・荒木隆男

雪 腐 黒 色 小 粒 菌 核 病 菌Typhula ishikariensis S. Imaiを 北 海 道 各地 よ り採集 し,各 菌 株 の類 別を 試 み た 。 そ の 結果,本 菌 に は 互 い に 交 配 しな い 二 つ の 群 が あ り,さ らに 一 方 の群 は培 養 形 態 の 異 な る二 つ の小 群 に 分 け られ た 。 これ らを 隼 物 型A, Bお よびCと した 。 生 物 型Aは 生 物 型Bあ るい はCと 交 配 せ ず,生 物 型Bと Cは 交 配 可 能 で あ った 。 生 物 型AはT. ishikariensisあ る い はT. ishikariensis var. ishikariensisと 同 一 で あ り,生 物 型CはT. ishikariensis var. canadensisと 類 似 で あ った が,生 物 型BはT. idahoensisあ るい は T. ishikariensis var. idahoensisと は 異 な った 。生 物 型AとBの 分 布 は 異 所 的 であ り,生 物 型Aは 多 雪 地 帯, 生 物 型Bは 寡 雪 地 帯 に 分 布 して いた 。T. ishikariensis菌 株 の 分 化 に っ い て論 議 した 。