Induction of Tomatine in Tomato Plant by an Avirulent Strain of Pseudomonas Solanacearum
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日 植 病 報 60: 288-294 (1994) Ann. Phytopath. Soc. Japan 60: 288-294 (1994) Induction of Tomatine in Tomato Plant by an Avirulent Strain of Pseudomonas solanacearum Triwidodo ARWIYANTO*,**, Kanzo SAKATA***, Masao GOTO***, Shinji TSUYUMU*** and Yuichi TAKIKAWA*** Abstract Tomato plants inoculated with an avirulent strain of Pseudomonas solanacearum (strain Str-10 op type) produced a growth inhibitory substance at a higher level than those of uninoculated control in root system but not in stem. The substance was identified to be tomatine by Mass Spectrometry, TLC, UV spectrometry and chemical component analysis. The tomatine contents five days after inoculation were 113 and 152ƒÊg/g fresh root when Str-10 op type was inoculated at the inoculum concentrations of 108 and 109cfu/ml, respectively. Tomatine content in the uninoculated control plants was 65.9ƒÊg/g. Tomatine content nine days after inoculation reached 450ƒÊg/g. In an in vitro experiment, 100ƒÊg/ disk of authentic tomatine and approximately 150ƒÊg/disk of extracted tomatine were sufficient for growth inhibition of P. solanacearum. Both extracted and authentic tomatine exhibited bacteriostatic activity against P. solanacearum. Treatment with heat-killed cells or culture filtrate of Str-10 op type failed to increase tomatine content in the root tissue. (Received December 7, 1993) Key words: tomatine, induced resistance, Pseudomonas solanacearum. INTRODUCTION Bacterial wilt caused by Pseudomonas solanacearum is one of the most serious bacterial diseases of crops. The disease is usually difficult to control because of the soil-inhabiting nature of the pathogen as well as the root systems as the general infection sites. To overcome these difficulties, biological control has been tried with various microorganisms as antagonists. Mechanisms of the biological control were often addressed to the production of antimicrobial substances produced by antagonistic microorgan- isms3,6,9,15). The resistance induced by such a mechanism fully depends on the presence of the antagonis- tic strains either in the infection site or inside the host plant. There are, however, little information available on the mechanisms of induced resistance of tomato plant (Lycopersicon esculentum Mill.) against P. solanacearum regardless of biotic or abiotic elicitor. It has been documented that susceptibility of tomato plants to Fusarium wilt varies with cultivars which are different in the content of inherent steroidal glycoalkaloid, tomatine or lycopersicin in tissue16). There is another article described that bacterial wilt-resistant tomato plants contain significantly higher concentrations of tomatine than susceptible ones and it may be responsible for the resistance to P. solanacearum14). We have proved that a spontaneous avirulent mutant of P. solanacearum (strain Str-10 op type) effectively prevented wilting of tomato plants which were challenged by the virulent strains of the * The United Graduate School of Agricultural Science , Gifu University, Yanagido 1-1, Gifu 501-11, Japan 岐 阜大学大学院連合農学研究科 ** Present address: Laboratory of Plant Pathology , Faculty of Agriculture, Gadjah Mada University, Sekip Unit 1, Yogyakarta, Indonesia *** Faculty of Agriculture , Shizuoka University, 836 Ohya, Shizuoka 422, Japan 静 岡 大 学 農 学 部 Ann. Phytopath. Soc. Japan 60 (3). June, 1994 289 bacteria2). When tomato plants were inoculated with the avirulent mutant strain, tomatine content of suscep- tible plant increased to the levels that were inhibitory to P. solanacearum. MATERIALS AND METHODS Plants. Tomato plants of the cultivar •ePonte Roza•f were used throughout the experiments. Four plants were grown in a vinyl pot (12cm in diameter) in a greenhouse at 29•Ž. Tomato plants were inoculated at the four full leaf stage. Bacteria. Bacterial strains used in this study were obtained from the Culture Collection of Plant Pathogenic Bacteria at the Laboratory of Plant Pathology, Shizuoka University. Bacteria were grown on yeast extract-peptone agar plates (YPA: yeast-extract 5g, peptone 10g, agar 15g, distilled water 1,000ml, pH 6.8)7) at 30•Ž for 48hr. A single colony was selected and grown on YPA slants for 24 hr at 30•Ž before use. Preservation of the bacterial cultures and preparation of inoculum were carried out by the method described elsewhere2). Inoculation method. Root systems of tomato plants were immersed in a suspension of Str-10 op type (109cfu/ml) for 30min. For control, root systems were immersed in distilled water for 30min. The plants were then replanted to the pots and kept in the greenhouse at 29•Ž. Detection of tomatine. Tomatine was extracted separately from root systems and stems five days after inoculation by the method of Roddick and Butcher17) with some modifications. Plant tissues were homogenized in a mixture of MeOH-AcOH-H2O (94:2:4, v/v) at the ratio of 10ml/g fresh weight. The homogenate then was kept at 4•Ž for 18hr. After filtration, the residues were treated twice with 64% MeOH for 5hr and 2hr, respectively. For the preparation from stem tissue, pigments and lipids were removed by extraction with petroleum ether. The residual extracts were combined and evaporated to dryness under reduced pressure at 45•Ž. The residues were dissolved in hot MeOH and then applied as a band on a TLC plate (Merck, silica gel 60 F254, 0.5mm in thickness). The plate was developed first in water, dried and re-developed in iso-PrOH-HCOOH-H2O (IFW, 73:3:24). Tomatine was detected by spraying one edge of the plate together with a spot of authentic tomatine with Dragendorff reagent10). The tomatine zone was then scraped off from the plate and then dissolved in hot MeOH (50ml, 4 times). The eluate was separated from silica gel by centrifugation at 3,000•~g for 20min. The eluates were combined and evaporated to dryness under reduced pressure at 45•Ž. The residue was dissolved in hot MeOH. Identification of tomatine. Tomatine was identified as follows: 1. Comparison of Rf values of both tomatine (glycoside) and tomatidine (aglycone obtained by refluxing tomatine in 1.0N HCl for 60min) on TLC in four kinds of developing solvents (1. IFW as stated above; 2. EtOAc-MeOH-HCOOH-H2O=30:20:10:1; 3. n-BuOH-HCOOH-H2O=4:1:5; and 4. MeOH-NH4OH=200:3). 2. Color reactions of tomatine (black) and tomatidine (green) on TLC plates after spraying with 50% (v/v) H2SO4 and heating for 10min at 100•Ž. 3. Ultraviolet spectroscopy of tomatine (ƒÉmax 322nm in conc. H2SO4). 4. Fast Atomic Bombardment Mass Spectrometry of tomatine. 5. Paper chromatography of the sugar moiety of tomatine in n-BuOH-toluene-pyridine-H2O (BTPW)=5:1:3:3 (The Rf values of glucose, galactose and xylose were 0.24, 0.21, and 0.35, respective- ly); n-BuOH-HCOOH-H2O (BFW)=4:1:5 (The Rf values of glucose, galactose and xylose were 0.12, 0.12, and 0.20, respectively); n-BuOH-EtOH-H2O (BEW)=4:1:2 (The Rf values of glucose, galactose and xylose were 0.16, 0.16, and 0.26, respectively) and phenol saturated with water (The Rf values of glucose, galactose and xylose were 0.34, 0.38, and 0.43, respectively)10). Estimation of tomatine content in plant tissue. Tomato plants were inoculated with strain Str-10 op type or treated with heat-killed cells or culture filtrate of strain Str-10 op type with the procedure as described before2). The plants inoculated with strain Str-10 op type were then kept in a greenhouse at various temperatures. The plants treated with heat-killed cells and culture filtrate were 290 日本植 物病 理 学 会 報 第60巻 第3号 平 成6年6月 kept in the greenhouse at 29•Ž. Root systems and stems were separately sampled five days after inoculation and subjected to extraction of tomatine by the method described above. Each extract was then applied on precoated TLC plate (Merck, silica gel 60 F254, 0.5mm in thickness, 5•~20cm) as a band and developed in IFW (solvent 1). The plates carried a spot of authentic tomatine which was located by spraying with Dragendorff reagent11). Tomatine eluted from silica with hot MeOH (50ml, 3 times) was evaporated to dryness at 50•Ž. After cooling, the residue was dissolved in 5ml of conc. H2SO4 and the mixture was incubated at 40•Ž for 24hr. The absorbance of the H2SO4 chromogen of tomatine was measured at 325nm and the amount of tomatine was determined by referring to a calibration graph. Bioassay of extracted tomatine. For the bioassay of extracted tomatine, P. solanacearum strain T11 was used as the indicator strain. Two-tenths ml of the bacterial suspension (107cfu/ml) was added to 4ml of 0.6% melted water agar at 50•Ž and poured over the bottom agar layer. The medium used as the bottom layer was Ayer, Rupp and Johnson's synthetic medium4) added with glucose as a carbon source (NH4H2PO4 1g, MgSO4 0.2g, KCl 0.2g, glucose 10g, agar 15g, H2O 1000ml, pH 6.8). The tomatine prepapration was applied to paper disks (Advantec Toyo, 1mm in thickness, 8mm diameter), dried in vacuo to remove the solvent and put on the agar-double layer. After incubation at 30•Ž for 24 hr, inhibition zones around paper disks were recorded. Bioassay of authentic tomatine. Tomatine (Sigma Chemical Co.) was dissolved in MeOH and the pH was adjusted to 4.5 with 1N HCl. Paper disks were loaded with the solution at the desired concentration. The disks were then dried to remove the solvent and put on the agar-double layer described above. After incubation at 30•Ž for 24hr, inhibition zones around disks were recorded. RESULTS Detection and identification of tomatine The Rf values and color reactions were presented in Table 1. When tomato tissue was subjected to the extraction of tomatine, each preparation gave only one Dragendorff-positive spot (bright orange color) on TLC developed with various solvents.