日 植 病 報 60: 288-294 (1994) Ann. Phytopath. Soc. Japan 60: 288-294 (1994)

Induction of in 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 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 (ămax 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 , and 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. The Rf value of extracted tomatine was identical with those of authentic tomatine in various solvents. When TLC was treated with sulfuric acid followed by heating at 100•Ž for 10min, tomatine gave black colored spot, while tomatidine green colored one.

A solution of sugars released by hydrolysis of tomatine was analyzed by paper chromatography

(n-BuOH-AcOH-H2O=4:1:5 or n-BuOH-EtOH-H2O=4:1:2). Only two spots were observed. One of them migrated in parallel with authentic glucose or galactose and another with xylose. The color developed with aniline hydrogen phthalate was red for xylose and brown for glucose and galactose. The

Table 1. Rf values of authentic and extracted tomatines and their aglycones

a) Solvent 1, Iso-PrOH-HCOOH-H2O=73:3:24; 2, EtOAc-MeOH-HCOOH-H2O=30:20:10:1; 3, n-BuOH- HCOOH-H2O=4:1:5 and 4, MeOH-NH4OH=200:3 b) a. Plates were dried in air for 24hr followed by spraying 50% H2SO4. The plates were heated at 100•Ž for 10

min. b.. Plates were dried in air for 24hr followed by spraying the Dragendorff reagent. c) Product of Sigma Chemical Co.

d) Extracted from tomato roots as described in the text. e) Aglycone prepared from the authentic tomatine. f) Aglycone prepared from the extracted tomatine.

g) not determined. Ann. Phytopath. Soc. Japan 60 (3). June, 1994 291

Fig. 1. Ultraviolet spectrum of tomatine. Lower: tomatine isolated from inoculated plant. Upper: authen- tic tomatine.

Fig. 2. Fast atomic bombardment mass spectrometry spectra of an authentic tomatine and an extracted

tomatine. Matrix: p-nitrobenzylalcohol (NOBA); positive MS. The presence of abundant Na+ in the extracted sample gave only [M+Na]+.

sugars were separated on ascending paper chromatography (n-BuOH-toluene-pyridine-H2O=5:1:3:

3) and descending paper chromatography (phenol saturated with water). Three spots were clearly

detected with the aniline hydrogen phthalate that migrated in parallel to those of authentic glucose, galactose and xylose. Ultraviolet spectroscopy of both authentic tomatine and extracted tomatine showed a single

absorption maximum at 322nm (Fig. 1). Fast Atomic Bombardment Mass Spectrometrum of an

authentic sample of tomatine showed [M+H]+ at m/z 1034 as well as [M+Na]+ at m/z 1056. The extracted sample gave [M+Na]+ at m/z 1056 because of the presence of excess Na+ in the sample (Fig.

2).

On the basis of the foregoing, the extracted sample was identified as tomatine.

Estimation of tomatine content in the plant tissue

Living cells but neither heat-killed cells nor culture filtrate of strain Str-10 op type induced tomatine production in root tissue above the inherent level of control. When the plant were inoculated with living cells of strain Str-10 op type, however, tomatine content in the root tissue became significantly higher than control. As the inoculum concentration of strain Str-10 op type increased from 108 to 109cfu/ml, the tomatine content also increased from 113.9 to 152.2ƒÊg/g fresh root. However, when the inoculum of strain Str-10 op type was increased to 1010cfu/ml, the tomatine content dropped to 102.0ƒÊg/g fresh root.

Tomatine content of the untreated control was 65.9ƒÊg/g. When the plants inoculated with strain Str-10 op type were kept under different temperatures, the plants maintained at higher temperature (30-37•Ž) did not increase the tomatine content. At lower temperature (18-25•Ž), however, the tomatine content 292 日本植 物 病 理 学 会報 第60巻 第3号 平 成6年6月

Table 2. Tomatine content in tomato tissues after treatments with cells of strain Str-10 op type, its culture filtrate and heat-killed cells

a) ƒÊg/g fresh weight. Means of 2 replications. Number in parenthesis is standard error. *: Significantly higher than control.

Fig. 3. Increase of tomatine contents in tomato tissues with or without inoculation of Str-10 op type.

(a) roots, (b) stems. •› inoculated with Str-10 op type. •œ not inoculated with Str-10 op type.

significantly increased by inoculation with strain Str-10 op type (Table 2).

Since only living cells of strain Str-10 op type could induce tomatine production in the root tissue, it was examined whether the tomatine content increased when the sampling date was extended. The results indicated that tomatine concentrations in root system increased as the time after inoculation of strain Str-10 op type elapsed. The maximum tomatine content 450ƒÊg/g was obtained nine days after inoculation. In contrast, the tomatine content in the roots of control plants was not detected in the third Ann. Phytopath. Soc. Japan 60 (3). June, 1994 293

Table 3. Effect of authentic tomatine on growth of P. solanacearum

*: no inhibition zone around paper disk .

day and 150ƒÊg/g in the 9th day of inoculation (Fig. 3).

Bioassay of extracted tomatine

Approximately 150ƒÊg/disk of extracted tomatine showed inhibition zone against P. solanacearum on defined agar media. The mechanism of inhibition was bacteriostatic because bacterial growth was obtained when agar containing inhibition zone was aseptically pick-up, suspended in YP broth medium, and then incubated at 29•Ž for 24hr.

Bioassay of authentic tomatine

Authentic tomatine showed growth inhibition against all bacteria tested (Table 3). The amount of

100ƒÊg authentic tomatine per paper disk was enough to inhibit growth of P. solanacearum in vitro.

Strain Str-10 op type was the most resistant compared with the other strains tested.

DISCUSSION

An alkaloid isolated from tomato root tissue showed the inhibitory activity against P. solanacea- rum. The data of TLC, UV spectroscopy, mass spectrometry and chemical component analysis indicat- ed that the inhibitory substance was tomatine. Following the inoculation with strain Str-10 op type, tomatine concentration increased in root tissue but not in stem tissue. The strain Str-10 op type failed to induce tomatine production at higher temperatures. This fact may explain, at least in part, why strain

Str-10 op type protected tomato plants at moderately higher temperature but not at higher temperature of 30•Ž or more2).

Heat-killed cells or culture filtrate of strain Str-10 op type could not induce tomatine production in plant. Presence of bacteria was demonstrated in the transverse sections of tomato roots infected with Str-10 op type. Slow irritation caused by the strain could trigger the defense mechanism in plant.

Tomatine concentration in the root nine days after inoculation (450ƒÊg/g fresh weight) (Fig. 3) was much greater than that needed for growth inhibition of P. solanacearum in vitro (100ƒÊg). Mohanakumaran et al. reported that 400ppm of authentic tomatine was needed to prevent in vitro growth of this bacterium14). This discrepancy might be resulted from the differences of assay procedures including media.

The involvement of tomatine in resistance have mostly been studied with plant pathogenic fungi, especially Fusarium oxysporum f. sp. lycopersici1,5,8,11,13,18).

P. solanacearum is a soil-borne pathogen and infects plants through root system12). Plant resistance against such soil-borne pathogens, therefore, may involve the defense mechanisms primarily taken place in root system. The increase of tomatine content induced by infection of strain Str-10 op type (Table 2,

Fig. 3) seems to make tomato plant more resistant against P. solanacearum by its bacteriostatic activity.

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和 文 摘 要

T. ARWIYANTO・ 坂 田完 三 ・後藤正 夫 ・露無 慎 二 ・瀧川雄 一:非 病 原性 青枯 病 菌 の接 種 に よ る トマ トの トマチ ン生 産 に よる病害 抵 抗性 の発 現

ス トレ リチア(Strelirtziareginae Banks)か ら分離 され た青 枯病 菌 の非 病 原 性 菌 株(Str-10 op型)を 接種 した トマ ト の 根 系 か ら,非接 種対 照 植 物 の根 系 よ り も多量 の トマチ ンが検 出 され た。茎の組 織 内で は この よ うな トマチ ン濃度 の 上 昇 は見 られな か った。Str-10 op型 の 接 種 源濃 度 を108か ら109cfu/mlに 増 加 す る と,接 種5日 後 に お け る根部 組 織 内 の トマチ ン含量 は113μg/g根 か ら152μg/g根 まで増加 した。接種9日 後 に は トマチ ン含量 は450μg/g根 まで増 加 し た 。この濃度 は青枯病 菌 の病 原性 菌 株 の増 殖 をin vitroで 抑 制 す るの に十 分 な濃 度 で あ った。トマチ ン に よ る青枯 病 菌 の発 育 抑制 は静菌 的 で あ った 。一 方,Str-10 op菌 株 の培 養 ろ液 及 び加 熱 死 菌 で処 理 し た トマ ト根部 で は トマチ ン濃 度 の増 大 は見 られ なか っ た。