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日植 病 報 52: 801-808 (1986) Ann. Phytopath. Soc. Japan 52: 801-808 (1986)

A Whole-Leaf and Clearing Procedure for Analysing Cytological Aspects of Interaction

between Rice Plant and Rice Blast

You-Liang PENG, Jiko SHISHIYAMAand Masaki YAMAMOTO

Abstract

In order to analyse the cytological aspects of interaction between rice plant and blast fungus, a new whole-leaf staining and clearing procedure has been developed, which includes following treatments of the infected tissue as bathing at 95C in chlorite solution acidified with acetic , dehydrating in series, staining in autoclave at 121C with blue dissolved in alcoholic lactophenol and clearing with lactophenol. In a comparative study with present procedure, acidic . especially aniline blue, were found to be much more effective than basic ones to stain the hyphae of rice blast fungus in epider mal cells of cleared rice leaves. was better than for acidfying rice leaf tissue. Critical proportions of in staining solution contributed to the stain ing of infection hyphae and prevented rice leaf tissue from collapse by autoclaving. Staining in autoclave was effective and shortened staining time. With such superiority, this stain ing and clearing procedure allowed infection hyphae, even those beneath appressoria, to be easily observed and kept infected rice leaf tissue microscopically intact. This procedure should be adaptable for exactly investigating the infection behavior of rice blast fungus and the cellular reactions occurred in rice leaves during infection. (Received June 23, 1986)

Key words: rice blast fungus, rice plant, whole-leaf staining and clearing procedure, cel lular reactions, infection behavior.

Introduction

Whole-leaf clearing and staining techniques have been particularly applied to analyse cytological events associated with host-parasite specificity1,2,19). Several such techniques have been sucessfully applied, some to observe the behavior of pathogenic fungi before penetration7,12), others to observe the development of pathogenic fungi at different stages after penetrating inside plant tissue2,3,5,13,15,19) However, a suitable clearing and stain ing procedure has not been established yet for cytological examination on rice plant parasite interactons in rice leaf blades. Consequently, cytological studies on rice plant -Pyricularia oryzae interactions had been mainly carried out so far with inner epidermis of leaf sheath14,17,18) or by observing paraffin sections of leaf blades4,8). In the case of leaf sheath, fungal development and host cellular reaction have been demonstrated to differ from those in leaf blades, and when paraffin sections are used, the infection pro

Laboratory of Plant Pathology, Faculty of Agriculture, Kyoto University. Kyoto 606, Japan

京 都 大 学 農 学 部 802 日本植物病理学会報 第52巻 第5号 昭和61年12月

cess of pathogen can seldom be observed to an appreciable extent. Accordingly, both paraffin sections of leaf blades and inner epidermis of leaf sheath give an inadequate conception on the overall relationship between rice plant and blast fungus. It is neces sary, therefore, to develop a staining and clearing procedure applicable for rice leaf blades infected by rice blast fungus, and several attempts have been made6,9,16,21) but none of those are satisfactory. In this paper, a whole-leaf staining and clearing pro cedure, found to be effective to stain infection hyphae and clear rice leaf tissue, is de scribed.

Materials and Methods

Fungus and plant. Two cultivars, Tsuyuake and Ishikarishiroke, of rice plant, Oryza sativa L. at the 5th leaf stage, were used to inoculation with race 13120) of rice blast fungus, Pyricularia oryzae Cavara. Both rice seeds and pathogen were kindly supplied by Dr. R. Yoshino and Dr. H. Koga, Hokuriku National Agricultural Experiment Station. With race 131, Tsuyuake and Ishikarishiroke shared susceptible and resistant relation ships, respectively. The inoculation was performed by spraying conidia suspension onto leaf blades. The inoculated seedlings were maitained at about 25 C under 5,000lux in candescent light for 12hr every day. Whole-leaf staining and clearing procedure. The inoculated fifth leaves were sampled at the prescribed intervals after inoculation, fixed and decolorized in lactophe nol, then treated in the following procedure. Decolorized leaf blades were first put into a jar containing 100ml of 0.75% sodium chlorite solution acidified with 5ml of 15% acetic acid, and bathed at 95C. This took about 2hr to complete. Then, the leaf blades were transferred and were dehydrated in 50, 70 and 95% ethanol series for 5min, respectively and finally in absolute ethanol for 10min. Before staining, the leaf blades were cut into 1cm length segments and subdivided longitudinally by half in order to facilitate permeation of dyes into leaf tissue. Staining was accomplished by autoclaving the leaf pieces immersed in 0.01% (w/v) staining solution at 121C (1atm) for 15min. The solution was prepared by dissolving dyes in the mixture containing equal volumes of absolute ethanol, lactic acid and phenol (ALP). Finally, above stained leaf pieces were cleared for 5min in the clearing solution composed of phenol and lactic acid with volume ratio of 4 to 1. Microscopic observation. The stained and cleared specimens were mounted on a glass slide in the medium of above clearing solution and observed under Nikon light microscope with emphasis on the temporal changes in the penetration frequency and development of infection hyphae and on the types and percentage of host cellular re action. The penetration frequency was expressed by percentage of the appressoria with visible infection hyphae to the observed appressoria. The growth of infection hyphae was evaluated by Takahashi's scales17) and was expressed by average hyphal indices which means hyphae indices per appressoria with visible infection hyphae. Comparison of staining ability. Acid fuchsin (Nakarai Chemicals. Ltd.), cotton blue (Schmid & Co.), aniline blue (E. Merck), safranin (G. Grubler & Co.) and crystal Ann. Phytopath. Soc . Japan 52 (5). December, 1986 803 violet (E. Merck) were used to compare their staining ability for the infection hyphae inside the cleared leaf tissue at the same concentration (0 .01%) in ALP solution. Also, hydrochloric acid was compared with acetic acid to examine their effects on the staining of infection hyphae and the host leaf tissue at different concentrations. Critical ratio of ethanol to lactic acid and phenol in the staining solution was also investigated.

Results

Staining ability of different dyes for the infection hyphae inside leaf tissue Table 1 shows the staining ability of five kinds of dyes for the infection hyphae inside host leaf tissue and the epidermal and mesophyll cells. Two basic byes, safranin and , stained neither infection hyphae nor host leaf cells. The acidic dyes, aniline blue, cotton blue and acid fuchsin showed distinguishable staining ability for the infection hyphae in rice leaf epidermal cells and aniline blue appeared more effective than acid fuchsin and cotton blue. Plate I shows the infection hyphae stained by ani line blue in bulliform cells of rice leaves. In contrast to normal infection hyphae in compatible cells (Plate I-A), those in incompatible ones were evidenty inhibited and malformed (Plate I-B, C, D). Temporal changes of penetration frequency and development of infection hy phae Race 131 of rice blast fungus began its penetration into both susceptible and re sistant cultivars 18hr after inoculation. The penetration frequency increased with time and was over 30% in both Tsuyuake and Ishikarishiroke 72hr after inoculation. Race 131 was observed to be much more inhibited not in the penetration frequency but in the growth of infection hyphae in resistant Ishikarishiroke than in susceptible Tsuyuake (Fig. 1). Cellular reactions of rice leaves Five types of responses of penetrated cells were observed in leaf blades of either resistant Ishikarishiroke or susceptible Tsuyuake, which are shown in A to E of Plate 1. Here, these reactions of infected epidermal cells are referred to as type A, B, C, D and E, respectively. Type A is a representative of the infected cells without any light microscopic change. Type B is the infected cells with light brown and stained cell wall. Type C and D are well-debated fine cytoplasmic granulation and cell necrosis, respec

Table 1. Staining ability of different dyes

a) Dyes are dissolved in ALP as indicated in the text. b) -: less stained; +: stained; _??_: well stained. 804 日本 植 物病 理 学 会 報 第52巻 第5号 昭 和61年12月

Fig. 1. Penetration frequency (-) and growth of infection

hyphae (---) of Pyricularia oryzae Cavara in rice leaves. •› and •œ indicate combinations of race 131

with Tsuyuake and Ishikarishiroke, respectively.

Fig. 2. Cellular reactions of rice plant to infection of Pyricularia oryzae Cavara at 72hr after inoculation. _??_ and _??_ indicate combinations of race 131 with Tsuyuake and Ishikarishiroke, respectively.

tively. Type E is the coarse cytoplasmic granulation similar to those in infected leaf sheath reported recently by Tomita and Yamanaka18). Fig. 2 is the percentage comparison of these cellular reactions between Tsuyuake and Ishikarishiroke at 72hr after inoculation with race 131. Although Tsuyuake was characteristic of high per centage of type A, Ishikarishiroke showed high percentage of type D. And also type B occurred more frequently in Ishikarishiroke than in Tsuyuake. Ann. Phytopath. Soc. Japan 52 (5). December, 1986 805

Table 2. Effect of and ethanol on the staining of infection hyphae and the rice leaf tissue

a) +: collapse; -: not collapse. b) and c) are referred to Table 1.

Effects of acids and ethanol on the staining of infection hyphae and on host leaf tissue Hydrochloric acid caused collapse of rice leaf tissue even when it was used at lower concentration, instead of acetic acid, for acidifying rice leaves. Autoclaving caused col lapse of host leaf tissue and infection hyphae were less stained when ethanol was not added into staining solution. Ethanol contributed to specific staining of the infection hyphae and prevented leaf tissue from collapse by autoclaving when it was mixed with lactic acid and phenol at equal volume ratio, but higher proportion of ethanol led to staining of host leaf tissue (Table 2).

Discussion

Differential staining of internal infection hyphae and no breakout of plant cells are critical to cytological studies of plant-parasite interactions. Yosh-ino21-23) stained spe cifically the infection hyphae of rice blast fungus inside rice leaf epidermal cells by using hydroxide to remove host cytoplasm. For this reason, his method is evidently inadequate for examining host cellular reactions. In the present procedure, phenol was used for clearing leaf tissue together with lactic acid and kept leaf tissue microscopically intact. Koga and Kobayashi9) reported that crystal violet dissolved in methyl salicylate was a good to stain the infection hyphae. But the authors failed to stain them with basic crystal violet and safranin. In contrast, acidic dyes, especially aniline blue, were more effective. These seem that basic dyes would be improper to stain internal infection hyphae of rice blast fungus, at least in the present procedure. Acidifying treatment of infected leaves appears to be necessary for staining of the infection hyphae. This is consistent with Yoshino's view21). But, the present results turned out to be contrary to his another conclusion that hydrochloric acid was more safe than acetic acid for rice leaf tissue21). As to the reason for different effect of acetic acid on rice leaf tissue, the concentration he used, which was 30% and far higher than the present 0.007%, may be attributable. But what is responsible for breakout of rice leaf tissue by hydrochloric acid in the present, not in Yoshino's experiments21), may 806 日本植物病理学会報 第52巻 第5号 昭和61年12月 be based on the different temperature. Ethanol seems to enhance staining of the infection hyphae, which is in favour of Hering's observation3). But, excessive proportion of ethanol in the staining solution possi bly results in staining of host leaf tissue. In addition, ethanol prevents rice leaf tissue from collapse. As shown in Table 2, staining in autoclave caused disorganization of leaf blades and the infection hyphae were less stained when no ethanol was put into the staining solution. The improved staining of infection hyphae is also probably due to, in part, autoclave treatment. High temperature usually helps staining of the in fection hyphae, therefore, autoclave staining at 121C was employed into the present procedure and showed its effectiveness. At room temperature, the staining will take over 12hr in present procedure, while use of autoclave shortens the staining time to only 15min. This may be that autoclaving forces dyes into leaf tissue. The above-mentioned superiority of this procedure over others allowed the infection hyphae easy to be observed, even those beneath appressoria besides cellular reactions of host. The penetration of rice leaves by blast fungus in the present study, therefore, was far higher and earlier than that reported by Yoshino21-23) and Koga and Kobaya shi10,11). It is suggested that this newly developed whole-leaf staining and clearing method is adaptable for analysing cytological aspects of interactions between rice plant and Pyricularia oryzae Cavara.

The authors are indebted to Dr. R. Yoshino and Dr. N. Koga, Hokuriku National Agricultural Experiment Station, for their kindly supplying with rice seeds and isolate of rice blast fungus.

Literature cited

1. Hansan, H. (1981). Ann. appl. Biol. 99: 119-124. 2. Heath, M. C. (1971). Phytopathology 61: 383-388. 3. Hering, T. F. and Nicholson, P. B. (1964). Nature 201: 942-943. 4. Ikata, S., Matsuura, K. and Taguchi, S. (1931). Noji Kairyo Shiryo 20: 1-140 (in Japanese). 5. Isaac, P. K. (1960). Phytopathology 50: 474. 6. Ito, S. and Shimada, S. (1937). Noji Kairyo Shiryo 120: 1-190 (in Japanese). 7. Janes, B.S. (1962). Nature 193: 1099-1100. 8. Kawamura, E. and Ono, K. (1948). J. Natl. Agr. Expt. Sta. 4: 13-22. 9. Koga, N. and Kobayashi, T. (1980). Ann. Phytopath. Soc. Japan 46: 679-681. 10. Koga, N. and Kobayoshi, T. (1982). Ibid. 48: 506-513 (in Japanese). 11. Koga, N. and Kobayashi, T. (1982). Proc. Assoc. Plant Prot. Hokuriku 30: 12-18 (in Japanese). 12. Mclean, R. L. and Byth, D. E. (1981). Aust. J. Agr. Res. 32: 435-443. 13. McBryde, C. (1936). Am. J. Bot. 23: 686-688. 14. Ohata, K., Goto, K. and Kozaka, T. (1963). Ann. Phytopath. Soc. Japan 28: 24-30 (in Japanese). 15. Shipton, W.A. and Brown, J. F. (1962). Phytopathology 52: 1313. 16. Suzuki, H. (1940). Nogyo Oyobi Engei (Agriculture and Horticulture) 15: 1999-2010 (in Jap anese). 17. Takahashi, Y. (1956). Bull. Yamagata Univ. Agr. Sci. 2: 37-51 (in Japanese). 18. Tomita, H. and Yamanaka, S. (1983). Ann. Phytopath. Soc. Japan 49: 514-521 (in Japanese). 19. White, N. H. and Baker, E. P. (1954). Phytopathology 44: 506-513. 20. Yamada, M., Kiyosawa, S., Yamaguchi, T., Hirano, T., Kobayashi, T., Kushibuchi, K. and Watanabe, S. (1976). Ann. Phytopath. Soc. Japan 42: 216-219. 21. Yoshino, R. (1971). Proc. Assoc. Plant Prot. Hokuriku 19: 14-17 (in Japanese). 22. Yoshino, R. (1972). Ibid. 20: 4-9 (in Japanese). 23. Yoshino, R. (1979). Bull. Hokuriku Natl. Agr. Expt. Stn. 22: 163-221 (in Japanese). Ann. Phytopath. Soc. Japan 52 (5). December, 1986 807

和 文 摘 要

彰 友良 ・獅山慈孝 ・山本昌木:葉 身の一染色透明化法による イネといもち病菌との 相互作用の細胞学的 解析

イ ネ と い も ち病 菌 と の相 互 作 用 を 細 胞 学 的 に解 析 す るた め に葉 身 の 一 染 色 透 明 化 法 を 考 案 した 。 本 法 に よ る と,イ ネ葉 身 に おけ る い もち病 菌 は 接 種18時 間 後 に侵 入 を始 め,そ の 後 の 侵 入 率 は,従 来報 告 され て き た値 よ り も高 く,接 種72時 間 後 に 親 和 性 組 合 せ お よ び 非親 和性 組 合 せ の い ず れ に お いて も30%を 越 え た 。 また,い も ち病 菌 の 侵 入 し た イ ネ葉 身 表 皮 細 胞 は そ の反 応 に よ って5型 に 分 け られ た 。感 染 菌 糸 の染 色 に は 塩 基 性 色 素 よ り酸 性 色 素,と くに ア ニ リ ンブ ル ーが 効 果 的 で あ っ た。 ま た,染 色 前 に イ ネ葉 身 を酸 性 に 保 つ 必 要 が あ り,そ の処 理 は 塩 酸 よ り酢 酸 の方 が よ く,イ ネ葉 身 細 胞 を壊 す こ とが な か った 。 染 色 液 に適 量 の エ タ ノ ー ル(染 色 液 の1/3)を 加 え る こ と に よ っ て感 染 菌 糸 の染 色 が促 進 され,オ ー トク レー プ に よ る加 熱 染色 に よ って もイ ネ葉 身細 胞 は 崩 壊 し なか っ た。 以 上 に 述 べ た方 法 に よ れ ば イ ネ 葉 身 に お け る い も ち病 菌 の感 染 行 動 と イ ネの 細 胞 反 応 を よ り詳 細 に調 べ る こ とが で き る と考 え られ た。

Explanation of plate Plate I Light micrographs of infection hyphae and cellular reactions. A and C occurred in combination of race 131 with Tsuyuake and B, D and E in combination of race 131 with Ishikarishiroke. A: Cellular reaction type A and smart growth of infection hyphae at 40hr after inoculation. B: Cellular reaction type B and inhibited infection hypha at 48hr after inoculation. C: Cellular reaction type C and inhibited infection hypha at 40hr after inoculation. D: Cellular reaction type D and malformed infection hypae at 72hr after inoculation. E: Cellular reaction type E at 72hr after inoculation.

Abbreviations AP: appressorium BSW: brown and stained cell wall CCG: coarsely cytoplasmic granulation CN: cell necrosis FCG: finely cytoplasmic granulation IH: infection hypha Each bar indicates 25ƒÊm 808 日本植物病理学会報 第52巻 第5号 昭和61年12月

Plate I