[Agr. Biol. Chem., 32, No. 7, p. 816821, 1968]

Studies on Hypertrophic Disease of Cherry (Genus ), So-called "Witch's Broom" Caused by wiesneri Part II. On the Phenolic Acids and Their Related Compounds, Especially on Melilotic Acid in Infected Japanese Flowering Cherry Leaves* By SatoshiFuni, HirooAOKI**, Masahiko KoMOTO and KatsuraMUNAKATA** TheHyogo University ofAgriculture, Sasayama, Hyogo**; NagoyaUniversity, Faculty of Agriculture,Nagoya ReceivedSeptember 13, 1967 Phenolicacids and their related compounds inJapanese flowering cherry leaves infected by Taphrinawiesneri were examined. In theacidic and neutral extracts of infectedcherry leaves(I), eighteen compounds positive to diazotizedsulfanilic acid and two fluorescent compoundswere detected by paperchromatography. Of these compounds, coumarin, 3,4- dihydrocoumarin,melilotic acid, o- and p-coumaric acids, p-hydroxybenzoic melilotic acid, ferulicacid and caffeic acid were identified. Melilotic acid and coumarin were obtained in crystallineform. The amount of meliloticacid in I washigher than that in healthy leavesindependent of sample source, although increased with the growth of cherryleaves.

In the previous paper, the authors" reported composition of invaded tissues. It has been on the products formed in vitro by Taphrina recognized that infected cherry leaves are wiesneri (Rath.) Mix. which causes witch's unusually small and fragile, and that the edge broom disease. Since no study had been of the leaves become brown over the end of published on the chemical composition of the spring. This work was performed to de tissues of Japanese flowering cherry infected termine what constituents were present in by T wiesneri, the authors were interested in infected Japanese flowering cherry leaves and this subject. Similar studies on virus-infected whether there were significant differences in peach and cherry2•`5) and (T these constituents between healthy and infected deformans)-infected peach" have shown that leaves or not. the microbial infection alters the chemical EXPERIMENTAL AND RESULTS * This paper was presented at the Annual Meeting Leaves of Prunus yedoensis, Somei Yoshino , of the Agricultural Chemical Society of Japan, Tokyo, were chiefly used as the material . April 1967. 1) S. Fujii, H. Aoki, M. Komoto, K. Munakata and I. Paperchromatographic comparison of chemi the late T. Tamura, Agr. Biol. Chem., 32, 803 (1968). cal components between infected and healthy 2) R. C. Lindner, Science, 107, 17 (1948). cherry leaves. 3) T.E. Rawlins and H.E. Thomas, Phytopathology, 31, 916 (1941). Infected and healthy leaves were sampled 4) T. O. Diener and C. A. Decker, ibid., 643 (1954). at various periods and places at Sasayama 5) T. A. Geissman, Arch. Biochem. Biophys., 60, 21 , H (1956).6)A yogo: the campus of the Hyogo University . Schneider, Compt. Rend., 248, 442 (1959). of Agriculture, June 20, August 18 and Studies on the Hypertrophic Disease of Cherry (Genus Prunus). Part II 817

September 21, 1965 and July 15, 1966; the No. 50 filter papers. The solvents used were Kawashiro park, September 24, 1965; and n-butanol-acetic acid-water (4: 1 : 1), n-pro Ojiyama park, September 25, 1965. The panol-ethyl acetate-water (7:1:2) and leaves were air-dried in the shade and stored. isopropanol-28% aq. ammonia-water (8: 1 : 1). The samples were powdered and then Ninhydrin, benzidine, Doragendorff's reagent, extracted in five steps with different solvents ferric chloride and diazotized sulfanilic acid according to the procedure shown in Chart 1. were used as spray reagents. Fluorescent sub- Fifteen grams of each sample was successively stances were detected under ultraviolet light. extracted with petroleum ether, ethyl ethanol There was no appreciable difference bet by using a continuous extractor. Extraction ween the chromatograms of the extracts from was continued until the solvent layer was no infected leaves and those from healthy ones more colored. Finally the residue was except that a spot (P-3, in Fig. 1) in the extracted once with 50 ml of water under chromatogram of the ether extract of the reflux for an hour. Each extract was con former was more intense than that of the centrated to 5 ml under reduced pressure and latter. P-3 had an Rf value of 0.61 (iso- an aliquot of the solution was subjected to paper chromatography by using Toyo Roshi

FIG. 1. Phenolic Compounds in the Ether Extracts of Infected and Healthy Cherry Leaves. Solvent: Isopropanol-28% aq. Ammonia-Water CHART I. Procedure for Extraction of Chemical (8:1:1) Components of Cherry Leaves. Spry Reagent: Diazotized Sulfanilic Acid 818 Satoshi FUTII, Hiroo AOKI, Masahiko KOMOTO and Katsura MUNAKATA propanol-28% aq. ammonia-water, 8 : 1 : 1) material was extracted with ether by using a and gave a positive reaction with diazotized continuous extractor. The extract was sulfanilic acid. These results were obtained routinely fractionated into three parts-acidic, independently of sample source. neutral and basic. The acidic and neutral II. Chromatographic examination of phenolic fractions were paper-chromatographed. One- acids and related compounds in infected cherry or two-dimensional ascending chromatogra leaves. phies were carried out by using Toyo Roshi Infected cherry (Prunus yedoensis) leaves No. 50 filter papers. The solvents and the were collected at the campus of the Hyogo methods for the detection of compounds are University of Agriculture on June 20, 1965, given in Table I. As shown in Fig. 2, air-dried in the shade and powdered. The eighteen substances were detected by spraying

TABLE I. PAPERCHROMATOGRAPHIC BEHAVIOR OF PHENOLIC COMPOUNDS IN INFECTED CHERRY LEAVES Solvent: A, Iso-PrOH-28% NH4OH-H2O (8:1:1); B, 20% KCl; C, Benzene-AcOH-H2O (7:3:1); D, 2% AcOH; E, H2O. Color Reaction: A, Fluorescence under UV Lamp; B, with Diazotized Sulfanilic Acid; C, with Diazotized p-Nitroaniline. Studies on the Hypertrophic Disease of Cherry (Genus Prunus). Part II 819

treated as described in the experiment II. Two kilograms of powdered, air-dried material was obtained from 7 kg of fresh leaves. Isolation of P-3 was carried out according to the procedure as shown in Chart 2. The sample was extracted with ether by using a large continuous extractor for 35 hr. The extract was evaporated in vacuo to give 68g of dried residue. The sirupy material was suspended in 1.7 liters of warm water (50°C) and stirred mechanically for 5 hr. The suspension was filtered and washed with warm water. The filter cake was treated twice as above. The filtrates were combined with the washings, concentrated under reduced pressure to 50 ml and then reextracted FIG. 2. Two-dimensional Chromatogram of Phenolic with ether. The extract was routinely Compounds in Infected Cherry Leaves. fractionated into three parts, acidic (3.3g),

Spray Reagent: Diazotized Sulfanilic Acid neutral (0.4g) and basic (trace). The acidic fraction was subjected to cellulose column diazotized sulfanilic acid solution together chromatography by using 20% aq. potassium with two fluorescent substances. The Rf chloride solution. Fractions were collected by values and the color reactions of these each 10 ml. Fractions No. 35-65 correspond- compounds are listed in Table II. Of these ing to P-3 were evaporated under reduced compounds nine were paper-chromatographi pressure to dryness and triturated with cally identified as following substances: P-1, methanol. The methanolic solution was coumarin; P-2, 3, 4-dihydrocoumarin; P-3, separated by filtration from pottassium chloride melilotic acid; P-4, o-coumaric acid; P-5, and concentrated to give 450 mg of sirup. The sirup was applied to fifteen filter-paper p-coumaric acid*; P-6, p-hydroxybenzoic acid; P-7, vanillic acid; P-8, ferulic acid*; and P-9, sheets (40 x 40 cm) and chlomatographed in caffeic acid* P-1 and P-2 were found in the isopropanol-28% aq. ammonia-water (8: 1 : 1). neutral fraction. Location of P-3 was detected with the Examination of the components in healthy diazotized sulfanilic acid-stained guide strips. leaves was carried out in the same manner The area of P-3 was cut off and eluted with as that for infected leaves. The chromato methanol. The eluates were combined, decolorized with a small amount of active grams were qualitatively similar to that of the infected leaves though a spot (P-3) was carbon, and evaporated under reduced smaller. A typical chromatogram is shown pressure to a sirup. After standing the sirup in Fig. 1. for several days, colorless prisms crystallized out. The yield was 68.5 mg. Successive III. Isolation and identification of P-3. recrystallizations from benzene and n-hexane Infected cherry (Prunus yedoensis) leaves were collected at the campus of the Hyogo Uni gave colorless prisms which melted at 85--- versity of Agriculture on June 20, 1965 and 86°C. Anal. Found: C, 64.98; H, 6.05. Calcd. for C9H10O3 : C, 65.05; H, 6.07 ~o. The UV and the IR spectra and the paper chromato * These compounds were kindly supplied from the late Prof. Teiichi Tamura, Nagoya University. grams of P-3 were all identical with those 820 Satoshi FUJII, Hiroo AOKI, Masahiko KOMOTO and Katsura MUNAKATA

CHART 2. Procedure for Isolation of Melilotic Acid and Coumarin from Infected Cherry Leaves. of authentic melilotic acid. The melting IV. The amount of Melilotic acid in infected and healthy leaves. point of P-3 was not depressed by admixture with authentic melilotic acid. Thus P-3 was Infected and healthy leaves of P. yedoensis identified as melilotic acid. growing on the campus of the Hyogo Uni Colorless needles the same Rf value as that versity of Agriculture were collected on the of P-1 was deposited from the neutral fraction following days; May 22, June 20, August 18, (Fig. 2). Recrystallization of this compound and September 10, 1965. For each sample from water gave colorless needles which 10 g of air-dried powdered leaves was continu melted at 6870°C. On the basis of several ously extracted with ether for 35 hr . The physical and chemical properties P-1 was extract was evaporated to dryness under identified as coumarin. reduced pressure. Thirty milliliters of water Studies on the Hypertrophic Disease of Cherry (Genus Prunus). Part II 821 was added to the oily residue. The mixture sample source, and the growth and the senes was stirred for 30 min and then filtered. An cence were too early as compared with the acidic fraction from the filtrate was evaporated, healthy leaves. It is well known that cherry and dissolved in methanol, the total volume contains large ammounts of coumarin. Some being 3 ml. Every 44.4 pl of the methanolic studies point out"' that coumarin is converted solutions was applied to a filter-paper sheet in vitro or in vivo into melilotic acid. On the with a micropipette and developed in isopro other hand, Taphrina wiesneri also converted panol-28% aq. ammonia-water (8: 1 : 1). The coumarin into melilotic acid"' Although it area corresponding to melilotic acid was cut off has remained unexplained whether melilotic and eluted with water until the volume of the acid is attributable to witch's broom or not, eluate amounted to about 9.5 ml, the final it is of great interest that the acid is produced volume of the eluate being adjusted to 10 ml. by a host-fungus interation. The solution was subjected to colorimetric In the infected cherry wood, vanilic acid, determination according to the Griffiths's p-hydroxybenzoic acid, o- and p-coumaric acid method" As shown in Table II, the amount and several unkown acids were detected but melilotic acid was not. It seems that the TABLE II. CHANGESIN THE AMOUNTOF accumulation of melilotic acid would occur MELILOTICACID DURINGTHE GROWTH only in the infected cherry leaves. A studies OF CHERRYLEAVES. on the acid contents in fresh cherry leaves is under examination. Among the three phenolic acids which were produced in vitro by T. wiesneril1), p-hydroxy phenylacetic acid could not be detected in both of the leaves. of melilotic acid in infected leaves was higher than in healthy leaves, and in both leaves it Acknowledgements. The authors are indebted increased with the growth of the leaves. to Dr. Eijiro Hamaguchi, Emeritus Professor of the Hyogo University of Agriculture, for DISCUSSION his helpful guidance in this work. The authors Nine phenolic compounds identified are wish to thank Misses Mieko Hamaji and widely distributed in plant kingdom. Un- Kiyoko Nakane for microanalysis and are familiar phenolic compounds were not detected. grateful to Mr. Teruo Matsushita for his The most noteworthy finding was the obser technical assistance. One of the authors vation that the infected leaves showed higher (S. F.) thanks Dr. Saburo Okamoto, the content of melilotic acid than the healthy Hyogo University of Agriculture, for his leaves. Schneider6) found that some amino helpful criticism on the manuscript. acids were more accumulated in peach trees parasitized by as compared 8) T. Kosuge and E. E. Conn, J. Biol. Chem., 234, with healthy ones and discussed a possible 2133 (1962). relation between the accumulation of the acids 9) J. Kaji, M. Hara and K. Tamari, read at the and the physiology of the infected trees. In Annual Meeting of Agricultural Chemical Society of the case of the infected cherry leaves melilotic Japan, Tokyo, April, 1967. 10) S. Fujii, H. Aoki, M. Komoto and K. Muna acid contents were higher independent of kata, ibid. 7) L. A. Griffiths, J. Expl. Bot., 13, 169 (1962).