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In an incompatible combination of rice cultivar and blast fungus race, the rapid increase of perexidase activity of rice leaves occured within 24hr after inoculation, while a little or no increase was observed in a compatible case at early stage of infection. However, sudden increase occured at 3-5 days after inoculation in a compatible combination and the activity exceeded that of an incompatible combination. These alteration of activities occured more rapiclIy in aged leaves. The highest activity was observed at t.he time of rapid lesion enlargement in compatible case. The isezymes activated by the infection were iixed, and no new isozyme speeific to cornpatible or incompatible combination was detected. The distinct difference between both combinations was the time of increase started, The arnple supplies of nitrogenous fertilizer reduced the degree of resistance to rice blast disease. However, peroxidase activities increased with the doses of N-fertilizer and the highest activity was observed at the highest dose, i,e., under the most susceptible condition. These results indicate that the increase of peroxidase activity is possibly the reflection of host cell collapse or physiological disorder and may not be directly involved in the resistance mechanisrn. (Received June 8, 1981) Introduction The shift of oxidative enzyme activities reflects a rapid physiological response of host cells in infection site and is commonly observed in various plant diseasesJ The increased activity of peroxidase after infection have been well documented5,iS,37,4i,4Sny50). AIthough the role and site of action of peroxidase are still in arnbiguity, some close relations with physiological processes have been clarified. Peroxidase plays as a catalyst of ethylene,47) lignini3,33,34) and phytoalexin4) formation, further it may, in some cases, participate in the oxidation of phenolics20,54) and direct inhibition of parasite's growth23) through such as myeloperoxidase-halide-hydrogen peroxide antibacterial systemi9). Therefore, the implication of peroxidase in resistanee to various plant diseases have been much stressed. Some reports indicated that the qualitative and quantitative changes of peroxidase isozyrnes may assoeiate with determining resistance or susceptibilityi2,Z2i45,5i). However, in some cases it was indicated that the increase of peroxidase aetivities or appearance of new isozymes occured as a result of syrnptom response, or may be related with the degeneration of host cell$ caused by infectionii,ai:2"). The critical reports describing the increase of peroxidase activities unrelated with the resistance have been ''i Fa'c' ulty of Agriculld'r'e, Kyushu University, Fukuoka 812, Japan th.Y'Hjc*va#V,B ** Research Laboratory of Japan Plant Protection AssociatiQn, Kodaira, TQkyQ 187, Japan H* reimMnvaskMXwt The Phytopathological Society of Japan NII-Electronic Library Service he hytopathologica S ciety f apan Ann. Phytopath. Soc. Japan 47 (5). ?ecember, 1981 655 recently accumulatedi,e,6LiO,i4"'i7i24,8t,S6,3S,40,42,52,53). These re$ults, however, were mainly on unrelated increase of activity with race-specific resistance and not with the general resistance which changes according to the ageing of leaf tissues or various environmenta! conditions, In this report the alteration of total and individual isozyme aetivity of peroxidase in compatible and incompatible combinations in riee blast disease caused by Ryricularia el:y2ae Cav, was compared and, furthermore, the increase of activity which does not correlate with the general resistance in cornpatible combination will also be reported, The abstracts and similar data have partly been repDrted elesewhere25ny2S). Most experiments were carried out at National Institute of Agricultural Sciences, Tokyo, Materials and Methods ISolates. The isolates of PJ,ricularia oryxae Cav. used in this experiment were Ken 68-17 (NIAS) and TH 65-105 (Tohoku National Agricuitural Experiment Station)・ ExP・ 1Rice cultivar Kanto 51 (Pi-k) which is susceptible to Ken 68-17 and resistant to TH 65-105 was sown in soil mixture of Nishigahara soil and paddy field soil (6:4) fertilized with 1g of pre-mixed fertilizer (Chiyoda Kasei Co, Ltcl.) in plastic pots (15× 6 × 10cm). At 5-Ieaf stage, spray inoculation was reutinely carried out with a spore suspension (ca. 10 spores under × 150microscopic field). PIants sprayed were incubated in a moist chamber at 24 C for 17 hr. Then, inoculated plants were transferred to a green house air-eonditioned around 27C. Sampling of 4th and 5th ieaves was carried out at O, 1, 3, 5 and 7th day after inoculation. Samples eollected were wrapped with aluminium foil and stored at -10C until the final sampling at 7th day, The preparation of peroxidase from every sample was earried out at the same day. Time courses of total and isozyme activities of peroxidase after infection in compatible and incompatible combinations of host and parasite were compared. Exp, 2. Cultivar Aichi Asahi (Pi-a) was preliminarily raised on the mesh-screen floated on tap water which was adjusted to pH5.0-5.5. At 2-Ieaf stage, seedlings of uniform size were selected and transplanted to vermiculite in 15cm porcelain pots, six seedlings per each pot, One-fourth strength of Kasugai's culture solution2) was watered every 4days for 8days after transplanting and followed by 1/2 strength solution. When 6th leaves cleveloped fully, the ammonium sulfate solutions containing 188 and 376mg of (NH4)2S04 respectively were added to some pots. These treatments were designated as Tr-1 and Tr-2 respectively. The ample supplies of ammonium sulfate was repeated once more at 5th day after the first reatment When 7th leaves developed fully, 6th and 7th leaves were sampled separately. The alteration of peroxidase activity in amply N-fertilized rice p]ant which reduced its resistance to rice blast was examined. P}'eparation of peroxidase. In Exp. 1, lg of leaf sample was homogenized in 5 ml of 12.5% glucose containing 1115M phosphate buffer (pH7.4) with the addition of quartz sand in a mortar. The homogenate was centrifuged at 12,OeOxg for 10min. Then, 2ml of cold acetone was added to 1ml of the supernatant in a small centrifuge tube and centrifuged at 6,OOOxg for 10min, Resulting pre ¢ ipitate was resuspended to 3ml of cold acetone and centrifuged as stated above. The washing was repeated once more and remaining acetone was completely eliminated in vacuo. The precipitate was transferred to a Tefron homegenizer with distilled water, homogenized slowly to make The Phytopathological Society of Japan NII-Electronic Library Service he hytopathologica S ciety f apan 656 Hdstsimfien\ftiR as47ts ee5e HllEll.56ff12A m.r a emulsion in ice cold, The homogenate was finally adjusted to 60ml with distilled water and used as the enzyme solution. The supernatants before acetone treatment were used as the enzyme preparations for gel electrophoresis. In Exp, 2, the preparation of enzyme was conducted as stated above except for using 1/60M phosphate buffer (pH 7.4) and addition of equal volume of cold acetone for precipitation of enzyme. All procedures were carried out volumetrically in the cold. Peroxidase activity. Five ml of O,2M acetate buffer (pH4,7), lml of 1% o-phenylenediamine ethanol solution, lml of 0.3% H202 solution and 200pt1 of the enzyme solution were ineubated at 30C for 5min. Then, lml of saturated sodium bisulfite solution was added to stop the reaction, The activity was measured colorimetrically by O. D, at 430nrn with a spectrophotometer (Beckman Co.). Thin tager electrophoresis. The enzyme preparations were subjected to polyacrylamide gel thin layer electrophoresis. Nitto GE (Acrylamide 92% and ethyleneureabisacrylamide 8 %) was used as a supporting medium with DMAPN (Dimethylamino propionitrile) and ammonium persulfate as catalysts, A 4% gel was used with discontinuous buffer system (Gel buffer : O.038M Tris hydroxymethylaminomethane-O.05 M NaOH, pH8.2). Ten pt1 of each sarnple was poured into gel slots, The samples frem compatible and incompatible combinations were always run on a same gel sheet with the check sample from healthy leaves. The running was conducted at ca. 5C for about 100 min at constant 1 mAlcrn (width of the gel). Dletection and densitometry. o-Dianisidine which is the most stable electron donor and was recommended for the detection of peroxidase35) was used. One hundred mg of o-dianisidine was dissolved in 50ml of 95% ethanol and mixed with 49ml of O.2M acetate buffer (pH4,7). One ml of O.3% H20z (final coneentration is ca. O.O03 %) was added to the mixture stated above and poured in the tray. Gel was dipped in and the tray was floated on 30C water bath. Fifteen min after dipping, 1ml of saturated sodium bisulfite was added to stop the reaction. The gel was immediately transferred to Chromoscan densitometer (Joyce Loebl Co,) and the activity of each isozyme was measured colorimetrically by O,D, at 430nm. Results The alterations of peroxidase activities after inoculation were compared in compatible and incompatible combinations of rice cultivar and races of Pyricularia ory2ae Cav, In the incompatible combination, the total activities at 5th leaves increased within 24hr after inoeulation and successively inereased. While in the compatible combination, the activity was stable until 3rd day and followed by rapid increase, reaching its maximum at 5 th day and then slightly decreased toward 7 th day, The activities in the compatible case at 5-7th day were higher than that ef the incompatible case (Fig. 1-a). The highest increase was observed at the time of rapid enlargement of blast lesions in the compatible combination. The increase of activities occured more rapidly at 4th leaves (second leaf from the top) (Fig. 1-b). The gradual increase was observed in intact eheck leaves. AIthough total activity is the reflection of each isozyme activity, total activities were affected by activities of the major isozymes. Isozyme

Increase of peroxidase activity unrelated with resistance to rice blast disease.