日植 病 報 65: 553-556 (1999)

Ann. Phytopathol. Soc. Jpn. 65: 553-556 (1999)

短 報

Phytopathological Note

Mode of Infection of Echinochloa oryzicola by Exserohilum monoceras

Hiroshi TSUKAMOTO*, Mitsuya TSUDA** and Takane FUJIMORI*

Key words: Exserohilum monoceras, Echinochloa oryzicola, appressorium, penetration, submergence, myco-herbicide.

Exserohilum monoceras (Helminthosporium mono-

ceras) has been investigated as a mycoherbicidal agent

to control Echinochloa weed species, with particular

attention to the influence of environmental factors and

water management on its herbicidal activity2,5,8,9,11,13,14).

Different methods for applying the fungal conidia, such

as spray inoculation of aerial plant parts and drop

inoculation of plants submerged by flooding, have also

been investigated. Herbicidal activity after spray inocu-

lation is influenced greatly by dew duration whereas

activity after drop inoculation is affected by flooding

conditions9,14). In either case, stable and high herbicidal

activity requires a continuous water supply from

conidial germination through lesion development, simi-

lar to most fungal foliar pathogens. However, the cytol-

ogy of the infection has not been reported.

Here, we report on the establishment of E. monoceras

in E. oryzicola using light microscopy to determine the

initial histological events at the infection site, such as

fungal appressorium formation and penetration into the

host under dew conditions and submergence. Moreover,

we discuss the mode of infection in the case of E.

monoceras and E. oryzicola compared to other Helmin-

thosporium pathogens and their hosts. A preliminary

report of this work was presented at the Annual Meet-

ing of the Phytopathological Society of Japan in 19977).

Conidia of E. monoceras JTB-808 prepared as de-

scribed in previous reports8,9) were washed by centri-

fugation at 1600•~g for 10min and resuspended with

distilled water in the usual manner. This procedure was

repeated three times. The resultant conidial suspension

was adjusted to 104conidia/ml with a haemacytometer

(Fucks-Rosenthal, Kayagaki Irikakogyo, Japan) and

used as inoculum. Echinochloa oryzicola C type plants at

the first-leaf stage were grown in pots as described in a

previous report8). Plants were taken from the pots and

their roots were covered with wet absorbent cotton.

They were then laid on petri dishes and secured with

tape. The plants being used for the drop inoculation

experiment were then submerged in distilled water.

Four hundred ƒÊl of the inoculum was pipetted onto the

water surface above the submerged leaves. In the case of

the dew treatment, 4 drops (3ƒÊl/drop) of the inoculum

were pipetted onto the upper surface of the first leaves

which were not submerged. The petri dishes in both

treatments were sealed with Parafilm (American Nation-

al Can, WI, USA) and kept at 25•Ž in the dark for 36,

48 or 63hr after inoculation. After incubation, the first

leaf was excised from the sheath. The leaves were

cleared by soaking them in an ethanol-lactophenol solu-

tion (1ml phenol, 1ml lactic acid, 1ml glycerol, 1ml

distilled water and 8ml ethanol) for 4 to 6 days. The

leaves were then mounted in distilled water containing

20% (v/v) glycerol and examined at •~400, •~630 and •~

1000 (Axioskop, Carl Zeiss, Germany) to locate sites of

attempted penetration. The proportion of cuticular to

stomatal sites of penetration was evaluated. Penetration

was distinguished by a visible connection between the

appressoria and intra- or intercellular hyphae. Moreover,

the proportion of sites at which infection hyphae elon-

gated intra- or intercellularly was evaluated.

Most conidia germinated monopolarly or occasionally

bipolarly on the host. Many of the germ tubes extended

along the surface of the junction of the epidermal cells.

The germ tubes formed a unicellular saccate appresso-

rium on the epidermal surface (Plate I, A-C, E, F). The

appressoria formed and started penetrating at three

different sites: direct penetration through the upper

layer of the junction of epidermal cells (Plate I, C, E-H),

through a site over the cell (Plate I-A), and through

stomata (Plate I-B). A primary infection vesicle some-

times formed beneath the appressorium (Plate I-E, G).

Infection hyphae extended intercellularly (Plate I-C, D)

or intracellularly into the epidermal cell (Plate I, A, E-

H). The intracellular hyphae grew well, ramified the

epidermal cell and often entered adjacent cells (Plate I-

G). No papillae-like structures were observed beneath

the appressoria except in one case, under dew condi-

tions, when the appressorium succeeded in penetrating

(Plate I-H). By 63hr after inoculation under dew condi-

tions, fungal mycelia had covered most of the host

surface, many conidiophores protruded out of stomata,

and conidia had formed (Plate I-I). Conidia did not form

under submergence.

* Plant Protection Research Laboratory, Japan Tobacco Inc., Umegaoka, Aoba-ku, Yokohama 227-8512, Japan 日本 た

ば こ産 業 株 式 会 社 植 物 保 護 開 発 セ ン タ ー** Graduate School of Agriculture

, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan 京

都 大 学 大 学 院 農 学 研 究 科

554 日本植物病理学会報 第65巻 第5号 平成11年10月

Table 1. Infection sites of Exserohilum monoceras JTB-808 on Echinochloa oryzicola C type under submergence and dew conditions

a) Conidial suspension was applied to leaves under submergence or in dew conditions. Observed 48hr after inoculation.

b) Penetration directly into site on periclinal wall of an epidermal cell but not on the juncture of the epidermal cells.

Infection hyphae elongated intracellularly into the cell in all penetrations.

c) Penetration through upper layer of the juncture of epidermal cells. Infection hyphae elongated intra- or intercellularly.

d) Penetration through stomata. Infection hyphae elongated intercellularly in all penetrations.

e) Mean•}standard error from ten leaves, ca. 30 penetrations/leaf. There were no significant differences in the rate of

penetration sites between the two treatments, submergence and dew, according to Student's t test (p=0.05).

According to Student's t-test, there were no signifi-cant (p=0.05) differences between dew conditions and submergence conditions in terms of the percentage of

penetrations in each of the three sites: i.e. cuticular penetration through the upper layer of junctions of host epidermal cells; penetration through a site on the peri-clinal cell wall; and penetration through stomata (Table 1). Following cuticular penetration, the percentage of hyphae elongating intra- or intercellularly was also not significantly (p=0.05) different between the two condi-tions (Table 1). Therefore, we suspected that dew and submergence were equivalent in the sense of supplying water to the fungus from conidial germination to lesion

development. Further studies will encompass the time-course from conidial germination, via appressorium formation to penetration to elucidate the differences in lesion development under dew conditions and

submergence9).Most (>90%) appressoria formed over junctions

between host epidermal cells, so that they penetrated directly through the cuticle (Table 1). Other f oliar

graminicolous pathogens belonging to the Helmintho-sporium genus also favor the junction in forming an appressorium. Eighty-one to 93% of appressoria of H. carbonum (Bipolaris zeicola) are formed over the cell

junctions of maize6). Moreover, H. turcicum (E. tur-cicum) on maize4), H. dictyoides (Drechslera dictyoides) on ryegrass1) and H. oryzae (B. oryzae) on rice3), princi-

pally form appressoria on junctions, although the fre-quency was not mentioned. Direct penetration through a site above the epidermal cell has not been reported in Helminthosporium pathogens, except in this study of E.

monoceras.A few appressoria (<10%) of E. monoceras formed on

cells adjacent to stomata, then penetrated through stomata (Table 1). With H. carbonum, about 1% of the appressoria formed over stomata6). H. turcicum penetrat-ed occasionally through stomata4), while H. oryzae did not form appressoria over or enter through the sto-mata3), and H. dictyoides passed over and rarely pene-trated stomata1). Stomata do not seem to be attractive to these pathogens. However, in H. maydis (B. maydis)

at least two-thirds of penetrations occurred through the

junctions adjacent to stomata, then the hypha penetrat-ed into the stomatal cavity. About 16% of the penetra-tions were stomatal10).

Appressoria of E. monoceras on upper layers of junc-tions directly penetrated either intracellularly or inter-cellularly. During intracellular penetration in an epider-mal cell, the hyphae expanded into a spherical vesicle and colonized the cell, similar to H. catenarium (D. catenaria) on reed canarygrass12) and H. carbonum6) and H. turcicum4) on maize. A high frequency of hyphae

progressing intercellularly was a distinctive characteris-tic of E. monoceras after penetrating a site over a

junction of epidermal cells. After the fungus penetrated through the junction and grew intercellularly in the epidermal cells, it progressed into the mesophyll cells. However, subcuticular hyphae were not observed in the case of E. monoceras although they did occur in most cases of penetration by H. carbonum6), and occasionally in H. dictyoides1) and H. turcicum4).

The authors are grateful to Dr. Y. Yamasue (Kyoto Uni-versity) for providing the Echinochloa oryzicola seeds used in this study. We also thank Ms. K. Shimizu for culture mainte-nance and acknowledge the assistance of Ms. A. Mackie in the English revision of this manuscript.

Literature cited

1. Cromey, M.G. and Cole, A.L.J. (1985). Cytology of the host-pathogen interactions between Lolium perenne and Drechslera dictyoides. Plant Pathol. 34: 83-94.

2. Gohbara, M. and Yamaguchi, K. (1992). Biological control agents for rice paddy weed management in

Japan. In Proceedings of International Symposium on Biological Control and Integrated Management of Paddy and Aquatic Weeds in Asia, National Agriculture Research Center, Tsukuba, pp. 353-366.

3. Hau, F.C. and Rush, M.C. (1982). Preinfectional inter-actions between Helminthosporium oryzae and resistant and susceptible rice plants. Phytopathology 72: 285-292.

4. Knox-Davies, P.S. (1974). Penetration of maize leaves by Helminthosporium turcicum. Phytopathology 64:

Ann. Phytopathol. Soc. Jpn. 65 (5). October, 1999 555

1468-1470.

5. Morita, H. (1996). Morphological characteristics of spikelets and panicles of Echinochloa colonum (L.) Link and lack of establishment in Kyushu island at low tem-

peratures. Weed Res., Japan 41: 90-97 (in Japanese with English summary).

6. Murray, G.M. and Maxwell, D.P. (1975). Penetration of Zea mays by Helminthosporium carbonum. Can. J. Bot. 53: 2872-2883.

7. Tsukamoto, H., Gohbara, M. and Fujimori, T. (1997). Effect of flooding on infection, lesion development and mycoherbicidal activity of Echinochloa spp. pathogen, Exserohilum monoceras. Ann. Phytopathol. Soc. Jpn. 63:

216 (Abstr. in Japanese).8. Tsukamoto, H., Gohbara, M., Tsuda, M. and Fujimori,

T. (1997). Evaluation of fungal pathogens as biological

control agents for the paddy weed, Echinochloa species by drop inoculation. Ann. Phytopathol. Soc. Jpn. 63:

366-372.9. Tsukamoto, H., Tsuda, M., Gohbara, M. and Fujimori,

T. (1998). Effect of water management on mycoher-bicidal activity of Exserohilum monoceras against

Echinochloa oryzicola. Ann. Phytopathol. Soc. Jpn. 64: 526-531.

10. Wheeler, H. (1977). Ultrastructure of penetration by

Helminthosporium maydis. Physiol. Plant Pathol. 11: 171-178.

11. Yabuno, T. (1975). The classification and geographical

distribution of the genus Echinochloa. Weed Res., Japan 20: 97-104 (in Japanese).

12. Zeiders, K.E. (1976). A new disease of reed canarygrass

caused by Helminthosporium catenarium. Plant Dis. Rep. 60: 556-560.

13. Zhang, W.M., Moody, K. and Watson, A.K. (1996). Responses of Echinochloa species and rice (Oryza sativa)

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和 文 摘 要

塚 本 浩史 ・津 田盛也 ・藤森 嶺:Echinochloa oryzicola(タ イ

ヌ ビエ)へ のExserohilum monocerasの 感染 様 式

Exserohilum monocerasは,水 田 に発 生す るノ ビエ類 に対 す

る生 物 除草 剤 の活性成 分 として,有 用 な 糸状 菌 で あ る。本研究 で

は,タ イ ヌ ビエ に対 す るExserohilum monocerasの 感 染様式 を

調 べ た 。す なわ ち,1葉 期 のタイ ヌ ビエ の葉 の表 面 に本 菌胞子 懸

濁 液 を接種 した36, 48あ るい は63時 間後 に,接 種 葉 を ラク トフ

ェノー ル-エ タノー ル水 溶 液 に浸 漬 し,固 定 ・脱 色 した後,顕 微

鏡下 で本 菌 の感 染行動 を観 察 した。 ほ とん どの胞 子 は発芽 管 を

伸 長 させ た後,表 皮細胞 の縫 合部 の上層 に付 着器 を形 成 し,角 皮

侵 入 した。侵入 菌 糸 は表 皮細胞 中 に侵入 す るか,あ るいは細胞 間

隙 を伸 展 した。また,ご く一部 の付着 器 は表 皮細 胞上 あ るい は気

孔孔 辺細 胞 上 に形成 され,そ れ ぞれ 角皮 侵入 あ るい は気 孔侵 入

を行 った。さ らに,結 露 お よび冠 水条 件下 の宿主 葉上 にお ける本

菌 の侵入 部位 お よび侵 入菌 糸の伸 長部 位 を比較 した が,重 要 な

違 い はな か った。

(Received December 14, 1998; Accepted June 9, 1999)

Explanation of plate

Plate I. Conidial germination, appressoria formation, penetration, elongation of intra- and intercellular hyphae and sporula-

tion, of Exserohilum monoceras JTB-808 on Echinochloa oryzicola C type under submergence or dew conditions.

Bars represent 20ƒÊm.

A: Direct penetration into an epidermal cell through a site over the cell under dew conditions, 48hr after inoculation. Note

the conidium (c), the appressorium (a) and intracellular hypha (ah).

B: Penetration through the stomata (s) under submergence, 48hr after inoculation.

C: Appressorium (a) on upper layer of the junction of epidermal cells under submergence, 63hr after inoculation.

D: Intercellular hypha (eh) from the appressorium (a) (out of focus) shown in Plate I-C.

E: Primary infection vesicle (v) between two host cells beneath the appressorium (a) under submergence, 36hr after

inoculation.

F: Infection into an epidermal cell through the junction of epidermal cells, ramified intracellular hyphae (ah) under

submergence, 48hr after inoculation.

G: Primary infection vesicle (v) between a cell wall and an invaginated membrane, and hypha entering neighboring cell

under dew conditions, 36hr after inoculation.

H: The papilla-like structure (p) beneath the appressorium (a) and intracellular penetration through the papilla-like

structure under dew conditions, 36hr after inoculation.

I: The conidiophore (cp) protruding from the stomata (s) and the regenerated conidium (c) under dew conditions, 63hr after

inoculation.

556 日本植物病理学会報 第65巻 第5号 平成11年10月

Plate I