日植 病 報 40: 412-418 (1974) Ann. Phytopath. Soc. Japan 40: 412-418 (1974)

Diplocarpon mali, sp. nov., the Perfect State of Blotch Marssonina coronaria

Yukio HARADA*, Kenzo SAWAMURA* and Koki KONNO*

原 田幸 雄 ＊・沢村健 三 ＊・今野 宏 規 ＊:リ ン ゴ褐斑 病 菌 の 完 全時 代 ( mali, sp. nov.) に つ い て

Abstract

An apothecium was found on apple leaves infected with blotch which had been overwintered out of doors on the ground. The ascospore grown on a culture medium produced Marssonina-type conidia. The ascospore caused typical symptoms of blotch when inoculated to apple leaves. The apothecium produced on overwintered diseased leaves is the perfect state of the causal fungus of blotch, Marssonina coronaria (Mars- sonina mali). After Nannfeldt's classification, the perfect state of the fungus belongs to the genus Diplocarpon of , . Morphological characters and host plants of the present fungus are distinct from those of the species known hitherto in this genus, hence Y. Harada et K. Sawamura, sp. nov., is proposed for it. A possible role of the ascospores as primary inocula in spring to early summer is suggested. (Received February 4, 1974)

Introduction

Blotch of apple caused by Marssonina mali (P. Henn.) S. Ito was first reported by Miyake1) in

1907 as a new apple disease in Japan. The disease was very destructive in the 1910's, but since that

time its occurrence has gradually decreased, probably owing to the general use of Bordeaux mixture.

Although blotch is at present of minor importance in apple diseases, the damage is likely to increase

in some districts of northern Japan.

In 1971 Parmelee8) published a paper entitled •gMarssonina leafspot of apple•h, in which he adopted

Marssonina coronaria (Ell. et J.J. Davis) J.J. Davis as the correct name of the causal fungus, re- garding Marssonina mali as one of its synonyms. Therefore, blotch of apple in Japan seems to be the same disease as Marssonina leafspot of apple in Canada. Parmelee further states that the perfect state of that fungus is unknown; possibly a discomycete related to Diplocarpon.

A microscopic examination of the infected leaves placed out of doors on the ground was carried

out by the authors during the dormant season of 1972-1973 in Hirosaki, Aomori Prefecture, to clarify the life history of the causal fungus of blotch of apple. The results of this study will be reported

here.

Development of the perfect state

Certain changes in the production of conidia of the fungus occurred in the leaves still on the

trees in autumn. The acervuli formed in summer contained only the conidia (Plate I, 2, 3), while

those in autumn had numerous rod-shaped structures besides the conidia, or often the former alone

* Faculty of Agriculture , Hirosaki University, Hirosaki, Japan. 弘前大学農学部 Ann. Phytopath. Soc. Japan 40 (5). December, 1974 413

(Plate I, 4, 5). These structures resembling Miura's5) young conidia or Parmelee's8) microconidia, were

considered to be the spermatia known in other allied species1,2,12).

On November 8, 1972, about 200 leaves bearing the fungus were collected and placed in a wire

cage to overwinter out of doors. At about 2-week intervals, some of these leaves were brought into

the laboratory, carefully washed free of soil particles, and hand-sectioned. The sections were mounted

in water or in lactophenol containing 0.1% cotton blue and examined microscopically. Initials of

the apothecium appeared as small mycelial masses between the epidermis and palisade tissue of the

infected leaves in late autumn and gradually enlarged during the winter (Plate II, 3), the diameter of

which attaining to 150-200ƒÊm by the end of December. The formation of asci and paraphyses

within the initials was apparent in late February the following year, and the mature ascospores were

seen from the latter part of April (Plate II, 1, 4, 5). Some apothecia containing viable ascospores were observed even in late July, suggesting the importance of those spores as inocula over an extended period from spring too early summer.

Isolation and culture of ascospores

The ascospores taken from the apothecium were suspended in sterilized distilled water, which

were then poured into Petri dishes containing a thin layer of water-agar. The dishes, after the

removal of excess water from agar surface, were incubated at 20C under a 12 hr-photoperiod (daylight-

type fluorescent light at an intensity of about 500lux) and 12 hr-dark. After two days of incubation, when the ascospores were germinating with a germ tube 40-60ƒÊm long, ten monosporic isolations were made onto potato saccharose agar slants, which were grown at 15C under the same light con- dition as above.

Seven out of the ten cultures were contaminated by bacteria within 10 days, but the remaining

three yielded small colonies (0.5-1mm in diameter) in 2 weeks of growing. After one month of growing, the colonies were subgloboid, with somewhat wrinkled surface, 2-3.5mm in diameter and

1.5-3mm in height, and dark brown in color. A microscopic examination revealed that these colo- nies contained many Marssonina-type conidia and some spermatia.

Inoculation experiment

Young leaves of potted apple seedlings were sprayed with the ascospore suspension in a green-

house. The plants were immediately covered with a belljar, to keep the leaf surface wet, and

allowed to remain covered for 7 days. The temperature in the greenhouse was 15-25C during the

inoculation experiment. Typical symptoms of blotch having Marssonina-type conidia appeared on

the leaves 10-20 days after inoculation. Also the conidia obtained from the pure cultures of ascospores

caused similar symptoms in this series of the experiment.

In another series of inoculation experiments, with conidia produced in pure culture from the conidial isolates, the fungus showed pathogenicity to apple, but not to pear, rose, and strawberry.

Morphology of the present fungus

From the results of preceding experiments, the authors consider that the apothecium found on

the overwintered apple leaves is the ascigerous state of the causal fungus of blotch of apple.

The morphological characters of the present fungus are as follows.

Ascigerous state: Apothecia fleshy, cup-shaped, 120-220ƒÊm in diameter, 100-150ƒÊm in height;

asci broad-clavate to oblong, 55-78•~14-18ƒÊm, 8-spored; ascospores oblong-elliptical, usually 1-septate, 414 日本 植物 病 理 学 会 報 第40巻 第5号 昭和49年12月

straight or somewhat curved, slightly constricted at the septum or not so, acute or rounded at the ends, hyaline, 23-33•~5-6ƒÊm; paraphyses filiform, 1-2 septate, almost the same height as the asci,

2-3ƒÊm in width, slightly broadened at the apex.

Conidial state: Spots usually epiphyllous, circular, 5-10mm in diameter, often enlarged and

coalesced to form an irregular shape, grayish brown, having a purple tinge at the periphery; acervuli

occurring in somewhat concentric rings, subcuticular, lens-shaped, pitch-dark, 100-200ƒÊm in diameter,

35-45ƒÊm in height; conidia born on small clavate conidiophores, gourd- or ampoule-shaped, 1-septate,

constricted at the septum, hyaline, guttulate, 20-24•~6.5-8.5ƒÊm; spermatia produced in the acervuli,

solely or mixed with the conidia, rod-shaped to elliptical, continuous, hyaline, 4-6•~1-2ƒÊm.

Taxonomic discussion

After Nannfeldt's classification7), the morphological characters of the perfect state of the present

fungus agree with those of the genus Diplocarpon of Dermateaceae, Helotiales. As far as the authors

are aware, the following four species of the genus Diplocarpon have been described on plants:

Diplocarpon agrostemmatis (Fuck.) Nannf., D. earliana (Ell. et Everh.) Wolf, D. maculatum (Atk.)

Jorstad (=D. soraueri (Kleb.) Nannf.), and D. rosae Wolf. Morphological characteristics of these spe-

cies and the present fungus are summarized in Table 1.

Table 1. Morphological characteristics of Diplocarpon on Malus and four species of the genus Diplocaron hitherto described

a) The side of leaf surface on which apothecia are formed.

As shown in Table 1, Diplocarpon maculatum is quite unique in the conidial type, Entomosporium. Among the remaining three species possessing Marssonina-type conidia, D. agrostemmatis differs from the present fungus in having somewhat larger apothecia on both upper and lower surfaces of the leaf and smaller ascospores with a septum at a third to quarter part of the cell3). D. earliana is not accordant with the present fungus in that the conidia are strongly curved10) and that larger apothecia develop on the lower surface of the leaf. In a narrower width of conidia as well as a shorter length of ascospores, D. rosae is distinct from the present fungus. The host range of these fungi seems, too, to afford an additional support to the view that the present fungus is different from the other known related species. Ann. Phytopath. Soc. Japan 40 (5). December, 1974 415

In view of the morphological features and the parasitism of the fungus the authors consider it

a new species of the genus Diplocarpon and propose the following name:

Diplocarpon mali Y. Harada et K. Sawamura, sp. nov.

Syn. Marssonina coronaria (Ell. et J.J. Davis) J.J. Davis, Trans. Wisc. Acad. 17: 881, 1914.

Marssonina mali (P. Henn.) S. Ito, Bot. Mag. Tokyo 32: 206, 1918.

(For other synonyms, see Parmelee, 1971)

Apotheciis epiphyllis, disciformibus, 120-220ƒÊm diam., 100-150ƒÊm altis; ascis oblongis vel sub- clavatis, 55-78•~14-18ƒÊm, 8-sporis; ascosporidiis oblongo-ellipticis, communiter 1-septatis, non vel vix

constrictis ad septa, hyalinis, 23-33•~5-6ƒÊm; paraphysibus filiformibus; 2-3ƒÊm crassis, septatis.

Status conidicus: Acervulis epiphyllis, lenticularibus, piceis, 100-200ƒÊm diam. et 35-45ƒÊm altis; conidiis ampulliformibus, 1-septatis, constrictis ad septa, hyalinis, guttulatis, 20-24•~6.5-8.5ƒÊm; sper-

matiis bacillaribus vel ellipticis, continuis, hyalinis, 4-6•~1-2ƒÊm.

Hab. in foliis Malorum spp.

Type specimen: On leaves of Malus pumila Mill. var. domestics Schneid., Hirosaki, Aomori

Prefecture, April 23, 1973, by Y. Harada, deposited in the Herbarium of the Faculty of Agriculture,

Hirosaki University, Japan.

The authors wish to express their thanks to Dr. T. Kobayashi, Government Forest Experiment

Station, Meguro, Tokyo, for his suggestions on the of the present fungus and reading of the manuscript.

Literature cited

1) Dodge, B.O. (1931). Mycologia 23: 446-462.

2) Higgins, B.B. (1936). Amer. J. Bot. 23: 598-602.

3) von Hohnel, F. (1917). Sitzb. Kais. Akad. Wiss. Wien, Math.-naturw. Klass. Abt. I. 126: 333-335.

4) Magnus, P. (1906). Hedwigia 45: 88-91.

5) Miura, M. (1915). Byotyugai Zassi 2: 398-402. (in Japanese)

6) Miyake, I. (1907). Bot. Mag. Tokyo 21: 49-53. (in Japanese)

7) Nannfeldt, J.A. (1932). Nova Acta Reg. Soc. Sci. Upsal., Ser. IV, Vol.8, No.2. Almqvist &

Wiksells Boktryckeri-A.-B., Uppsala. pp. 81-173.

8) Parmelee, J.A. (1971). Can. Plant Dis. Surv. 51: 91-92.

9) Seaver, F.J. (1951). The North American Cup-fungi (Inoperculates). Hafner Publ. Co., New

York. pp. 189-191.

10) Stone, R.E. (1922). Phytopathology 12: 375-380.

11) Stowell, E.A. and Backus, M.P. (1966). Mycologia 58: 949-960.

12) Stowell, E.A. and Backus, M.P. (1967). Ibid. 59: 623-636.

13) Wolf, F.A. (1912). Bot. Gaz. 54: 218-234 & Plate XIII.

和 文 摘 要

リ ン ゴ 褐 斑 病 菌 の 完 全 時 代(Diplocarpon mali, sp. nov.)に つ い て

原 田幸雄 ・沢村 健三 ・今 野宏規

野 外 地 表 で越 冬 させ た 本 病 罹 病葉 上 に 春 季 子 の う盤 を発 見 し,子 の う胞子 の分 離 培 養 お よび接 種 試 験 の結 果 か ら,こ れ が 本病 の病 原 菌Marssonina coronaria (Ell. et J.J. Davis) J.J. Davisの 完 全時 代 で あ る こ とを確 416 日本植 物 病 理学 会報 第40巻 第5号 昭 和49年12月

認 し た 。 完 全 時 代 の 形 態 はNannfeldt (1932)の 分 類 に 従 う とHelotiales, DermateaceaeのDiplocarpon属 に

一 致 す る が ,本 属 の い ず れ の 既 知 種 と も 形 態 お よ び 寄 主 に お い て 異 な る の で,本 菌 を 新 種 と認 め てDiplocarpon mali Y. Harada et K. Sawamuraの 学 名 を 与 え た 。

Explanation of Plates

Plate I. Diplocarpon mali Y. Harada et K. Sawamura, sp. nov.

1. Lesions on an apple leaf (a) and surface view of an acervulus (b, •~25).

2. A cross section of acervulus containing conidia (•~300).

3. Conidia (•~600).

4. A cross section of acervulus containing spermatia (•~250).

5. Spermatiophores (a, •~830) and spermatia (b, •~830).

Plate II. Diplocarpon mali Y. Harada et K. Sawamura, sp. nov.

1. Surface view of apothecia (•~15).

2. Subcuticular mycelial strands on the upper surface of leaf lesions (•~500).

3. Apothecial initials in early December (•~130).

4. A cross section of mature apothecium (•~230).

5. Asci (a, •~450), ascospores (b, •~700), and a germinating ascospore (c, •~230).

6. Drawings of ascospores. Two spores on the left side seem to be premature ones (•~1000). Plate I Plate II