Isolation, Purification, and Biological Activity of an Inhibitor from Septoria tritici

Zelikovitch, N., Eyal, Z., and Kashman, Y. 1992. Isolation, purification, and biological activity of an inhibitor from Septoria tritici. Phytopathology 82:275-278. Methyl-3-indole carboxylate (3-indole carboxylic acid methyl ester Complete inhibition of S. triticiisolates ISR398 and ISR8036 was recorded [ICA. Me]) was identified in liquid cultures of Septoria tritici. For physioat 0.08 mg/ml of ICA.Me, with only partial inhibition of ISR7901 at logical studies, ICA.Me was synthesized from commercial 3-indole carthat concentration. The regulatory effects of these indole compounds were boxylic acid (ICA) by methylation with diazomethane. Inhibition of tested on wheat coleoptile and cucumber hypocotyl segments. IAA markgrowth of the melanin-producing isolate ISR398 by ICA.Me on thinedly increased growth of both coleoptiles and hypocotyls at 0.5-0.7 Ag/ layer chromatography (TLC) plates was recorded at a concentration of ml. Application of ICA and ICA.Me caused a slight decrease in hypocotyl 0.02 mg/ml. Inhibition by 3-indole acetic acid (IAA) was recorded at growth and a slight increase in coleoptile growth at concentrations ranging a concentration of 25-fold, whereas no inhibition was recorded for ICA from 0.4 to 20.0 lsg/ml. It is possible that ICA.Me may be involved at a concentration range of 0.02-0.5 mg/ ml. Growth of S. tritici on liquid in regulating the pathogen's population on the phylloplane and within media was differentially affected by different concentrations of ICA.Me. wheat tissue. Interactions between organisms are mediated in many instances MI) (4%), yeast extract (Institut Pasteur, France) (1%), and by secreted inhibitory products of diverse chemical nature (22). sucrose (4%). Cultures consisting mainly of conidia were cenSome of the inhibitory compounds are involved in antibiosis, trifuged for 15 min at 5,860 g. The supernatant was extracted namely, in which a metabolite produced by one organism has three times in ethyl acetate (200 ml of solvent per 100 ml of an harmful effect on the other (21). There are numerous examples culture filtrate). The fractions were evaporated to dryness under for antibiosis in fungi for both nonvolatile and volatile compounds vacuum and weighed. Extractions were made from 10-30 L of (3,4,5,19,23). In addition, fungi may produce compounds that growth medium in which conidia concentration was about 10' are self-inhibitors (autoinhibition), mainly involved in suppressing spores per milliliter. germination and production of appressoria (1). In Colletotrichum Rapid extraction. S. tritici isolates were grown for 4-5 days gloeosporioides, the self-inhibitor was chemically defined as dihyin shake liquid malt media, after which cultures were passed dro-5-hydroxy-5(8-pentyl-2-oxocanyl)-acetyl-2(3H)_furanone through Whatman No. 1 filter paper. One liter of culture filtrate, (gloeosporone) (16). In Dictyostelium discoideum, the self-inhibifree of conidia, was adjusted to pH 2.5 with HCl and was passed tor was identified as 2 -dimethylamine-6-oxypurineriboside (2). through a 1.0X 0.5-cm packed C18 reverse-phase column (SepInhibitory substances are specific to species from which they Pak C18 cartridge, Millipore Waters Associates, Milford, MA) have been identified and lack chemical similarities (1), with the that fitted to a syringe. The column was rinsed with distilled exception of different cinnamic acid derivatives found in several water followed by 10 ml of ethyl acetate. After the passage of rust fungi (1,9,12,18,20). Methyl 3,4-dimethoxycinnamate secreted the culture filtrate, the column was washed with 30 ml of distilled from urediospores of Puccinia graminis was reported to selfwater and with 30 ml of ethyl acetate. The water and ethyl acetate inhibit their germination (18). The same compound was involved fractions were evaporated in a vacuum and placed on silica gel in self-inhibition of Puccinia arachidis (9). As is the case with thin-layer chromatography (TLC) plates (Kiesel-gel 60F 254, E. antibiosis, the chemical natures of the identified self-inhibitors Merck, Germany). The TLC plates contained 1:2 (v/v) petroleum are rather diverse (1). ether/ethyl acetate mixture. The plates were dried, placed under In some cases, chemicals secreted by fungi have synergistic an 254or 366-nm ultraviolat lamp, and fluorescing spots were effects on germination and growth, as reported in the rusts for marked. The plates were then stained with vanillin (2% vanillin, 6-methyl-5-hepten-2-one and n-nonanal (10,11). Stimulating sub10% H 2SO 4, 70% methanol in H20). Comparable plates not stances in high concentration might cause self-inhibition of germitreated with vanillin were used for biological assays. nation. N-nonanal was found as the most effective substance in Biological assay using TLC. The silica gel TLC plates were these cases (10). It also has been suggested that substances exsprayed with a 4-day-old suspension of 1 X 10' conidia per millihibiting inhibitory effects are involved in regulating population liter of the melanin-producing isolate ISR398 (ATCC148507) of structure (16). S. tritici (17). The plates were incubated in a moist chamber under The purpose of this study was to elucidate the nature of chemical light for 48 h at 25 C. product(s) secreted by Septoria tritici in vitro and its possible Purification of antifungal fractions. Fractions exhibiting fungal involvement in regulating fungal growth. growth inhibition were scrapped from the vanillin-treated TLC plates, suspended in ethyl acetate, and shaken for 24 h. These MATERIALS AND METHODS fractions were applied on TLC plates and tested for growth inhibition of isolate ISR398. Weighed fractions were applied to a Organic extraction. S. tritici cultures were grown for 4-5 days 6X 2-cm silica gel column (Kiesel-gel 60H, E. Merck) connected in liquid medium consisting of malt (Difco Laboratories, Detroit, to a vacuum system. The column was washed with double its volume of petroleum ether (40-60 C, BDH, U.K.). The adsorbed © 1992 The American Phytopathological Society fractions were eluted with changing ratios of solvent mixture of Vol. 82, No. 3,1992 275 increasing polarity of ethyl acetate in petroleum ether, whereas spectrum of the purified compound in fraction no. 8 identified at the end it was washed with methanol. Fractions expressing by the 360-MHz Bruker is as follows: biological activity were repeatedly passed through fresh Kieselgel 60H columns and tested for purity under ultraviolet spectrums 6(CDC13; TMS = 0): of 254 and 366 nm and vanillin staining. Parallel-unstained TLC 8.20 br (NH,1H), 7.86 m (1H), 7.74 d (J = 8.5 Hz, 1H), 7.35 plates were tested for biological activity after each purification. m (11H), 7.20 m (2H), 3.54s(OCH 3) ppm. Purified fractions showing inhibition of S. tritici were analyzed by nuclear magnetic resonance (NMR) and mass spectra techThe mass spectrum (m/e EIMS) is as follows: 175 (87%, M+), niques. 'H NMR spectra were recorded on a Bruker AM-360 144 (100%, M-0Me), 116 (14%, M-CO 2Me), 89 (5%). The mass spectrometer (Bruker, Karlsrough, Germany) operating at 360 and NMR spectra of the extracted purified compound were idenMHz, and a Fenningan 4921 mass spectrometer (Quardopoletical to the synthetic ICA.Me synthesized from ICA according type Instrument) (Fenningan-Mat, San Jose, CA) was employed, to the methylation procedure described in the Materials and Methylation of indole-3-carboxylic acid (ICA). A methyl group Methods section. The extracted and synthetic fractions were also was added to ICA to yield indole-3-carboxylic acid methyl ester identical on TLC plates and differed from IAA and from ICA (ICA.Me) or methyl-3-indole carboyxlate. The methylation was (Fig. 1). S. tritici isolates ISR398, ISR7901, and ISR8036 all conducted as follows: solution A, 5 ml of water in which 1 g yielded ICA.Me after rapid extraction directly from the medium of KOH was dissolved was added to 16 ml of cold diethylene through a C18 reverse-phase column (Sep-Pak) and thereafter were glycol monomethyl ether; solution B, in a separate Erlenmeyer chromatographed on TLC silica gel plates and analyzed by NMR. flask, 3 g of diazald (N-methyl-N-nitroso-p-toluenesulfonamide, Biological activity. The organic extraction of purified ICA.Me Aldrich, Milwaukee, WI) was dissolved in 25 ml of diethyl ether yielded 0.04 A.g/ml of medium on which ISR398 grew. At high and was added to solution A. The upper fraction in solution concentrations, the inhibitor was not soluble in water and not B was moved to a distillation apparatus from which diazomethane diffusible in aqeous solution. ICA.Me inhibited S. tritici growth and purified ether were collected. One gram of commercial ICA on TLC plates (soluble in ethyl acetate or methanol) at a con(Sigma, St. Louis, MO) was dissolved separately in 10 ml of centration of 0.02 mg/ml, whereas IAA inhibited growth at 50% methanol and 50% diethyl ether to which the distilled diazo0.5 mg/ml. No inhibition was recorded for ICA at a range of methane was added until the solution attained a yellow color. 0.02-0.5 mg/ml. IAA and ICA had no inhibitory effect on growth The organic solvents were evaporated, and fractions were analyzed of ISR398 in liquid culture at concentrations ranging from 0.04 on TLC (compared to S. tritici product) and by NMR. to 0.08 mg/ml for 96 h, whereas ICA.Me expressed inhibitory Effect of indole compounds on fungal growth. S. tritici isolates, effects already at 0.04 mg/ ml, with complete inhibition of growth ISR398 (isolated from the bread wheat cv. Hazera 84, Israel), at 0.07 mg/ml (Fig. 2). Similar inhibitory effects were recorded ISR7901 (from cv. Shafir, Israel), and ISR8036 (from cv. Shafir, for isolate ISR8036. The same concentrations of ICA.Me had Israel) were grown in liquid malt medium in Erlenmeyer flasks less inhibitory effects on isolate ISR7901. The magnitude of fitted with side-attached test tubes. Concentrations of 0.04, 0.06, growth of the three isolates at the range of 0-0.04 mg/ml of and 0.08 mg/ml of indole-3-acetic acid (IAA) (Sigma), ICA, and the indole compounds was rather different. Application of IAA, ICA.Me were added separately to the medium after autoclaving. ICA, and ICA.Me to wheat seedling leaves at 1, 2, and 5 mg/ The inoculated preparations were shaken at 20 C on a rotary ml did not result in necrosis or chlorosis for a 10-day period. shaker. The growth of S. tritici was assessed daily by measuring Leaves treated with dimethyl sulfoxide showed a chlorotic turbidity with a Klett-Summerson photoelectric colorimeter (Klett response, whereas methanol had no visible effect. Mfg., NY) with a red filter. Growth of cucumber hypocotyls and wheat coleoptiles was Effect of indole compounds on cucumber and wheat. Seedlings affected by IAA at concentrations of 0.5-0.7 gg/ml (Fig. 3). A of cucumber (Cucumis sativus L.) cv. Delila (Hazera Seed Co., decrease in growth was recorded at concentrations greater than Israel) and of wheat (Triticum aestivum L.) cv. Shafir (Son64A/ Tzpp//Nai60/3/FA) (Hazera Seed Co.) were grown at 25 C in the dark for 5 days. Washed and weighed 1-cm-long cucumber hypocotyl segments were placed in test tubes (10 segments per test tube with three tubes per treatment) in a solution consisting of 3 ml of phosphate buffer (pH 7.0), 0.1 mM CaCI2, 2 mM KC1, and 10 mg/ L of chloramphenicol succinate. Concentrations of 0.5, 1.0, 4.0, 7.0, 10.0, and 20.0 •tg/ml of IAA, ICA, and ICA.Me were added to test tubes shaken for 5 h in the dark at 25 C. At the end of the incubation, the hypocotyl segments were dried and weighed. Wheat coleoptile segments pruned to a length of 1.2 cm were treated similarly to the cucumber hypocotyl segments. The effect of IAA, ICA, and ICA.Me on growth of cucumber hypocotyl and wheat coleoptile segments was calculated as follows: percentage of growth (Aweight/ weight at to) X 100, in which to = weight at time zero.