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OLIVE 香川大学学術情報リポジトリ

THE ROLE OF AVENALUMIN IN THE RESISTANCE OF OATS TO CROWN RUST

Shigeyuki MAYAMA

CONTENTS

I. GENERAL INTRODUCTION 11. THE PRODUCTION OF PHY

RESPONSE TO CROWN RUST, PUCCZNZA CORONATA F SP. AVENAE . . 4 111 CORRELATION BETWEEN AVENALUMIN ACCUMULATION AND THE

SPECIFIC RESISTANCE TO INCOMPATIBLE RACES OF T H ~ CROWN RUST FUNGUS 3

IV EFFECTS OF ELEVATED TEMPERATURE AND a-AMINOOXYACE- TATE ON AVENALUMIN ACCUMULATION AND HYPHAL GROWTH IN OAT LEAVES 8

V. LOCALIZATION OF AVENALUMIN AT TIS INFECTED WITH FUNGAL PATHOGENS 3

VI. MICROSPECTROPHOTOMETRIC ANALYSIS OF CELLULAR LOCATION

ABIOTIC ELICITORS VIII. DETECTION OF ACCELERATED ACCUMULATION OF CINNAMIC

ACID DERIVATIVES, THE PRECURSORS OF AVENALUMINS, AND THE AMOUNT OF AVENALUMINS ACCUMULATED IN THE RUSTED OAT LEAVES 45

IX. GENERAL DISCUSSION 50 X. SUMMARY AND CONC 5 3

ACKNOWLEDGEMENT 55 LITERATURE CITED . 56

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CHAPTER I

GENERAL INTRODUCTION

Plants in natural condition are exposed to many potentially parasitic microorganisms and insects, in addition to physical and chemical environmental stresses Each plant and parasite

interaction is a struggle for survival between two organisms; plants resist to protect themselves from the parasite invasion and parasites try to attack plants for obtaining food materials. If plants are defeated by their attacks, we will often lose our food stuffs It is evident historically that food starvation occurred many times due to the epidemic of plant diseases [ I , 361. The resistance and/or protection of plants against parasite infections are a n unavoidable and earnest desire of agricultural production.

The mechanisms for resistance of plants to parasite infection have been studied for a long time [22,23,47, 11 1, 137, 138, 1401. The resistance expression can be related either to the occurrence of physical structural barriers, or to the presence of antifungal compounds in plant cells. These mechanisms are also classified into constitutive or pre-infectional resistance and induced or post- infectional resistance, according to whether a resistance mechanism functions before or after infection. Plant defences against parasites appear to depend upon these various factors The major physical features for constitutive resistance have been indicated to be the thickness or hard- ness of the cuticle and cell wall, silicification of cell walls, stomata1 closure and the presence of schlerechyma [2], and for induced resistance, tissue lignification [3, 1321 and the formation of cork and papillae [64, 1321.

More attention in plant resistance has been given to the presence of antimicrobial compounds which are present pre-infectionally or induced post-infectionally in plant cells On the basis of their presence form in plants, Ingham [53] suggested that antifungal compounds should be classi- fied into four categories:

1. Prohibitins: pre-infectional materials which reduce or completely halt the in uiuo develop- ment of microorganisms

2. Inhibitins: pre-infectional materials which undergo a post-infectional increase in order to express fully their toxic potential

3. Post-inhibitins: post-infectional materials formed by the hydrolysis or oxidation of pre- existing substances.

4. Phytoalexins: post-infectional metabolites whose forn~ation involves either gene de-repres- sion or the activation of a latent enzyme system

Induced antifungal compounds like post-inhibitins or phytoalexins are considered to be important since their production in plant cells may relate to the specific interaction between both genotypes in host plants and parasites, which determines that a given plant is susceptible or resistant to different pathogen strains, and that a given pathogen strain is avirulent or virulent on different host cultivars. The genetical basis for the expression of the specific resistance in host cultivars and fungal race interactions has been known by the gene-for-gene concept postulated by Flor [34, 351,

and explained in detail by Day [22] Flor [34] summarized the evidence from flux rust in the

gene-for-gene hypothesis, postulating that during their evolution host plant and parasite develop

complementary genetic systems, and for each conditioning rust reaction in the host plant there is a

specific gene conditioning pathogenecity in the parasite. On the expression of the genes for

resistance, there is a recognition mechanism involving the interfaces between the two organisms

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that deter.mines whether disease reaction leads to induced resistance or induced susceptibility. It is now understood to be the key point for determination ofthis fundamental biological phenomenon, "specificity" [20, 21, 93, 1381

Up to the present time, many phytoalexins whose presence was originally hypothesized by Miiller and Borger [83] in 1941 have been formed in a t least 100 plant species representing 21 families [58]. Major phytoalexins in plants whose chemical structures have been identified are listed below (Table I)..

Table 1. Major phytoalexins in plants -.

Plant

Pea Soybean French bean

Broad bean

Clover Sweet potato Potato

Tomato Cotton Red pepper Grapevine

Phytoalexin (trivial name)

Pisatin Glyceollin Phaseollin Kievitone Wyer one acid Wyer one Medicar pin Ipomeamar one Rishitin Phytuberin Rishitin Hemigossypol Capsidiol a-Vinifer in

Chemical class

isoflavonoid isoflavonoid isoflavonoid isoflavonoid f uranoacetylene fur anoacet ylene isoflavonoid ter penoid ter penoid ter penoid ter penoid ter penoid ter penoid cyclic stilbene tr imer

Orchid Or chino1 dihydr ophenanthrene Safflower Safynol polyacetylene Carrot 6-Methoxymellein isocoumar in

- --

Relationships between phytoalexin production and the specific expression of resistance have been demonstrated especially in the interactions of potato-Phytophthora znfestans [ 1271, soybean-phy- tophthora megasperma f sp glycznea [56], and French bean-Colletotrlchum lzndemuthianum [6, 71. These findings provide a general understanding that phytoalexin production could be associated not only with the expression of disease resistance in plants but also with a mechanism through which the specific interactions of host plant and parasite could be mediated

Despite the widespread plant taxa known to produce phytoalexins and the many reports suggest- ing the production of phytoalexin in barley [90], corn [ 69, 701 and rice [89, 1301, the involvement of phytoalexins in antifungal resistance in the Gramineae has not been established [23, 58, 651. Although benzoquinone derivatives and momilactones were recently reported as phytoalexins of barley [32] and rice plants [13, 141, respectively, evaluation of these compounds in varietal resistance is still required Efforts for identification of phytoalexins from gramineous plants are

important not only to generalize phytoalexins in plant defences, but also to determine real anti-

microbial agents causing the specific resistance expression in major diseases of cereal plants such as

rust or powdery mildew of wheat and barley, where the genetical basis for disease resistance has

been most extensively studied [23, 31, 481

In the crown rust of' plants used in the present investigation, the ma,jor genes for resistance to

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the parasite have also been well analyzed [ 109,1101, and the mechanisms for the appearance of specificity in the interactions of oat cultivars and the rust races have been a subject to be explain- ed. A series of events in oat plants along with the incompatible reaction have been analyzed with special reference to cause or effect of resistance in oat plants [118, 120, 1431 No real antifungal agents effective for the rust invasion have yet been found as other rust or powdery mildew diseases of cereal plants. In any case, it is essentially important to identify antifungal agents associated with the specific resistance of oat to crown rust, as a prerequisite for understand- ing of the specificity of either resistance or susceptibility in the interactions of oat cultivars and the rust races

The present study was therefore conducted to examine whether the production of phytoalexin is involved in resistance of oat plants against crown rust invasion, as a step to detect the antifungal agents As a result, possible phytoalexins of oat plants, named avenalumins I, I1 and 111, were found to accumulate in the incompatible interactions In this paper, evidences for the detection and identification of avenalumins are described and the phytopathological significance of avena- lumin production is evaluated in relation to the expression of specific resistance in oat plants against crown rust fungi

CHAPTER 11

THE PRODUCTION OF PHYTOALEXINS, AVENALUMINS, BY OAT IN RESPONSE TO CROWN RUST, PUCCINIA CORONATA I?. SP. AVENAE

Using the disease system of oat cultivar Shokan 1 which responds resistingly to race 226 but susceptibly to race 203, it was pursued whether the production of phytoalexin was involved in the incompatible interaction The methanol extracts from the incompatible and compatible interac- tions as well as the uninoculated leaves were carefully analyzed to detect the antifungal compounds specific to the incompatible reaction A remarkable accumulation of three distinct antifungal compounds was found in leaves which were responding to the infection with the incompatible race. Based on the various facts which are described in this paper, the compounds were regarded as phytoalexins responsible for antifungal resistance in crown rust of oat plants, and given the trivial name as avenalumins I, I1 and 111 The isolation and the chemical and biological properties of avenalumins are described in this chapter

MATERIALS AND METHODS

Growth and inoculation 01 plants. Two oat (Avena sativa Lo) cultivars, Shokan 1 and a line containing PC 38 gene for resistance to the crown rust, were grown on vermiculite a t 20-21°C in a growth chamber under continuous fluorescent illumination a t 8,000 lux.. Primary leaves of' 7-day- old seedlings were inoculated by a spray method with uredospores of race 226 or 203 of' Pucciniit

coronata Cda, f:, sp, avenue Fraser et Led. Usually 100 mg of' spores were mixed with about 2.5 g of talc and dusted using a hand sprayer on the primary leaves of 10 to 15 pots (9 cm in diam.), each of which contained about 30 plants. The inoculated plants were then maintained under moist

conditions for 15 hours and returned to the growth chamber thereafter.. Race 226 was incompati-

ble with Shokan 1 and compatible with PC 38.. Infection types of' the two cultivars with race 203

were the exact reverse of' that with race 226. The disease reactions were assessed by measuring the

lengths of' all branched infection hyphae originating from substomatal vesicles, which were stained

with alcoholic lactophenol containing aniline blue and visible under a n ordinary light microscope.

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Detectron of antijungal compounds assocrated wzth resrstance reactron Forty-eight hours after inocu-

lation, the primary leaves (5g fresh wt) inoculated with either race 226 or race 203 and the uninoculated leaves were dissected into segments after eliminating the top 1 cm of the leaves and dipped without maceration in 200 ml of hot methanol kept in a water bath (80°C) for 10 minutes The extract was filtered through a filter paper and the f i l t~ate was then evaporated to dryness rn vacuo at below 40°C The residue was dissolved in methanol and fractionated into 90 fractions through a Sephadex LH 20 column (3 x 45 cm) with methanol as eluent. Each fraction (6 ml) was concentrated to 0 5 ml A 60~1-portion of each concentrated fraction was spotted onto silica gel plates (Merck GF,,,60; 0 25 mm) for thin layer chromatography (t 1 c ). Elution profiles of the fractions were obtained by measuring the u v -absorption at 340nm of each spot on the t 1 c plate The t. 1 c plates were then developed with a solvent system of chloroform: methanol : water (65 : 35 : 10, v/v) The spots were detected by scanning with u v light in the range of 250 to 350 nm by a Hitachi fluorescence spectrophotorneter or by spraying with 20% sulfuric acid and heating at 110°C for lominutes To detect antifungal compounds specific to the incompatible interaction, antifungal activity of all the fractions developed on the t 1 c plates was screened by using the t 1 c plate bioassay method as described below Isolation and spectral analyszs o j avenalumtns A large number of primary leaves of Shokan 1 were inoculated with uredospores of incompatible race 226 About lO0g (fresh wt) of leaves were harvested 5 to 6 days after inoculation The methanol extract of the leaves was applied to a Sephadex LH 20 column (3 x 35 cm) and a crude fraction containing avenalumins was isolated This fraction was concentrated, and then applied to a longen column (3 x 120 cm) of Sephadex LH 20 Fractions containing avenalumins I and 11, I and 111, II and 111 were collected by slowly eluting with methanol The individual avenalumins were then isolated on 0 35 mm t 1 c plates. The isolated avenalumins were further purified on the t 1 c and dissolved in methanol The u v. and fluorescence spectra were analyzed by a Hitachi u v spectrophotometer and a Hitachi fluorescent spectrophotometer MPF-4, respectively Determination of chemlcal structures of avenalumrns I , II and III The chemical structures of purified avenalumins I , I1 and III were elucidated[80]. Physicochemical analyses based on ultra violet, nuclear magnetic resonance (NMR) and mass spectra (MS) were conducted and the structures of avenalumins were proposed The proposed structures of avenalumins I , I1 and 111 were synthesized for confirmation of the structures Physicochemical data as well as antifungal activities of synthe- sized avenalumins 11, II and III were compared with those of isolated avenalumins I , I1 and 111 Bioassay for anttfungal actrvlty The antifungal compounds were detected on t 1 c plates using uredospores of P coronata f sp avenae race 226 After the developed t I c plates were well mois- tened by spraying with water, uredospores were dispersed heavily on the plates and brushed to make a uniformly dense layer The plates were again sprayed with a mist of water and incubated in a dark chamber at 20°C for 16 hours The presence of compounds active against uredospore germination as well a s germ-tube growth was recognized as orange-yellow spots contrasting to the background where the uredospores germinated and their original color became fainter

The antifungal activity of isolated and synthesized avenalumins and some phenylpropanoid compounds in aqueous solution was also assayed by the microscope-slide technique Uredospores

of P coronata f sp avenae races 226 and 203 and Puccinla gramrnts f. sp trztrci Erikss et Henn.

race 56 were floated on a droplet and allowed to germinate a t 20°C for 10 hours. The rate of

germination and the length of germ-tube were measured

Quantrtative assay of avenalurnrn Leaf pieces of 5 cm length (1 to 6 cm portion from the leaf tip)

were taken and subjected to extraction with hot methanol and then fractionated through a

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Sephadex LH 20 column (1 x 25 cm). The fraction containing avenalumins was diluted [with ap- propriate amount of methanol, depending on the concentration of avenalumins, to give complete separation of their spots each other on the t 1 c. The fractions were then chromatographed on the silica gel plates (0 25 mm) with the same solvent system as above and scanned under 340 nm as exitation wavelength by the Hitachi fluorescence spectrophotometer Avenalumins were detected as dark spots against fluorescence from the silica gel layer The amount of avenalumins separated on the t 1. c plate was estimated by u v -absorption and calculated by comparing with that of synthesized standard compound whose absorbance a t 340 nm responded linearly within the range of 0 5 to 3 0 pg The amount was expressed as pg per g fresh weight of leaves

RESULTS

Detect~on o j antijiungal compounds in incompat~ble znteraction A comparison was made among the

extracts of healthy leaves of Shokan 1 and the leaves inoculated with incompatible race 226 and compatible race 203 Elution profiles from the Sephadex columns bearing each of the extracts were examined under the u v wavelengths from 250 to 350nm The results indicated that u. v -absorption a t 340nm in fractions 75 to 85 was characteristic of the incompatible interaction

(Fig 1) The portion corresponding to this peak was visualized on the column as a yellow-green fluorescence zone by irradiation with long wavelength u v -light and was referred to as Fr. G

All of the fractions eluted from the column were further compared by t 1 c Three major and two minor compounds were detected in Fr G from the incompatible sample Of particular inter-

est were the major spots a t Rf 0 30, 0 34 and 0 36, designated as avenalumins I, I1 and 111, respectively (Fig. 2) There was another blue-fluorescing spot detected in fractions 42 to 48 of the incompatible sample This spot was also absent in the fractions from susceptible and healthy leaves

Screening for antifungal compounds was conducted by the t 1 c. plate bioassay method on all the fractions shown in Fig. 1 from the incompatible and compatible interactions and the uninoculat- ed. Although the number of antifungal compounds detected by this method depended on the quantity of the fraction applied to the t 1. c. plates, it was clear that the major antifungal com-

Fractlon number Fig 1 Elution profiles from Sephadex LH 20 column of methanol extracts of oat leaves (cv Shokan

1) 48 h after inoculation with incompatible race 226 (-) or compatible race 203 ( ) of Puccznza coronata f sp avenue and uninoculated control leaves (-.-----) The bar indicates the fractions that contain avenalumins and is referred to as the FI G fraction

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Avenalumin Avenal.umin

Avenalumin

Time after inoculation ( h )

Fig 2 Thin-layer chromatogram of Fr G from oat leaves (cv Shokan 1) inoculated with compatible race 203 (left) or incompatible race 226 (right) of Pucclnla coronata f sp avenae Avenalumins were detected as dark spots under u v light; only the lower half of the chromatogram was photographed To demonstrate the presence of avenalumins I and I1 at 28 h after inoculation, a large amount of Fr G was applied to the t 1. c. plate; thus, avenalumins I1 and 111 are overlapped in this experiment

pounds which accumulated in the interaction were the avenalumins I , I1 and 111 The antifungal spot was also found in the fractions 42 to 48 and this antifungal spot, B1, was not found in either uninoculated or compatible race-inoculated leaves The spot fluoresced blue under eradiation with u v -light. However, antifungal activity seemed to be inferior to those of the avenalumins No antifungal spot was detected a t Rf 045 and 055 from fractions 15 to 38 of the incompatible interaction on the t 1 c plates where the preformed antifungal substances of oat leaves, 26-desglu- coavenacosides A and B (26-DGAs) would be expected to be located Chemical properties and structures of avenalumlns The pure avenalumins are white powders and heat stable but, unlike other phytoalexins, they are highly water soluble.. The methanol solutions of the avenalumins have their maximum u. v -absorbance near 336 nm while the maximum fluores- cence emission spectra of avenalumins I , I1 and 111 are 490, 510 and 560 nm, respectively (Fig 3). The possibility that the avenalumins are present as glycosides in the leaves and they are released by enzymatic action was ruled out by the fact that homogenization of leaf' tissues did not result in the rapid release of' avenalumins. Avenalumins are highly aromatized compounds containing nitrogen and phenolic hydroxy groups.. Molecular fbrmulae were I : C16Hl,N0,, 11: CI7Hl3NO5, and 111: ClsH,,N04. Methanolysis of avenalumins showed that they are composed of' the f'ollow-

ing structural moieties : 5-hydroxy-anthranilate in I, I1 and 111, p-coumarate in I , f'er ulate in I1 and p-hydroxyphenyl-pentadienate in 111. Acetyl derivatives of' avenalumins I , I1 and 111 were formed and used for spectrophotometric investigation with NMR and MS. The chemical structures of'

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230 270 310 350 390 Wavelength (nm) Wavelength (nm)

Fig 3 U. v absorption (a) and fluorescence emission (b) spectra of avenalumins I (-), I I ( ) and I11 (---.--) in methanol. Excitation light for fluorescence emission, 410 nm.

0 Fig 4 The structures of avenalumins I, I1 and 111.

avenalumins were characterized as shown in Fig 4. The proposed structures were confirmed by total synthesis [80]. Avenalumin I was 2-[2-(4-hydroxyphenyl)ethenyl]-6-hydroxy-4H-3, 1- benzoxazin-Cone Avenalumin I1 was 2-[2-(3-methoxyphenyl)ethenyl]-4H-3, l-benzoxazin-4- one Avenalumin I11 was 2-[4-(4-hydroxypheny1)-1, 3-butadienyll-4H-3, 1-benzoxazin-4-one. Antcjungal actzvzty of avenalumzns The antifungal activity of the Fr G fractions was found to be present only in those leaves inoculated with the incompatible race (Table 2) The reduction of germ-tube growth increased as the time after inoculation increased. Since avenalumins were the major compounds in Fr. G, inhibitory activity of isolated avenalumins was examined using two rust fungi (Table 3) Inhibition of germination and germ-tube growth was observed to a similar extent on droplets of avenalumins I, I1 and 111 a t concentrations of 200pg/ml ar greater. About 50% inhibition of germ-tube growth was obtained a t 200-250 pglml There was no obvious differ- ence in sensitivity among P coronata f sp avenae races 226 and 203 and P gramznzs f. sp trzticl.

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Table 2. Germ-tube growth of'Pucciizih coronata f sp avenae race 226 uredospores on solutions of Fr G which was extracted from oat leaves (cv Shokan 1) uninoculated and inoculated with incompatible race 226 or compati- ble race 203 of' Puccinia coronata f' , sp avenae

Germ-tube length (,urn) of race 226 -

Time after inoculation Uninoculated Inoculated

(h) Race 226 Race 203 -- (incompatible) (compatible)

28 515 354 49 7

Fr G from 1 5 g fresh weight leaves was dissolved in 1 ml of water Uredospores were allowed to germinate for 16 h at 20°C Data are means of 100 uredospores observed in each of three replicates

Table 3 Germination and germ-tube growth of Puccin~a coronata f sp avenae and Pucciniagramlnls f sp t r ~ t ~ c ~ uredospores on different concentrations of avenalumins

-- - Germination (94) Germ-tube growth (pm)

Concentration (vglml) P coronata P coronata

P graminis P graminis Race 226 Race 203 Race 226 Race 203

Water control Avenalumin I 200

300 500

Avenalumin I1 200 300 500

Avenalumin I11 200 300 500

Uredospores were allowed to germinate for 10 h at 20°C Data are means of 100 uredospores observed in each of three replicates

It was confirmed that synthesized avenalumins I, I1 and 111 were also antifiingal at equal level to those of natural avenalumins (Table 4).. The ED,, values of avenalumins I, I1 and I11 for the germ-tube growth were about 200pg/ml, respectively.. The antifungal activities of' avenalumins were then compared with those of' phenylpropanoid compounds which are possible precursors of avenalumins. The result showed that avenalumins were antifungal at about equal level to those of' phenylpropanoids though the latter compounds showed higher activity by about 20-30% than those of avenalumins..

In the t. 1.. c. plate bioassay with the uredospores of race 226, avenalumins I, I1 and I11 gave clear spot at the sites of corresponding Rf' values (Table 5). In this method, the minimum amount detecting the antifungal activity was 5 pg per spot for both avenalumins and the phenyl- propanoids, The acetylated derivatives of avenalumins had no antifungal activity, however.

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Table 4 Antifungal activity of synthesized avenalumins I, I1 and I11 and phenolic acids against the germination and germ-tube growth of Puccinra coronata f sp avenae in the micro- scope-slide technique

- .- -- -- -

Compounds Concentration Germination Germ-tube Inhibition rate (pglml) (%) growth (pm) (%)

Water contr 01 Avenalumin I

Avenalumin I1

Avenalumin I11

Cinnamic acid

p-Coumar ic acid

Caff eic acid

Ferulic acid

Table 5 Antifungal activity of synthesized avenalumins I, 11 and 111, their acetylated deriva- tives and phenolic acids in the t 1 c plate bioassay with Pucclnia coronata f sp avenae

--

pglspot Compounds --

1 2 5 5 10 15 20 25

Avenalumin I - Avenalumin I1 - Avenalumin I11 - Acetylated avenalumin I - Acetylated avenalumin I1 -.

Acetylated avenalumin I11 - Cinnamic acid + p-Coumar ic acid - Caff eic acid - Ferulic acid -

Accumulation of avenalumins during the rnfection process The amount of avenalumins in the prima- ry leaves of Shokan l following inoculation with incompatible race 226 or compatible race 203 was estimated during disease development (Fig 5) In both incompatible a n d compatible interac- tions, avenalumins were detected as early a s 10 to 12 hours after inoculation, when substomatal vesicles were formed in the stomata1 cavities The amounts detected were very low a t this stage, and a slow increase was observed up t o 24 hours after inoculation for both incompatible a n d compatible interactions

I n the incompatible interaction, avenalumins I a n d I1 accumulated rapidly after 28 hours from

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Fig 5 Time course of accumulation of avenalu- mins I (8) , I1 (0) and 111 ( A ) in oat leaves (cv. Shokan 1) after inoculation with incompatible race 226 (-) or com- patible race 203 (- .--) of Puccznia coronata f sp. avenae

'Time after inoculation (h )

0 21 18 52 96 0 21 18 '72 96 'Time after inoculatior~ (h)

Fig, 6,. A correlation between avenalumin accumulation and fungal growth in primary leaves of' oat infected with Puccinih coronata f' sp auenae xaces 226 [(a), (c)] and 203 [(b), (d)] respectively.. Race 226 is incompatible with cv. Shokan 1 (@) and compatible with the PC 38 line (0). Disease reactions of the two cultivars to race 203 are the reverse of'those to race 226..

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inoculation and avenalumin 111 after 36 hours At 48 hours after inoculation, when fungal growth ceased completely, about 400 pg of total avenalumins per gram fresh weight had accumulated. In contrast to the rapid accumulation of the avenalumins in the incompatible response, avenalumins I and I1 decreased after 28 hours in the compatible interaction and no avenalumins were detected from 2 to 6 days after inoculation. Avenalumin 111 was not detected in the compatible interaction throughout the experimental period.

The accumulation of avenalumins was also examined in the oat line possessing resistant gene PC 38 whose infection types with races 226 and 203 were the opposite of those of Shokan 1. The contents of total avenalumins increased in this oat line after inoculation with race 203 but not with race 226 The result, thus, indicated that the accumulation of avenalumins was associated with race-cultivar resistance (Fig 6) In both incompatible combinations, the time of rapid accu- mulation of avenalumins coincided with the time of retardation of growth of infection hyphae. The fact that the amount of avenalumins in leaves of the line possessing the PC gene was consid- erably lower than in Shokan 1 may reflect differences in the degree of resistance between the cultivars. In the PC 38 line, the cessation of growth of intercellular hyphae of the incompatible race occurred between 48 to 60 hours after inoculation, while in Shokan 1 it occurred between 28 and 36 hours after inoculation (Fig 6).

DISCUSSION

The data presented in this chapter demonstrate that the avenalumins are phytoalexins of oat plants. Avenalumins are extensively accumulated in the incompatible host-parasite combinations.. The time of' major accumulation of the compounds coincided with the time of cessation of fhngal growth within tissues. No major compounds other than avenalumins I, I1 and 111 seemed to participate in antifungal activity of Fr.. 6, since the antifungal activity in Fr. G (Table 2) is rough- ly in accord with that of total avenalumins accumulated in vi'vo (Fig. 5), as estimated by the in

vitro data in Table 3 Avenalumins are unique phytoalexins in that they are highly hydrophilic and contain nitrogen Avenalumins I, I1 and I11 are the first chemically-identified phytoalexin of' cereal plants infected with rust fbngi.

It has been demonstrated that the cereals, rye, wheat and maize contain glucosides of dihydroxy- benzoxazolinone and glucosidases, which interact in homogenized tissues resulting in the rapid release of' antifungal aglucones [I351 In a wheat-stem rust system, Deverall [23] found a n antifhngal compound in wheat seedlings which had expressed to P..graminis f: sp.. tritici: The com- pound was identified as benzoxazolinone which was released fiom a glucosidic precursor in hypersensitively responding host cells.. However, formation of avenalumins is not the same as that of' the antifhngal substance described by Deverall [23], since the homogenized leaves of' uninoculated oat leaves did not produce avenalumins. Also, the slow accumulation of' avenalu- mins during the process of' infection may indicate that they are not released from immediate precursors but are synthesized in response to inkction..

In oat leaves, the preformed antif ungal compounds, 26-DGAs referred as post-inhibitin, have been shown to be activated from the immediate precursors, avenacosides A and B, by /3-glucosidases in injured leaf' tissues [55, 721 .. The possible significance of' these preformed substances has been

discussed for the host-parasite interactions of' oat leaves [ 1061. In this study, however, no post-,

infectional increase of 26-DGAs was detected in oat leaves which had responded to the incompati-

ble race of the crown rust fungus. This suggests that the formation of' 26-DGAs from the

immediate precursors is not involved in rust resistance, contrary to the case of' stem rust resistance

in wheat.

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It is of special interest that avenalumins are highly hydrophilic; it is thus considered, as Daly postulated [ 191, that these avenalumins may have more contact with invading organisms in infect- ed host tissues than other reported phytoalexins which are generally low in water solubility

There are two phases of avenalumin accumulation during the infection process The first phase is the prehaustorial stage of fungal development when no visible host responses such as cellular collapse are observed. In this phase, a small amount of avenalumins was detected in both incom- patible and compatible combinations The second phase is the period of early haustorium formation and the initial period of host cell collapse at infected sites Rapid accumulation of the avenalumins is observed in this stage but only in the incompatible combinations In the compatible combina- tion, the amount of avenalumins decreased as haustorial formation proceeded It appears therefore that avenalumin accumulation could be controlled in at least two phases during infection; one is non-specific induction as the stage of stomata1 penetration and the other is race-cultivar specific regulation during the process of fungal development within leaf tissues

CHAPTER 111

CORRELATION BETWEEN AVENALUMIN ACCUMULATION AND THE SPECIFIC RESISTANCE TO INCOMPATIBLE RACES OF THE CROWN RUST FUNGUS

As described in the previous chapter, avenalumins, the phytoalexins of oat plants, were accumu- lated in the incompatible oat-rust interaction Chemical analysis of avenalumins showed that they are highly hydrophilic, luminescent nitrogen-containing phenolic compounds, and the chemical structures were elucidated One may ask if the production of avenalumins is really associated with the mechanisms specifying the varietal resistance in the crown rust of oat

Phytoalexins have been considered to have a n important role in disease resistance on the basis of their fungitoxicity and the site, time and concentration of their accumulation in infected tissues in relation to restriction of fungal development [6, 7,8, 56, 104, 105, 1451 The role of avenalumins in resistance of oats to crown rust should also be evaluated in regard to those properties of phytoalexin accumulation

In oats many major genes for resistance to crown rust have been identified and studied by Simons and coworkers [ 109, 1101 In this study, 21 oat PC lines each possessing a single resistance gene and two crown rust races were employed. The relationship between resistance gene expres- sion and the rapidity and amount of avenalumin accumulation was examined in detail in the 42 combinations between the PC lines and fungal races The role of avenalumins in expression of PC genes against crown rust is discussed in this chapter

MATERIALS AND METHODS

Plants and fungi': Twenty-one cultivars of' oat whose genes fbr resistance to crown rust had been identified [ 1101, and cv. Shokan 1 were employed for the present study.. The identified resistance genes and reactions to races 203 and 226 of P. coronata f.. sp, avenae are listed in Table 6. In this study, these oat varieties are referred to as PC lines, and are designated by the gene number

instead of the code for the varieties.. PC lines 2, 14, 51, 52, 53, 57, 58, 59 and 61 were supplied by

Dr. M. D. Simons, USDA, Iowa State University and PC 54, 55 and 56 by Dr. D. E.. Harder, Agri-

culture Canada Research Station, Winnipeg, Canada. PC 35, 38, 39, 40, 45, 46, 4'7, 48 and 50,

originally from Dr. J. W. Martens, Agriculture Canada Research Station, Winnipeg, Canada, were

donated by Dr. S , Sato, Tsukuba University, Japan. All the PC lines were grown in the university

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farm of Kagawa University and the seeds were yielded and supplied for the present study. Among

the PC lines, PC 35, 38, 39, 40, 45, 46, 47, 48, 50, 54, 55 and 56 are all near-isogenic to each other, and PC 51 and 52 are also near-isogenic (Dr M D Simons, personal communication) Growth and znoculation of plants Dehusked seeds were soaked in water for 2 hours and incubated on wet filter papers in Petri dishes for 36 hours About 200 uniformly germinated seeds were selected and sown on vermiculite in a planter (15x 25x 10cm) and grown in a growth chamber at 20-21°C under illumination for 16 hours daily with 10,000 lux fluorescent and incandescent lamps.. Seven day-old seedlings of each PC line were inoculated as stated in the previous chapter except that 90mg spores mixed with 3g of talc were dusted onto the primary seedling leaves in each planter About 15% of stomata were infected by inoculation with this mixture Obsevvatlon o j fungal development Intercellular hyphae of crown rust in infected leaves were observed using a modification of the Calcofluor technique employed by Rohringer et a1 [ loo] Preliminary tests inidicated that 5 minutes staining with a Calcofluor White (Kayaphor FB R08618, Nippon Kayaku Co , L'PD Japan) solution (0 1%) facilitated simultaneous observation of infec- tion hyphae and host cell collapse as shown in stem rust-infected wheat leaves [loo] ;however, 16-20 hours staining with the fluorescence developer was necessary for complete visualization of all branched hyphae developing from substomatal vesicles of crown rust fungi Leaves stained for 20 hours were observed under a Zeiss research microscope equipped with a epifluorescence condenser IIIRS and a mercury high-pressure lamp, HBO 100 W/2 using a Zeiss HBO 100/2 power supply The filters used were as follows: BP400-440 excitation filter with peak transmittance of 400-440

Table 6 Oat varieties carrying identified genes for crown rust resistance and their infection types with races 203 and 226

- --. - .- .- -- -- . . -.--p.---p----..--.......-.

Infection type Variety PC gene - . .- -

Race 203 Race 226

Victor.ia Ascencao D-137 CW-491-4 F-366 F-83 F-169 F-290 C. I.. 8081A F-158 CW486 Iowa X434 Iowa X421 H441 CAV 1830 CAV 4963 CAV 1964 H555 TAM-0-301 TAM-0-312 Coker 234 Shokan 1

2 14 35 38 39 40 45 46 47 48 50 5 1 52 53 54 55 56 57 58 59 61

Unknown

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nm, chromatic beam splitter FT460 and barrier filter LP470. The lengths of stained infection hyphae developing horizontally from a vesicle were measured; the average lengths from 100 infec- tion sites were obtained for all samples and these were statistically compared

The classification of infection type was done in this investigation, partly with some microscopical observation, as follows: Immune to highly resistant: (0)-almost no visible symptom, no infection- hyphal mat, some small necrotic fleck. Moderately resistant: (1)-no uredia, few small infection- hyphal mat, necrotic or chlorotic fleck present (2)-few small uredia, plentiful small infection- hyphal mat, some necrotic areas seldom without uredia (3)-plentiful small-size uredia; necrotic areas seldom without uredia Susceptible: (4)-abundant medium to large uredia without necrosis or chlorosis Quantztatzve estzmatlon of avenalumln accumulatzon The quantitative assay for avenalumins was carried out as described in the previous chapter Segments 5cm long cut from 1 cm behind the tip of thirty infected primary leaves were used for the quantitation The methanol-soluble fraction containing avenalumins was separated on a Sephadex LH 20 column, evaporated to dryness under vacuum and usually redissolved in 1 ml of methanol The methanol-soluble fraction was then

chromatographed on a silica gel GFZs4 (Merck) thin-layer plate (0 35 mm) The amount of avena-

lumins I and I1 on the plate was estimated by scanning under a Hitachi fluorescence spectrophoto- meter MPF-4 Although avenalumin I11 was detected as a trace in certain PC lines, the amount was negligible as compared to avenalumins I and I1

RESULTS

Correlation between avenalumin accumulation and resistance expression in various PC llnes. Analysis

of the relationship between resistance expression and avenalumin accumulation in rust-infected leaves of'the PC lines was conducted by separately inoculating with both races 203 and 226.. The experiments were done in two series, one with PC 35 to 50 and the other with PC 2, 14 and 51-61. The oat cultivar Shokan 1 which was highly resistant to race 226 and susceptible to race 203 was used in both experiments as a reference line.. The results are summarized together in Tables '7 and 8 for race 203 and 226 respectively. The degree of resistance of PC lines toward races 203 and 226 of 'P. coronata f: sp.. avenue was determined by the length of intercellular hyphae and final infection

type. Although hyphal length was roughly correlated with the infection type, a more detailed order of the resistance of' PC lines within the same infection type was obtained by comparing the hyphal lengths. Up to 24 hours after inoculation, no significant variance in the growth of'intercel- lular hyphae was found among the race-PC line combinations However, retardation of' hyphal

growth in leaves possessing the genes PC 14, 48, 50 and 59 for race 203 and PC 48, 50, 5 1, 52 and 53 fbr race 226 was observed by 36 hours after inoculation.. At 36 and 48 hours after inoculation, average lengths of'intercellular hyphae in the PC lines diff'ered significantly and it was possible to classify the reactions approximately into three groups: immune to highly resistant, moderately resistant and susceptible.. In Tables 7 and 8, the PC lines were grouped in the order of' resistance, based on the length of intercellular hyphae at 48 hours after inoculation and final infection types. The orders of the PC lines assessed by both markers were parallel with each other except for a few lines.. The approximate time to detect retardation of growth of' inftction hyphae in each PC line

was also indicated.. Restriction of' fhngal growth in highly resistant lines was observed at 36 and

48 hours after inoculation, whereas retardation of' the growth in moderately resistant lines occurred

72 to 92 hours after inoculation.. The completely susceptible lines, infection type 4, were assumed

to show no retardation of'hyphal growth during the experimental period up to 144 hours.

The accumulation of' avenalumins I and I1 during inftction in the PC lines was then contrasted

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Table 7.. Resistance as measured by three parameters and avenalumin accumulation in primary leaves of PC lines infected with Puccznza coronata f sp avenue race 203 ---- - -- -- - - - -- --

Hyphal length" Time of Avenalumins I & I1 (,~g/g fresh wt) & growth -- --

PC lines infection type retarda- Time after inoculation (h) (in parentheses) tion (hr) 24

- - - - -- - - - 36 48 72 96 144

(Highly resistant) 14 165 (0) 36 22 83 198 238 363 401 50 178 (0) 36 25 90 190 278 347 479 5 1 189 (0) 36 12 40 113 290 196 209 53 193 (0) 36 10 63 282 351 371 463

(Moderately resistant) 38 243 (1) 72 tr tr 21 113 156 86

(Susceptible)

Shokan 1 426 (4) - tr t r tr tr t r tr

a The average length (pm) of intercellular hyphae developing from each substomatal vesicle 48 h after inoculation

b The remarkable retadation of growth of the intercellular hyphae was observed at indicated time after inoculation tr =trace

to the ordei of resistance to race 203 (Table 7) and race 226 (Table 8) There appears to be a relationship between avenalumin accumulation and degree of resistance associated with various genes In lines with resistance genes causing earlier restriction of rust development, the production of avenalumins was generally more rapid and abundant than in lines with resistance genes which retarded fungal growth at a later stage The clear relationship was evident between the highly and moderately resistant groups; the rapid accumulation of avenalumins was observed a t the early infection period of 24 to 48 hours post-inoculation in the former group, whereas little accumulation of avenalumins was detected in the latter one. In moderately resistant group, the accumulation

of avenalumin was observed after 72 hours post-inoculation though the amount of accumulated

avenalumins seemed to be those of the highly resistant lines. The time of rapid accumulation of

avenalumins corresponded to the time of retardation of growth of infection hyphae In a strict

sense of the correlation between the amount of avenalumin and the hyphal length, there seemed

to be some apparent exceptions such as for PC 40 and 48 to race 226 and PC 35 to race 203.

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Table 8 Resistance as measured by three parameters and avenalumin accumulation in primary leaves of PC lines infected with Puccznia coronata f sp avenae race 226

- -- .- -- --

Hyphal length" Time of Avenalumins I & I1 (pglg fresh wt) & growth

PC lines infection type retarda- Time after inoculation (h) (in parenthesis) tion (hr) 24 36 48 72 96 144

(Highly resistant) 51 150 (0) 50 160 (0) Shokan 1 168 (0) 53 171 (0) 14 172 (0) 52 181 (0) 59 186 (0) 58 205 (0) 61 224 (0) 48 314 (0-1)

(Moderately resistant) 5 7 230 (1) 55 244 (1) 2 312 (1)

40 374 (1) 56 340 (2) 35 390 (2) 54 392 (2) 46 436 (3)

(Susceptible) 4 7 452 (4) 39 460 (4) 38 528 (4) 45 560 (4)

a The average length (/*m) of intercellular hyphae developing from each substomatal vesicle 48 h after inoculation

b The remarkable retardation of growth of the intercellular hyphae was observed at indicated time after inoculation tr = trace

However, the retardation of growth of the intercellular hyphae seemed to be in accord with the time of avenalumin accumulation in these interactions It should be also pointed out that several lines such as PC 2,38, 39 and Shokan 1 responded oppositely to races 203 and 226, and avenalu- mins markedly accumulated only in the incompatible combinations Very little accumulation of avenalumins was found in any of the compatible interactions in the present study.

DISCUSSION

It was demonstrated in this investigation that in oat leaves production of' avenalumins was

directly proportional to the degree of resistance expression against crown rust; in other words,

susceptibility of' oat cultivars to races of' the rust was inversely related to the time and rate of

accumulation of' avenalumins.. These facts strongly suggest that avenalumins may be associated

with the specific resistance expression in the interactions between oat cultivars and rust races.

Resistance expression by the various genes, associated with the restriction of' the growth of the

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intercellular hyphae, occurred at different times after inoculation It appears, therefore, that in the expression of the various resistance genes a single mechanism associated with the production of avenalumins could be involved in restricting fungal development At present, there is no evidence in relation to regulation of differential time and rate of avenalumin production in the incompatible rust-oat interactions

For flax leaves infected with Melampsora lznz,Keen and Littlefield [62] presented similar results: production of phenolic phytoalexins, conyfer yl aldehyde was highly correlated with the expression of multiple allelic genes for resistance They suggested that in flax a single mechanism for expres- sion of resistance to M llnz may exist, and that the distinguishing character of the various resistance alleles is differential recognition efficiency of the incompatible fungus by plant cells. The present data obtained for crown rust of oat with the various gene combinations also suggest the presence of gene-specific recognition mechanisms that regulate differential avenalumin production, and that the production of avenalumin is regulated as a result of resistance gene expression.

CHAPTER I V

EFFECTS O F ELEVATED TEMPERATURE AND a-AMINOOXYACETATE ON AVENALUMIN ACCUMULATION AND HYPHAL GROWTH IN OAT LEAVES

Attempts were further made to ascertain the role of avenalumins in resistance by altering their zn vivo concentration by growing the plants at elevated temperatures and by treatment of the plants with a-aminooxyacetate, an inhibitor of phenylalanine ammonia-lyase

The effect of temperature on phytoalexin accumulation has been studied [18, 56, 60, 841 ; for instance, glyceollin was not detected at 50°C or above 40°C [56, 841 and resistance in soybean to P megasperma f sp glycznea was lost at temperatures above 45°C [ 15, 16, 561. When cv. Harosoy soybean leaves inoculated with Pseudomonas syr ingae pv glycznea race 1 were maintained at '31°C, no glyceollin was produced and a fully compatible reaction occurred [60] In potato tubers either infected with P znfestans [ 181 or treated with the fungal wall-released elicitor, rishitin accumulation was inhibited by incubating at 25 to 35OC and the tubers exhibited decreased resist- ance to Phytophthora capszcl and P znfestans [ 1241 In rust diseases of cereal plants, normal resistance reactions are diminished by incubation at elevated temperatures [ 10, 77,82, 119, 1481. In cv Shokan 1 oat leaves inoculated with race 226, resistance and hypersensitive necrosis were reduced when inoculated seedlings were incubated at high temperatures [ 1191.

a-Aminooxyacetate is a competitive inhibitor of phenylalanine ammonia-lyase [75]. Since this enzyme is associated with the production of avenalumins, it was determined whether a-aminooxy- acetate would block avenalumin production and promote fungus development within leaf tissues.

MATERIALS A N D METHODS

Planfs and pathogen culture and inoculation methods: The cultivar of' oat used in the present study was Shokan 1, which is highly resistant to P.. coronata f', sp avenue race 226.. The oats were grown on vermiculite in pots (diam.. 9 cm) in a growth chamber at 20°C by illuminating for 16 hours

daily with 10,000 lux fluorescent lamps. Inoculation of 7-day-old seedlings was achieved by dusting

with a mixture of' talc and uredospores and incubating in a moist chamber for 16 hours as de-

scribed in Chapter 11..

Effects ojgrowth temperature.:. 'To examine the effect of' growth temperature on avenalumin accu-

mulation and hyphal growth, plants were inoculated and, after 16 hours in a dew chamber held

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at 20°C, were grown at temperatures of 15, 20, 25, 30 and 35°C The infected plants were also

transferred to 30°C after the plants were incubated at 20°C for 36 or 72 hours after inoculation Treated leaves were collected at intervals during infection development and leaf segments 5 cnl long were excised I cm behind the tips and avenalumin accumulation and hyphal growth were determined as described in earlier chapters Treatment wzth a-amznooxyacetate (AOA) The infected leaves were cut at the stems 16 hours after inoculation and fed through their cut stems with Kasugai nutrient solution containing AOA at the concentrations of 10-200jlM or with the nutrient culture solution alone as control The pH of all the solutions was adjusted to 5 8 The duration of AOA feedings was 8 hours and it was carried out during the light period The leaves were then transferred to a fresh culture solution, pH 5 8 The quantification of avenalumins and observation of hyphal growth in the treated leaves were conducted at intervals during the next 48 hours Estzmatzon of avenalumzn content in treated leaves The quantitative estimation of avenalumin accumulation in treated leaves was conducted as described in Chapter I1 Twenty primary leaves were extracted with hot methanol and this was fractionated by Sephadex LH 20column chromato-

graphy The fraction containing avenalumins was then chromatographed on Silica gel (Merck GF,,,) t 1 c plates, where avenalumins were visualized as dark spots against background fluores- cence under a long u v -wavelength The a m w n t sf avenalumins was thus quantified by compar- ing negative fluorescence of the spots with that of a standard compound at an excitation wavelength of 340 nm with a Hitachi fluorescence spectrophotometer Quantztatlon of hyphal growth and fluorescent, collapsed host cells The intercellular hyphae in infected leaves were stained with the Calcofluor brightner for 16-20 hours The lengths of intercel-

lular hyphae developing horizontally from a substomatal vesicle were measured under a Zeiss epifluorescence microscope as described in Chapter III For observation of fluorescent collapsed cells, the infected leaves were fixed with an alcoholic lactophenol solution and stained with Calcofluor for 10 minutes to acertain the penetration points of crown rust The number of col- lapsed host cells was observed by fluorescence observation using BP400-440 excitation filter (peak transmittance 400-440 nm), chromatic beam splitter FT460 and barrier filter LP470

RESUL TS

Effect of temperature on avenalumzn accumulation, hyphal growth and cellular collapse Avenalumin

accumulation was dependent on the temperature for plant growth (Fig 7a) The optimal temper-

ature for avenalumin accumulation in oat plants was 20°C In oat leaves infected with race 226, avenalumin accumulation after 48 and 72 hours was largely reduced at elevated temperatures; a t 25°C the accumulation at 48 and 72 hours after inoculation was about half of that at 20°C and only traces of avenalumins were detected in plants kept at 30 and 35°C Slightly increased hyphal growth was observed at 25"C, but a considerable increase occurred in plants incubated at 30°C (Fig 7b) A large mycelial colony was found in the latter tissues, but uredospores rarely formed at 10 days after inoculation No promotion of fungus occurred a t 35°C

Avenalumin concentrations attained by incubation of race 226-inoculated leaves for 36 hours at 20°C decreased when the plants were transferred to 30°C (Fig 8a) Only small amounts of

avenalumins were detected at 48 hours after transfer, and this reduction of avenalumin content

coincided with the continuous growth of hyphae (Fig 8b) No subsequent growth of intercellular

hyphae was observed when plants were transferred from 20°C to 30°C at 72 hours after inoculation

At 20°C, hypersensitive collapsed cells were formed as early as 24 hours after inoculation and

observed a t all infection sites at 36 hours after inoculation The collapsed cells formed a t 36

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Fig. '7.

Temperature

Effect of temperature on (a) the accumulation of avenalumins I (-) and I1 (----) at 48 (9) and 72 ( 0 ) h after inoculation and (b) fungus growth at the corresponding times in oat leaves (cv.. Shokan 1) infected with Puccinia coronata f. sp. avenae race 226.

0 21 $8 '72 96 120 l i t Time after

0 24 48 72 96 120 144 inoculation (h)

Fig 8. Effect of temperature shift on (a) the accumulation of avenalumins I (-) and I1 (----) and (b) fungus growth in oat leaves (cv Shokan 1) infected with Puccinia coronataf sp avenae race 226 The infected plants were grown at 20°C (0) and transferred to 30°C ( 0 ) at 36 and 72 h post-inoculation

a n d 72 hours after inoculation a t 20°C remained after the plants were transferred t o 30°C. The average number of collapsed cells per infection site was 2.10 a t 20°C 36 hours after inoculation a n d it increased to 2.77 a t 30°C 24 hours after the transfer.. Effect of a-amiizoox,yacetate ( A O A ) on the production of avenalumins and hyphal growth.: In vivo

partial inhibition of' the production of avenalumins in leaves inoculated with the "incompatible"

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Time after inoculation (h)

Fig 9. Effect of a-aminooxyacetate on the accumulation of avenalumin I in oat leaves (cv Shokan 1) infected with Puccznia coronata f sp avenae race 226 The bar indicates the period of AOA feeding through the cut stems of infected plants. (0) AOA (200 pM), ( 8 ) control

Table 9. Effects of various concentrations of a-aminooxyacetate on inhibition of avenalumin accumulation after 48 h and fungus growth at 72 h after inoculation in oat leaves (cv Shokan 1) infected with Puccznia coronata f sp avenae race 226

Avenalumin (pg/g fresh wt) Hyphal length Treatment

I I1 & I11 Total % inhibition (pm)

Water contr 01 534 212 746 - 251 AOA 10pM 135 56 191 74 -

25 pM 110 56 166 78 37 1 50 pM 97 tr 9 7 87 3 75

100pM 87 tr 87 88 693

Table 10 Effect of a-aminooxyacetate treatment on the formation of fluorescent collapsed cells (FCC) in oat leaves (cv Shokan 1) infected with Puccinia coronata f sp avenae race 226

Total number of' FCC at 350 infection sites* % reduction in FCC

Water control AOA 10pM

25 pM 50 pM

100 pM

* Observed at 48 h after inoculation.

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race was associated with AOA treatment (Fig 9) The rapid accumulation of avenalumins be- tween 36 and 48 hours after inoculation was almost completely prevented when AOA was supplied at 16-24 hours after inoculation, after stomata1 penetration had occurred but before fungal pene- tration into mesophyll cells. At 72 hours after inoculation, the inhibition was reduced Inhibition was effective even when AOA was supplied at low concentrations; about 80% inhibition was obtained at 10-20pM and 90% was observed at concentrations of 50-100 pM (Table 9) In AOA-treated infected leaves, the promotion of hyphal growth was observed 72 hours after inocula- tion (Table 9) Small mycelial colonies developed a t each infection site in the treated leaves after the initial period of infection; however, the growth was retarded thereafter when the inhibition of avenalumin accumulation was reduced (cf Fig 9) and no uredia developed Hypersensitive autofluorescent collapsed cells were first observed at infection sites at 24 hours after inoculation. Treatment with AOA did not appreciably affect the formation of collapsed cells since only ca 20-40s inhibition was observed (Table 10)

DISCUSSION

The results in this chapter confirm the conclusions in the earlier chapters and further indicate that the production of avenalumins by oat leaves responding incompatibly to the rust race is directly involved in expression of resistance Increased susceptibility and suppression of avenalumin accumulation after temperature elevation and AOA application could result from either suppres- sion of phytoalexin production or promotion of phytoalexin degradation These treatments were applied after the determination of incompatibility as demonstrated in the incompatible race 226- Shokan 1 interaction [I201 Thus, increased susceptibility in this case implies that a metabolic control of accumulation of phytoalexins may affect disease reactions in normally incompatible host-parasite interactions where a recognition reaction towards incompatibility had already been triggered. Such a mechanism may also explain apparent cases of increased sllsceptibility in addi- tion to the type of suppression of incompatible recognition suggested elsewhere [29]. Similar experiments using an inhibitor of synthesis of precursors of the phytoalexin glyceollin have been conducted in soybean systems The results showed that the herbicide glyphosate, an inhibitor of the synthesis of chorismate, inhibited glyceollin synthesis in soybean leaves inoculated with P. syrcngae pv glycznea or hypocotyls inoculated with P megasperma f sp glycznea and greatly re- duced resistance expression in the soybean tissues to both pathogens [49]

In many reports with stem rust [77], leaf rust [ 111 and yellow rust [74] of wheat and crown rust of oats [96, 1201, it has been suggested that hypersensitive necrosis is not essential for inhibited rust growth The effect of elevated temperatures on the formation of collapsed cells in the leaves observed here also indicated that the promotion of fungal growth was initiated even though col- lapsed cells occurred in a similar way to that in leaves incubated at 20°C. This observation is basically similar to those with the temperature senstive Sr 6 gene of wheat to stem rust [77], and implies that hypersensitively-collapsed host cells are not themselves inhibitory to the development of race 226 in Shokan 1 leaves However, the accumulation of avenalumins appears to be required for resistance expression to rust infection. The severe damage to race 226 in Shokan 1 leaves appears to occur before 72 hours after inoculation under normal conditions (cf. Fig, 8), and this

may be due to prolonged exposure to the accumulated avenalumins

By transfering avenalumin-containing leaves from 20 to 30°C, it was found that the avenalmins

were metabolically degraded in the infected tissues, as reported for other phytoalexins such as

rishitin [52, 541, lubimin [52], capsidiol [I131 and glyceollin [146]. It is therefore likely that the

rate of avenalumin biosynthesis a t 20°C is higher than that for degradation; at 30°C, either the

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biosynthesis of avenalumin was prevented or the rate of biosynthesis was far less than that of biodegradation. The present data suggest that phenylalanine ammonia-lyase is involved in bio- synthesis of the avenalumins As shown in Fig 4, a part of the molecules of avenalumins are constructed by phenylpropanoid compounds; thus, the present results confirmed the probable involvement of phenylalanine ammonia-lyase in the biosythesis of the avenalumins.

CHAPTER V

LOCALIZATION OF AVENALUMIN AT TISSUE LEVEL IN OAT LEAVES INFECTED WITH FUNGAL PATHOGENS

It was shown in the previous chapters that the production of avenalumins could be closely asso- ciated with the expression of the genes for resistance to crown rust invasion However, the fact does not rule out the possibility that avenalumins might be accumulated in, the cells which locate far beyond the region of developing rust hyphae even though the apparent relationship was found. Thus, it is most important to know whether avenalumins occur a t infection sites where the phytoalexins might contact the infection hyphae and accumulate there into a concentration suffi- cient to inhibit the fungal development

Analysis of phytoalexin location in the micro-site of infected tissues has been carried out in detail in C lmdemuthzanum-infected bean [6 , 71, P megasperma f sp. glysinea-infected soybean [56, 1451 and Po znfestans-infected potato [ 104, 1051 Sato et al [ 1041 emphasized that rishitin was synthesized in the neighboring live cells adjacent to the infected cells and finally transferred to accumulate into the necrotic dead cells, as revealed by extractions of sliced sections of infected tubers In bean infected with avilurent races of the anthracnose fungus, phaseollin accumulation is restricted to small areas of necrotic tissues at infection sites. If the phaseollin is in the necrotic cells alone, its concentration in the cells greatly exceeds 10 pglml which prevents germ-tube growth in vltro [7] In soybean hypocotyls, Yoshikawa et al [ 1451 also showed by sliced sections of the infected tissues that glyceollin accumulated at the localized infection sites of the resistant soybean hypocotyls to concentration to exceed the ED9, value for inhibition of the fungal growth

Primary leaves of oat are thin tissues and the sites of rust penetration are restricted in the sites of stomata; thus, it is impossible to estimate avenalumin accumulation in the stomatal micro-sites by slicing or excision of the infected tissues as done in those plant tissues In this chapter, there- fore, the localization and concentration of avenalumins in the stomatal sites infected with crown rust fungus were analyzed a t tissue level without making the sliced sections to demonstrate if avenalumins accumulated are effective for preventing the hyphal growth m vlvo. It was also analyed whether avenalumins might accumulate in the epidermal cells of oat leaves infected with other fungal pathogens that could penetrate through the cuticular layer.

MATERIALS AND METHODS

Plants and fungi The incompatible combination between oat cv Shokan 1 and crown rust race 226 was used for the present study. Pyricularia or yzae and Pyricularla gr lsea, non-pathogens of oat

plant, and Drechslera avenae were also employed as pathogens which infect oat leaves through

cuticular layer The conidia of Pyricularla fungi and D. avencze were obtained by culturing on

oat-meal agar and V-8 juice agar media respectively.

Growth and lnoculat~on of plants The plants were grown as mentioned before and used for

inoculation 7 days after sowing To obtain different rates of stomatal infection by the fungus,

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inoculation was made by dusting various densities of spores diluted with talc powder The number of stomata penetrated by the fungus was counted for both abaxial and adaxial surfaces of leaves, and expressed as percentage of total stomata In order to assess the extent of avenalumin accu- mulation in infected and neighboring uninfected area, a partial inoculation was made A 1 cm- long region, 3 to 4cm from the tip, of the highly resistant Shokan 1 leaves was inoculated by brushing uredospores of race 226 onto the abaxial surface The rate of stomatal infection in the inoculated surface was adjusted to about 10 and 30%

The conidia of the saprophytic fungal pathogens were suspended in water and used for inocula- tion of the abaxial surface of the leaves by brushing After inoculation for 14 hours in a moist chamber, the plants were placed and grown in the growth chamber Quantztatzve estzmatzon of avenalumzn accumulatzon The estimation of avenalumins was carried out as described previously In the case of the partial inoculation, the inoculated region and the next two 1 cm regions towards the tip were excised 36 hours after inoculation; fifty excised segments for each sample were weighed and used for the avenalumin estimation

For the leaves infected with the saprophytic fungal pathogens, the lower layers were peeled out from the infected leaves 24 hours after inoculation The epidermal strips and the remaining epidermis-stripped leaves were supplied separately for estimation of avenalumins The concentra- tions of avenalumins accumulated in the stomata1 area infected by the rust fungi and the epidermal cells infected by the saprophytic fungal pathogens were estimated by dividing the amout of avenalumins accumulated in the infected tissues by the volumes of each infection sites, as described in the result section Detectzon of avenalumzn zn zntercellular fluid The intercellular Auid from rust-infected primary leaves of oat was collected by the method reported by Rathmell and Sequeira [98] Sixty infected leaves were collected a t intervals after inoculation and cut into 3 cm segments eliminating the tip 1 5 c m The cut ends were rinsed in water and the segments were wrapped in a layer of cheese cloth and then immersed in distilled water in a test tube Water was infiltrated zn vacuo for three periods of one minute each The leaf pieces were then removed from water and unwrapped and their surfaces were blotted dry with filter paper The leaf segments were again wrapped in cheese cloth, placed length-wise in a Spitz glass tube and centrifuged a t about 600xg for 10 minutes. About 0 2ml of intercellular fluid was obtained in the bottom of the tube Avenalumins contained in the fluid were separated by Sephadex LH 20column chromatography (1 x 25 cm) and quantified by t I c and fluorescence spectrophotometry as described above. The enzymatic activity of glucose-6-phosphate dehydrogenase in the fluid was assayed as described by Rathmell and Sequeira [98] in order to determine leakage of cell contents into the intercellular spaces

RESULTS

Dependency of avenalumin accumulation on znfectzon rate through stomata The experiments were made with the Shokan 1-race 226 system AvenaIumins accumulated in the partly-infected por-

tion of the primary leaves and none to little in the neighboring uninfected tissues (Fig 10) The results also indicated that the extent of avenalumin accumlation in the inoculated area was higher in leaves with higher infection rate In a subsequent experiment, amount of avenalumin was

analyzed in relation to stomatal infection rates (Fig 11) The results clearly showed that avena-

lumin accumulation in the infected leaves a t 36 hours after inoculation was directly related to the

proportion of stomata infected up to 50% The correlation coefficient between infection rate and

the amount of avenalumins was 0.98, and the regression line for the data of Figure 11 was Y =

23X, if the amount of avenalumins was dependent variable Y and the infection rate was the

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Leaf sectlons (cm) Fig 10 Localized accumulation of avenalumins in crown rust-infected areas of primary

leaves of oat inoculated with Puccinia coronata f sp avenae A portion of 3-4 cm from the tip of primary leaves of Shokan 1 was inoculated with race 226, afford- ing low (a) and high (b) infection rates, and the amounts of avenalumins in the inoculated and adjacent uninoculated area were estimated 36 h after inoculation Leaf sections: 1, infected region; 2, adjacent ~egion; 3, region nearer tip.

dependent variable X The data given in Figures 10 and 11, therefore, indicated that avenalumins could be produced

locally at each infection site Thus, it is plausible that, knowing the density of infection sites, the amount of avenalumins accumulated in one infection site could be estimated from the concentra- tion of avenalumins accumulated in a piece of infected tissue. For example, in Shokan 1 leaves inoculated with race 226, about 200pg/g fresh weight of avenalumins I and I1 were accumulated in the primary leaves when the rate of stomata1 infection was about 10% (Fig 11). The number of stomata calculated in both surfaces of the primary leaves of 1 g fresh weight was approximately 350,000; thus, 35,000 stomata (10%) were penetrated by the rust The amount of avenalumins at one infected site was estimated to be 5 7 x pg (200+35,000) It is known that infection hyphaeof race 226 are restricted within a volume measuring about 2 0 0 x 2 0 0 ~ 80pm in a n infec-

tion site in Shokan 1 leaves (Fig 12) Therefore, overlooking air-spaces within in the micro-site, the estimated concentration of avenalumins in one infected site would be 1,785 pg/cm3, which far exceeds the concentration known to inhibit rust growth [ 71 Detectzon of avenalumzns zn the zntevcellular fluzd of rust-znfected leaves The Shokan 1-race 226 combination was used to test whether avenalumins existed in or diffused into the intercellular

spaces of the infected leaves (Fig 13) The results obtained were similar to the results from methanol-extracts of whole leaves which were already described in Chapter 11. Twenty eight hours after inoculation, avenalumins were detected in the intercellular fluid obtained from the incompatible race-infected leaves, but not from the compatible race-infected ones. The concentra- tion of avenalumins I and I1 in the incompatible combination increased rapidly thereafter and reached up to 400pg/ml at 36 hours after inoculation when the growth of intercellular hyphae had

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Proportion of stomata infected (%)

Fig. 11. Dependency of the accumulation of avenalumins on the rate of stomatal infec- tion in primary leaves of oat (cv Shokan 1) inoculated with Puccznia coronata f. sp. avenae race 226 36 h after inoculation Y=23X (r =O 98), where Y= to- tal avenalumins, X= percentage stomatal infection.

Fig 12. Diagrammatic illustration of an infection site where the infection hyphae of Puccinia coronata f. sp avenae race 226 is restricted in oat leaves (cv Shokan 1) 36 h after in-, oculation

largely ceased. This concentration was much higher than required to give 50% inhibition of

germ-tube growth as described previously. Only a very small amount of glucose-6-phosphate

dehydrogenase which is confined to the cytoplasm was detectable in the fluids from both healthy

and incompatible race-inoculated leaves..

Detection of avenalumins in the epidermal cells infected wi'th saproph,yti'c fungal pathogen through

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Fig 13 Detection of avenalumins in the intercellular fluids from the primary leaves of oat (cv. Shokan 1) inoculated with Puccznza coronara f sp avenae race 226 ( 0 ) and 203 (0)

'Time after inoculation (h)

Table 11 The production of avenalumins in the epidermal cells of oat leaves (cv Shokan 1) infected with saprophytic fungal pathogens by cuticular infection

Avenalumins I & 11* Fungus % of avenalumins Concentration in

Mesophyll Epidermis 1n epidermal cells epidermal cells**

Drechslera avenae 159 Pyr icular ia or yzae 24 Pyr icular ia gr isea 24

* pgleach tissue in 1 g of' leaves * * pg/cm3 of the epidermal cells

cutzcular layer The rice blast fungus P. oryzae and P. grzsea isolated from Dzgztarza adscendens

Henr. could penetrate into the epidermal cells of primary oat leaves, but hyphal growth of P. oryzae

and P grzsea was restricted within the penetrated cells D. avenae, however, penetrated mesophyll cells beyond epidermal cells and caused necrotic lesions Avenalumins were inhibitory to the germ-tube growh when the conidia of the Pyricularza fungi were incubated in the aqueous solution placed on a slide glass; the ED,, values of avenalumins I and I1 were about 200 pg/ml D. avenae,

the pathogen of oat, was not inhibited a t concentration of 500pg/ml As shown in Table 11, avenalumins were detected by the extraction method in methanol-soluble fraction of the infected epidermal tissues. The most of avenalumin accumulation in infection with D avenae was found in the mesophyll cells, but a large amount of avenalumin was localized in the epidermal cells in case of Pyrzcularza infection. The concentration of avenalumins in the epidermal cells was estimated at 28 hours after inoculation to exceed 200pg/cm3 and 400pg/cm3 for infection with the Pyrzcularza fungi and D. avenae respectively

DISCUSSION

Avenalumins seem to accumulate exclusively in infected areas of leaves.. Little, if any, avena- lumin was detected in uninoculated area in partly-infected leaves and the amount of avenalumins

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in the inoculated portion was directly proportional to the density of stomatal infection with crown rust, when the infection rate was up to 50% There was no additive effect of inoculum dosage on avenalumin accumulation This indicates that avenalumins accumulate only in stomatal sites infected with the rust fungi and the amout accumulated in each infected stomatal site is the same regardless of the density of infection It is thus theoretically possible to estimate the conceqtration of avenalumins accumulated in an infected stomatal site In race 226-infected Shokan 1 leaves, the concentration of avenalumins at each infected stomatal site was about 1,800pg per cm3 as described in the result section From such calculations it appears that avenalumins accumulate to a concentration sufficient to inhibit the rust development This value is also comparable to other data of Tables 7 and 8 demonstrated in Chapter 111, since the infection rates in those experiments are almost the same In the combinations of immune to highly resistant, a range of about 400 to 1,000,ug avenalumins per cm3 are expected to be present in one infection site at the time when the fungal growth ceased In the moderately resistant combinations, the concentration is about 200 pg per cm3, yhen the growth speed of intercellular hyphae is apparently reduced

The detection of a high concentration of avenalumins in intercellular fluid suggests that either avenaIumin is secreted into intercellular spaces of infected leaves, or the compounds diffuse from mesophyll cells into infiltrated water during the extraction process In any case, there is evidence that avenalumins are capable of diffusing into intercellular spaces Thus, as indicated by Daly [19], it is likely that the highly hydrophilic avenalumins could occur at the infected micro-site in a form able to inhibit growth of intercellular hyphae of crown rust

In case of cuticular infection by saprophytic fungal pathogens, avenalumins could also accumu- late in the infected epidermal cells into a high concentration which might inhibit the growth of some saprophytic fungal pathogens such as the Pyrzcularza fungi used in this study Studies on the role of avenalumin production in other diseases of oats other than crown rust are desirable.

CHAPTER VI

MICROSPECTROPHOTOMETRIC ANALYSIS O F CELLULAR LOCATION O F AVENALUMIN IN OAT LEAVES IN RESPONSE TO FUNGAL INFECTION

In the previous Chapter V, it was demonstrated that the amount of accumulated avenalumins was linearly correlated with the infection rate through stomata exceeded the inhibitory level for fungus growth in the infected stomatal area However, the exact distribution of avenalumins in the infected stomatal area was not examined The present investigation, using a microspectropho- tometric method monitoring the fluorescence emission and u v -absorption spectra characteristic of avenalumins, was carried out to detect the phytoalexins directly within cells in the infection court. The location of avenalumins, accumulated in the epidermal cells in response to cuticular infection by the saprophytic fungal pathogen, P oryzae, was also analyzed by spectrophotometry.

MATERIALS AND METHODS

Plants and fungz 'The cultivar of oat used was Shokan 1 The plants were grown on vermiculite

and Fday-old seedlings were inoculated with uredospores of P. coronata f sp avenae races 203 and

226 as stated previously As described in the previous Chapter V,the seedlings were also inoculated

with conidia of P oryzae which is a nonpathogen of oat, but is capable of invading the epidermal

cells by cuticular penetration. Inoculated plants were incubated for 16 hours under moist condi-

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tions and grown thereafter in a growth chamber a t 20 to 21°C under illumination of 1 0 , 0 0 0 1 ~ ~ for 16 hours with fluorescent lamps Preparation o j znfected leaves Fresh or fixed samples of crown rust-infected leaves were used for preparation of thin sections Fresh infected leaves were sectioned a t given times after inoculation by a Lab-line Hooker Microtome; about 30-40 pm thick sections were prepared Infected leaves were fixed in 3% glutaraldehyde in 0 1 M phosphate buffer, pH 7 2 for 2 hours a t 4°C and embed- ded in Spurr resin after dehydration with ethanol Sections (10pm) of fixed leaves were made with a Sorvall MT-2 ultramicrotome using glass knives In leaves infected with P. oryzae, the infected epidermal cells were peeled off from the leaves Sections of fixed samples were floated on a drop of water on a quartz slide and heated over the flame of an alcohol lamp in order to stretch them out, and then mounted in glycerol The fresh sections and stripped epidermal cells were placed on a quartz slide as soon as the specimens were prepared without use of any mouting solution When necessary, the fresh tissues were extracted with hot methanol and the loss of fluorescence emission as well as u. v -absorption was measured as described below Microspectrophotometry The monochromatic spectral analysis of u v -absorbance was made with a Zeiss u. v -scanning microscope photometer 03 with a Zeiss XBO 150 W/1 power supply The fluorescence emission spectra were also measured by the Zeiss photomicroscope equipped with an epifluorescence condenser I11 RS and a mercury high-pressure lamp, HBO 100 W/2 using a Zeiss HBO 100 W/2 power supply The filter sets used for incident-light fluorescence microscopy were as follow: (1) G365 excitation filter with peak transmittance 340-370 nm, chromatic beam splitter FT 395 and barrier filter 470; (2) BP 400-440 excitation filter with peak transmittance 400-440 nm, chromatic beam splitter FT 460 and barrier filter 470; (3) BP 450-490 excitation filter with

peak transmittance 450-490 nm, chromatic beam splitter FT 510 and barrier filter LP 520; (4) BP 510-560 excitation filter with peak transmittance 510-560 nm, chromatic beam splitter FT 580 and barrier filter 590 The fluorescence emission and u v -absorption spectra of crystals and aqueous solutions of authentic avenalumins I, I1 and I11 were first characterized on a quartz slide by the photomicroscope The sites absorbing u v -light as well as emitting fluorescence were surveyed in the prepared infected tissues and the u v -absorption and fluorescence emission spectra were compared to those of avenalumins Microphotographs for fluorescent and u v -absorbing images were taken using a Kodakolor I1 and Kodak Tri-X pan film, respectively Estzmation of avenalumins Extraction, separation and quantitation of avenalumins were carried out as described in Chapter I1 Observation oj jhorescent collapsed cells The primary leaves of Shokan 1 infected with races 226 and 203 were fixed with an alcoholic lactophenol solution and stained with the Calcofluor brightner for 10 minutes to visualize the penetration points of crown rust as described in Chapter 111. The formation of fluorescent collapsed cells in the infection courts was observed by the Zeiss fluorescence microscope, using a BP 400-440 excitation filter (peak transmittance 400-440 nm) and chromatic beam splitter FT 460 and barrier filter LP 470.

RESULTS

Fluorescence and u v -absorption of avenalumzns The fluorescence emission spectra of the crystals

as well as aqueous solutions of avenalumins I, I1 and I11 were about the same as those of avena-

lumins dissolved in pure methanol as described in Chapter I1 (Fig. 14). Crystalyzed avenalumin

I was fbund to be strongly fluorescent to white blue, emission maxima 490-500 nm, and yellow,

emission maxima 510-520 nm, for the filter sets 1 and 2, respectively (Fig. 14a) The aqueous

solution of 1000,ug per ml also emitted fluorescence with the same emission maxima under both

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Wavelength (nm) Wavelength (nm) Fig 14 (a) Fluorescence spectra of avenalumin I in aqueous (0) and crystal (a) forms and (b) avenal-

umins I1 (@) and 111 (0) in crystal form Filter sets 1 (----), 2 (-), 3 ( ) and 4 (-.-.-) transmit wavelengths of 340-370,400-440,450-490 and 510-560 nm respectively.

filter sets Avenalumin I1 in crystal form was much more fluorescent; the emission maxima were 480-490 nm and 510-520nm under filter sets 1 and 2, respectively (Fig. 14b) However, fluores- cence of avenalumin 111 in crystal form was rather weak for both filter sets Avenalumins I, I1 and 111 were poorly fluorescent with the filter sets 3 and 4 which transmit longer wavelengths (Fig 14a, b) Among the avenalumins, avenalumin I1 was most strongly fluorescent and avena- lumins I and I1 were found to be major fluorescent compounds under the filter sets I and I1

The u v -absorption maxima of avenalumins I, I1 and 111 in aqueous solutions placed on a quartz slide glass were observed to be about the same as reported in pure methanol solution in Chapter I1 The absorption maxima of avenalumins I , I1 and I11 were 310-340nm, 320-350nm

and 330-360 nm, respectively (Fig 15) Mlcrospectrophotometrlcal observation of infected leaves A survey of the cells showing the fluores-

cence spectra characteristic of avenalumins under filter sets 1 and 2 was conducted in fresh and fixed Shokan 1 infected leaves. Fluorescent cells were formed only in mesophyll tissues a t infect. tion sites of the incompatible interaction to race 226. The intense fluorescence appeared white blue and green yellow with filter sets 1 and 2, respectively (Fig 16a, b), whereas the cells were observed to be very weakly fluorescent using filter sets 3 and 4. The emission maxima of fluorescence from the cells were 490-500 nm and 510-530nm for filter sets 1 and 2, respectively (Fig. 1%); these were close to those from authentic avenalumins At 24 to 28 hours after inocula- tion, autofluorescent cells often appeared intact and the intensity of autofluorescence was low. At 36 to 48 hours after inoculation, the shape of the fluorescent cells became irregular and the cells collapsed away from the surrounding cell walls and the fluorescence intensity increased The autofluorescent cells absorbed u. v -light of 320-340 nm as did authentic avenalumins (Figs. 18b and 19). No fluorescent and u v.-absorbing collapsed cells were found in the compatible interac-

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0.05 -

0.04 -

Wavelength (nm)

Fig. 15. U. v.-absorption of avenalumins I (-), I1 (......) and 111 (----) in aqueous solutions. .- - a The aqueous solutions mounted between quartz slide and cover glasses were measured-

by a Zeiss UV-microspectrophotometer.

Figs. 16 & 17. Autofluorescen, l l lG~~phyl l and epidermal cells of oat leaves (cv. Shokan 1) infected with (Fig. 16) Puccinia coronata f. sp. avenue race 226 and (Fig. 17) Pyricularia oryzae respectively.

I a and b were irradiated by wavelengths of 340-370 nm and 400-440 nm respectively. Fig. 16, ~ 2 4 5 ; Fig. 17, x200.

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Wavelength (nm) Wavelength (nm)

Fig 18 (a) Fluorescence spectra and (b) u v -absorption spectra of autofluorescent mesophyll cells in oat leaves (cv. Shokan 1) infected with Puccinia coronata f sp avenue race 226 at 40 h after in- oculation. (a), filter sets 1 (0) and 2 (*); (b), fluorescent (0-0) and nonfluorescent (0-0) cells and n E (8--0)

tion with race 203 throughout the period of the vegetative development of the rust fungus. The rice blast fungus P. oryzae could penetrate directly into the epidermal cells of primary oat

leaves, but hyphal growth was restricted within the penetrated cells. Microspectrophotometry of the fresh epidermal strips showed that infected cells and often the cells adjacent to the infected ones were strongly fluorescent for filter sets 1 and 2 (Fig 17a, b) ; the emisson maxima from autoquores- cent epidermal cells were 490-500 and 510-530 nm which were close to those of avenalumins (Fig 21a). U v -absorption was found in the fluorescent cells at infection court of the incompati- ble interaction (Fig. 20), and the absorption spectra showed a maximum peak at 300-350nm (Fig 21b) Fluorescent and u v.-absorbing materials seemed to accumulate more in the infected cells than in the directly-adjacent cells (Fig 22) No other epidermal cells showed such fluorescence as well as u. v -absorption When the fresh peeled epidermis was soaked in hot methanol (50°C) for about 5 minutes, the intensities of fluorescence as well as u. v -absorbance were reduced by ca. 40% of the original (TablelZ), thus indicating the elution of the autofluorescent and u. v.-absorbing compounds However, some autofluorescence still remained and the cells could still be observed as fluorescent. Healthy epidermal cells became fluorescent when the cells were injected with an aqueous solution (1000 ,ug/ml) of avenalumin I (Fig. 23)

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Figs. 19 & 20. U. v. microphotographs of infection sites of primary leaves of oat (cv. Shokan 1) infected with (Fig. 19) Puccinia coronata f. sp. avenue race 226 and (Fig. 20) Pyricu- laria oryzae, absorbing monochromatic lights of 300 and 320 nm wavelengths re- spectively. Fig. 19, x 600; Fig. 20, ~ 2 0 0 .

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Fig. 22. U.. v.-absorbance (-) and fluorescence (----) emission of infected and the ad,jacent epidermal cells of oat leaves (cv. Shokan 1) inoculated with Pyr icular ia or:yzae at 30 h after inoculation.. IC, infected cell; Ad-1, -2, -3, adjacent uninfected cells.

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Table 12 Effect of methanol treatment on the intensity of autofluorescence of var- ious sites in epidermal cells of oat leaves (cv Shokan 1) infected with Pyr icular ia or y zae

-- ~ ~~

Relative fluorescence % reduction Cellular site - - .. .. - -- - . ..

Untreated MeOH-treated in fluorescence

Infected cells 62 36 42

Uninf'ected cells 14 14 0

Uninfected guard cells 23 22 5 - - - - . -- - - -- -

Wavelength (nm)

Fig 23 Fluorescence emission from oat epidermal cells injected with an aqueous solution of avenalumin I (1000,ug/mI) The spectra were observed under filter set 2 Treated ( 0 ) and non-treated (0) cells

DISCUSSION

The comparison of zn vzvo u v -absorption and autofluorescence spectra with zn vidro spectra of authentic avenalumins strongly suggests that avenalumins accumulate locally within infected and the adjacent fluorescent cells found at infection courts in oat leaves inoculated with crown rust or the rice blast fungus The results support previous conclusion of Chapter V that avenalumins may accumulate locally to a level inhibitory for fungal growth in the infected stomatal area The previously-estimated concentration in a n infected stomatal area was calculated with the inclusion of the nonfluorescent cells as well as air spaces in the micro-site; thus, the concentration in infected and adjacent fluorescent cells must be higher than that previously estimated. Preliminary work

showed that the formation of autofluorescent collapsed cells was more rapid and more extensive in highly resistant oat-rust fungus interactions than in moderately resistant interactions; thus, the degree of fluorescent cell formation seems to correlate with the amount of accumulated avena-

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lumins since the latter also paralleled the degree of resistance in various oat-rust interactions as shown in Chapter 111 It has been shown in P mnfestans-potato cultivar interactions that the amount of rishitin accumulated in a dead collapsed cell is about the same in the various incom- patible interactions [25] It is not known yet, however, whether the amount of avenalumins located in an autofluorescent collapsed cell is the same regardless of the various PC genes control- ling the response to crown rust infection

The weakly fluorescent cells observed in rust-infected leaves during the early stage of infection did not seem to be collapsed but fluorescent cells were found to be fully collapsed 36 hours after inoculation It is not known, however, if the fluorescent collapsed cells observed in this study were dead Tomiyama [26] and Sato and Tomiyama [ 1051 have emphasized that rishitin accumulated exclusively in the infected dead cells as a result of secretion from the adjacent live cells where rishitin was actively synthesized

The use of fluorescence and u v microspectrophotometry has been shown in other situations to be usefull in accurately locating inhibitory compounds within infected tissues [50, 73, 791 Mans- field et a1 [73] demonstrated fluorophotometrically that wyerone and wyerone acid were produced in living cells in bean leaves infected with Botryt~s clnerea Holliday et a1 [50] observed that the microscopic fluorescence of glyceollin was identical in color to the autofluorescent materials in soybean leaves and suggested that autofluorescence of the collapsed cells could be attributed to the accumulation of glyceollin In incompatible powdery mildew-barley interactions, epidermal and mesophyll cells undergoing collapse fluoresced [ 78, 1291 and unknown fluorescent antifungal compounds were isolated from them [ 1281

In oat leaves infected with Erys~phegramlnls f sp avenue, Kidger and Carver [63] demonstrated recently extensitve autofluorescence in the epidermal cells responding resistingly to the fungus. Since the present study suggests that avenalumins accumulate in the fluorescent epidermal cells of oat leaves invaded by fungal pathogens, it is most likely that the production of avenalumins, the phytoalexins of oat, is involved in resistance expression of oat leaves against powdery mildew infection.

CHAPTER VII

INDUCTION O F AVENALUMIN PRODUCTION BY BIOTIC AND ABIOTIC ELICITORS

The results obtained in the previous chapters strongly suggest that the production of avenalumins is directly involved in the varietal resistance of oat cultivars to crown rust races This conclusion may naturally lead to the next interesting problem of the recognition mechanisms that regulate differential avenalumin accumulation

Fungal metabolites regarded as recognition factors have been obtalned In 1968, Cruickshank and Perrin [ 171 first detected a fungal metabolite, designated as Monilicol~n A, from the fungus Monlllnza fiuct~cola, which was active at low molarities in inducing formation of phaseollin by seed cavities of bean pods Keen [57] reported in 1975 that culture filtrates from avilurent races of P megasperma f sp glyclnea stimulated the accumulation of more phytoalexin glyceollin in resistant

than susceptible soybean hypocotyls Based on these facts, he postulated the "elicitor hypothesis"

that suggests the presence of a specific inducer active only on host cultivars resistant to the avilurent

races. Kuc et al. [68] also found the active inducer of rishitin production from fungal mycelium

of P znfestans. On the contrary, Doke et a1 [24,26] and Oku et a1 [91] recently reported, besides

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elicitors, the presence of fungal metabolites which could suppress phytoalexin production It has been known that phytoalexins can be also produced by the ions of some heavy metals

such as mercury and copper. It is believed that the metals could cause cell damage and the live cells adjacent to that damaged ones were irritated to produce phytoalexins [45,46] The experi- ments described in this chapter provide some characteristics of avenalumin production by biotic and abiotic elicitors such as wall materials from the crown rust fungi and heavy metals

MATERIALS A N D METHODS

Plants and fungz The oat cultivars used for the present study were Shokan 1 and the PC lines 35, 38 and 50 The primary leaves of the 8-day-old seedlings were obtained as described previously. The lower epidermal layers of the leaves were peeled away and exposed mesophyll cells of the 5 cm long-leaf segments were treated by either the uredospores of crown rust fungus race 226 or various elicitors described below Fifteen treated leaves were placed on a wet filter paper in a Petri dish (9 cm in diam ) and kept at 20°C with light illumination of 6000 lux, unless otherwise stated The uredospores sprayed on the mesophyll tissues germinated during incubation under moist condition Extractzon of bzotzc elzcztor fiom fungal walls o j crown rust fungus The uredospores of race 226 (10 g) were floated for germination on water kept in plastic trays (30x 70x 5 cm) for 5 hours The germinated spores were homogenized in a mill with 60ml of 005 M acetate buffer, pH 4 5; then the homogenate was sonicated a t 180 mA for 30 minutes The sonicate was centrifuged at 20,000 g for 30 minutes The pellet was washed twice with the acetate buffer The fungal walls were used in two isolation methods First, according to the method of KuC et a1 [ 681, the fungal walls (equivalent to 5 g fresh spores) were suspended in 60 ml of 0 05 M borate buffer, pH 8 8, and the suspension was heated under pressure at 120°C for 1 hour The heated fungal materials were then centrifuged at 2000g for 30 minutes and a clear supernatant was obtained The pellet was sus- pended in the buffer and recentrifuged to obtain the supernatant This process was repeated twice and the combined supernatant of 200 ml was dialyzed against 6 changes of 5 liters of water The dialyzed elicitor fraction, elicitor-K, was stored at -20°C

Another method of Ayers et a1 [4] was also used to isolate elicitors from the fungal walls The homogenized fungal materials from 5 g spores which were previously washed with the acetate buffer were successively washed with 100ml of water and 4 changes of 100 ml of chloroform-methanol

1 : 1 v ) The wall materials were then washed with 4 changes of 100ml of acetate and these purified fungal walls were air dried The purified walls were suspended in 100ml of water, and the suspension was heated under pressure a t 121°C for 3 hours The solubilized fraction was sepa- rated from the wall residue by centrifugation, concentrated to 20ml lit vacuo and dialyzed against water The dialyzed wall fraction, elicitor-A, was stored at -20°C The germination fluids of 5 g rust uredospores were concentrated to 20ml, followed by filtration through a Millipore membrane filter (0 45 pm) Fractzonatzon of wall-released fiactlon Fractionation of the heat-solubilized elicitor was conducted according to Ayers et a1 [4] A sample of the wall-released elicitor was applied to a column (1 O X 25 cm) of DEAE-cellulose which had been equilibrated with 10 mM potassium phosphate, pH 8 0 The column was washed with lOOml of the equilibration buffer, and the bound materials were

eluted from the column by 0 1 M NaCl in the same buffer The wall-released fractions were sepa-

rated into two, void and bound, fractions The protein content of the elicitors and the void and

bound fractions was determined by the method of Lowry et a1 [711 and the total carbohydrate

content in the fractions was estimated by the method of Dubois et a1 [ 301

Bzoassay ofelicrtor acrlvity for avenalumzn productzon Wall-released elicitors and the ions of copper

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and mercury of 10-90 lo-" M were assayed to determine their activities in avenalumin accumula- tion The elicitors were applied to each of a set of 15 leaf segments by floating the exposed mesophyll side onto the solutions. The treated leaves were incubated for 28 to 36 hours at 100% humidity at 20°C under light illumination of 2 , 0 0 0 1 ~ ~ Quantitative estimation of avenalumins in the elicitor-treated leaves was carried out as described in Chapter I1 It was also examined, by the t I c -plate bioassay, whether avenalumins could be the major antifungal compounds produced by oat leaves in response to the elicitors

RESULTS

Presence of b~otlc elrcltor In rust fungal walls The exposed mesophyll cells accumulated a large amount of avenalumins in response to the germinated rust spores of race 226 (Table 13) The experiment was then carried out to determine whether an elicitor for avenalumin production was present in the fungal walls of the germinated spores. The elicitors-A and -K showed clear activity to produce avenalumins in oat leaves (Table 14)

The methanol extracts of the leaves treated with elicitor-K were separated into three fractions by the Sephadex LH 20 column chromatography and each fraction was supplied for the t 1 c plate bioassay The result showed that avenalumins were induced, as major antifungal compounds, in oat leaves treated with the biotic elicitor (Fig 24) Another antifungal compound, B1 (cf Chapter II), was also induced more by the elicitor treatment, but the antifungal activity was inferior to that of avenalumins. Some characteristzcs of avenalum~n product~on by b~otlc elicitors The production of avenalumins in the leaves treated with biotic elicitors was dependent on the varying amount of elicitor (Fig 25) The accumulation of avenalumins increased almost linearly in response to the varying amount of

'Table 13. Induction of avenalumin accumulation by direct inoculation ofthe us- edospores of Puccinia coronata f sp avenae race 226 on mesophyll cells of epider mis-str ipped oat leaves

Avenalumin (pg/g fresh wt) * Cultivar Infection type

I I1 111

Shokan 1 (non-treated) Shokan 1 0 PC 50 0 PC 35 2 PC 38 4

* Estimated at 28 h after inoculation tr =trace

Table 14. Accumulation of avenalumins in epidermis-stripped oat leaves (cv. Sho- kan 1) treated with biotic elicitors

--

Concentration Avenalumin (pg/g fresh wt)* Tr eatment (pg glucose

equivalent) I I1 I11

Water contr 01 - 24 tr tr

* Estimated at 32 h after treatment. tr =trace

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Avenal

Avenal

umi

.mi

I C I C I C

1 2 3 Fig. 24. T I c -plate bioassay for antifungal activity of methanol extract from biotic elic-

itor-K-treated leaves against the germination and germ-tube growth of the uredo- spores of Puccinia coronata f sp avenae. 1,2,3: No. of fractions separated through Sephadex LH 20 column chromatography. I: elicitor-treated, C : untreated control Antifungal spots in the elicitor-treated of Fr. 3 are avenalumins and one in Fr 2 is unidentified Bl

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the elicitor-A A maximum amount of avenalumins was produced in the mesophyll cells in re-

sponse to the concentration of 3 to 4 mg glucose equivalents per ml In these experiments, absolute

amount of the biotic elicitor loaded on the leaves could not be estimated exactly; however, if the leaves were treated by the elicitor of 1 mg glucose equivalent per ml, the amount of elicitor supplied for the exposed mesophyll cells would be almost 5 pg glucose equivalent per cm2 of the leaf surface

The accumulation was dependent on the time after treatment (Fig 26) The mesophyll-exposed

Concentration (mg/ml)

Fig 25 Effect of concentration of biotic elicitor-A on avenalumin accumulation in epidermis-stripped oat leaves (cv Shokan 1). Avenalumins I ( 8 ) and I1 (0) were estimated at 28 h after treatment.

I lme after treatment (h)

Fig 26 Time course of accumulation of avenalumins I(.) and I1 (0) in ep- idermis-stripped oat leaves (cv Shokan 1) treated with biotic elicitor- A with the concentration of 1 mg glucose equivalent pex ml (-): elicitor-treated; (- - --): contr 01 (water-treated)

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leaves were treated with elicitor-A of 1 mg glucose equivalent and the content of avenalumins was estimated at various time intervals after treatment Avenalumins were detectable a t 12 hours after treatment and the level of avenalumins increased gradually over 60 hours. The level of avena- lumins in the untreated leaves was much less than that in the treated and gradually decreased after 36 hours post-treatment.

The activity of the biotic elicitors for avenalumin production was not specific, however, to the disease reactions in normal oat cultivar-rust race interactions (Table 15) The mesophyll-exposed leaves of the cultivar PC 38 accumulated a large amount of avenalumins in response to the biotic elicitor-A from race 226 by the application of 5 pg glucose equivalent per cmZ of the leaf surface, even though their normal interaction is compatible and little accumulation of avenalumins is observed in the race 226-infected leaves

Table 15. Non-specific elicitation of' avenalumin accumulation in oat cultivars, Shokan 1 and PC 38, by biotic elicitor-A originated from Puccinia coro- nata f: sp, avenae race 226

Infection type Avenalumin (pg/g fresh wt)* Cultivar to race 226

I I1 I11

Shokan 1 0

* Estimated at 36 h after treatment. tr=trace

Fraction Number

Fig 27 DEAE-cellulose chromatography of elicitora released from germinated uredospores of Puccinia coronata f sp avenae (a -a): Sugar (490 nm), (@---a): Protein (750 nm), (0-0) and (0---0): U. v.-absor bance at 260 and 280 nm respectively.

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Fractionatzon of wall-released elicitor Elicitors-A and -K isolated from 5 g each of fresh uredospores contained 36 and 92 mg of carbohydrate, and 79 and 248 mg of protein, respectively. The elicitor- A was separated into two fractions by DEAE-cellulose chromatography (Fig 27). The void frac- tion contained 54 and 15% of the original contents of carbohydrate and protein in the elicitor-A, respectively. The bound fraction contained 38 and 60% of total carbohydrate and protein in the same elicitor. Recovery of carbohydrate and protein of the elicitor-A after the chromatography was 92 and 7595, respectively

The void and bound fractions of the biotic elicitor-A were tested for their activity to induce avenalumin accumulation in the mesophyll cells (Table 16) Both the fractions induced the accu- mulation by the treatment with 1Opg glucose equivalent per cm2 of the leaf surface. At 48 hours after treatment, the void fraction seemed to stimulate the accumulation more than the bound frac- tion. Effects of abzotic elzcztor and llght illum~natzon on avenalumzn accumulat~on When the mesophyll cells were treated by the ions of copper and mercury a t concentration of lo-' M, no accumulation of avenalumins was observed; avenalumins were detected at concentration of M, but not stimulated to accumulate At concentration of lo-" M, slight stimulation of avenalumin accumu- lation was observed in comparison with the amounts of avenalumins in the water-treated leaves (Table 17). The biotic elicitor could stimulate the accumulation in the treated area, but not in the adjacent untreated tissue When the tissue was treated by M HgCl,, the treated area did not produce avenalumins, and the living tissues adjacent to the treated tissues could accumulate avena-

Table 16 Accumulation of avenalumins in epidermis-stripped oat leaves (cv Sho- kan 1) treated with void and bound fractions of elicitor-A separated by DEAE-cellulose column

Avenalumin (,ug/g fresh wt) * TI eatment

I I1 I11

Void fr .. * 241 161 tr

Bound fr * 185 128 tr

Water control 84 38 tr

* The concentration of each fiaction was 2 mg glucose equivalent per ml and avenalumins were estimated 48 h after treatment.. tr= trance

Table 17, Effect of heavy metals, abiotic elicitor, on avenalumin accumulation in epidermis-stripped oat leaves (cv Shokan 1)

Avenalumin (pg/g fresh wt) * Treatment

I I1 I11

HgCI, (10-2M) H ~ C I , (IO-~M) H ~ C I , (10 -"~) CuCI, (10-,M) CUCI, ( ~ o - ~ M ) CUCI, ( ~ o - ~ M ) Water contr 01

tr tr' tr tr tr ts tr

* Estimated at 36 h after treatment.. tr=trace

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lumins (Fig. 28). The production of avenalumins in the treated leaves seems to be dependent on light illumination

(Table 18). In mesophyll cells treated with the biotic elicitor and incubated in darkness, no obvious accumulation of avenalumins was detected though the stimulation of avenalumin accumulation was found in the elicitor-treated tissues under light illumination.

L,eaf sections (cm)

Fig 28 Location of avenalumin I accumulated in epidermis-stripped oat leaves (cv Shokan 1) treated with biotic elicitora (a) and abiotic elicitor HgCI, of lo-' M (b) Avenalumin I in the treated ( @ ) and adjacent untreated area (0) was estimated at 48 h after treatment

Table 18. Effect of light illumination on'avenalumin accumulation in oat leaves treated with biotic elicitor-A

Avenalumin (pg/g fresh wt)* Tr eatment

Elicitor-A* (light) 142 107 tr

Water (light) 73 59 tr

Elicitor-A (dark) tr tr tr

Water (dark) tr tr tr

* The concentration is 1 mg glucose equivalent per ml and avenalumins were estimated at 36 h after treatment. tr =trace

DISCUSSION

Elicitors of avenalumin accumulation were demonstrated in the wall-released materials from the germinated spores of crown rust fhngi. The t. 1. c, plate bioassay (Fig. 24) showed that the major

antifungal compounds induced in the mesophyll cells by biotic elicitors were also avenalumins as shown in the infected leaves.. These results as well as the fact of the induction of avenalumins by the abiotic elicitors suggest that avenalumins are synthesized in the host cells via the metabolic pathways which are activated after infection.

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The stimulation of avenalumin accumulation seemed to be dependent on the varying amounts of the elicitor; however, the stimulation rate was prevented by an excess of elicitor application (cf. Fig 25) This fact suggests that oat cells may have receptor sites for the wall-released elicitor.

The mesophyll tissues directly treated by lo-' M HgCl, did not accumulate avenalumins, but the neighboring live tissues were activated to accumulate the compounds This may indicate that the directly-treated tissues might be dead and not be able to produce avenalumins; however, the adja- cent live tissues might be activated for the production by the constitutive elicitor materials released from the injured, dead cells as postulated by Hargreaves and Bailey [46] The cells treated by

the biotic elicitor may not be dead and they are stimulated to produce the phytoalexins. Moesta and Griseback [81] examined the effects of biotic and abiotic elicitors on phytoalexin metabolism in soybean, and they observed that rates of synthesis of glyceollin as well as rates of conversion are very similar with the two types of elicitors From the relatively long half-time of conversion of glyceollin, they concluded that the accumulation of glyceollin isomers results mainly, if not exclusively, from the stimulation of their synthesis by the elicitors

Although the present results show only non-specific elicitor fractions, the existence of phytoalexin- eliciting metabolites specific to the incompatible interactions between oat cultivars and rust races is possible The methods used for separation of the elicitor fractions may not be so effective and elaborate as to detect a specific-elicitor, even it presented

An obvious mean of regulating specificity would be through selective stimulation or suppression of a key defence mechanism in oat plant Keen [57] has used the term, elicitor, to describe chem- ical signals that induce phytoalexin production in an incompatible response Keen and Legrand [ 611 recently demonstrated the data supporting the hypothesis that race specificity in the soybean P megasperma f. sp glycznea may be determined by specific plant rccognition of fungus surface glycoprotein. On the other hand, Oku et al [91, 1081 demonstrated the active suppressor of phyto- alexin production to account for the infection of pea leaves by Mycosphaerella pinodes Low molecular weight peptides were isolated from germination fluids and shown to suppress pisatin production by pea leaves in response to elicitors from the fungus The peptide suppressors also blocked phytoalexin production and thereby allowed colonization of the leaves by the normally non-pathogenic fungus Stemphyllzum sarcznaefrome Doke et al. [24,26] also demonstrated the suppressors of the hypersensitive reaction in the germinated fluids of P znfestans and postulated that race-cultivar specificity in the disease may be due to race-specific suppression of the hypersen- sitive response of host cells by fungal components rather than to specific elicitors of the hypersensi- tive response of host cells

Elicitor fractions have been also obtained from other rust fungi Hoppe et al. [51] reported that wall-released materials from the germ-tube of Uromyces phaseoll were effective as elicitors and induced the accumulation of the bean phytoalexin phaseollin, and that the infiltration of the elicitor into susceptible bean leaves induced protection against the bean rust fungus The elucidation of the mechanisms of specific recognition for avenalumin accumulation is absolutely important for understanding of the resistance in crown rust of oats.

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CHAPTER VIII

DETECTION O F ACCELERATED ACCUMULATION O F CINNAMIC ACID DERIVATIVES, THE PRECURSORS O F AVENALUMINS, AND THE AMOUNT O F AVENALUMINS

ACCUMULATED IN THE RUSTED OAT LEAVES

It is believed from the present study that phytoalexins are synthesized in oat plants after infection and play a n important role in the resistance of many plants against pathogenic fungi. Although a great number of studies on isoflavonoid and terpenoid phytoalexins are reported [66, 112, 114, 1331, little work has been done on the activity of the pathways of their synthesis in diseased plants Deverall [23] especially points out the lack of evidences that the biosynthesis of phytoalexins is initiated from remote precursors as distinct from conversion or hydrolitic release from close precur- sors and the role of particular enzymatic steps is evaluated in the pathways

Studies on the biosynthetic pathways of phytoalexins have been extensively conducted in the sweet potato root tissue infected with Ceratocystzs fimbrzata by Uritani and his colleagues [86, 87, 88, 92, 11 5, 1311. When acetate-2-14C, me~aIonate(MVA)-2-'~C, or far nesol-2-14C were applied to the infected tissue, the labeled compounds were incorporated into ipomeamarone The enzyme

system from acetyl CoA to 3-hydroxy-3-methyl-glutaryl(HMG) CoA, HMG CoA reductase [ 1151, and the enzyme system from mevalonate to isopentenyl pyrophosphate, pyrophosphomevalonate decarboxylase, were activated in the noninfected tissue adjacent to the infected region [86], pre- ceding the phytoalexin production in the infected region From these results, it was concluded

that ipomeamarone is produced by the acetate-MVA-farnesol pathway in the same way as sterol biosynthesis, and that the terpene biosynthetic system involving the acetate-MVA-farnesol pathway is activated in the sweet potato tissue in response to fungal infection In white potato infected with P znfestans, Sato et a1 showed that oxylubimin with a spirovetivane skeleton is a n intermedi- ate in the main biosynthetic pathway to rishitin with a noreudesmane skeleton, the phytoalexin of

potato [ 1031 On the other major flavonoid phytoalexin, Grieseback [41] demonstrated that the formation of

the basic flavonoid structure was derived from two metabolic pathways, the acetate-malonate and the shikimic acid routes Phenylalanine produced vza shikimic acid is deaminated by phenylalanine ammonia-lyase to cinnamic acid Cinnamic acid is then prepared for condensation with acetate units to form either a chalcone or its isomeric flavanone There have been abundant reports about the greatly enhanced activity of phenylalanine ammonia-lyase in relation to the production of flavonoid phytoalexins [43,97], and the enhanced rate of production of cinnamic acid was observed in infected plants where the isoflavonoid phytoalexin is produced

The most recent work of Gustine et al. [42] revealed the crucial terminal enzyme involved in biosynthesis of the legume phytoalexin, medicarpin They found that 0-methyl transferase activi- ty capable of methylating the 4'-hydroxyl of isoflavonoids increased following fungal infection, but not methyl transferases accepting hydroxylated cinnamic acid or flavones. This may suggest that a selective increase occurs in only that enzyme catalyzing the methylation of isoflavonoids

and this may be important in regulating induced medicarpin biosynthesis Recently Zahringer et

al, [ 1471 showed that the enzyme for prenilation of 6a, 3,9-trihydoxypterocarpan is inducibly

formed for the production of the immediate biosynthetic precursor of glyceollin isomers I1 and 111,

as a consequence of gene activation and de novo transcription and translation

In the case of avenalumin, the elucidation of the biosynthetic pathway is the future problems

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Based on the chemical structures of avenalumins I, I1 and 111 (Fig 4), however, it is naturally assumed that one of their direct precursors is the cinnamic acid derivatives produced vza shikimic acid pathway, that is,p-coumarate in avenalumin I, ferulate in avenalumin I1 and p-hydroxyphenyl- pentadienate in avenalumin 111 The results described in Chapter IV showed that a-aminooxyacetic acid, a n inhibitor of the synthesis of cinnamic acid, inhibited avenalumin synthesis extensively in oat leaves responding resistingly to crown rust These facts suggest that the synthesis of the phen- ylpropanoid compounds is deeply associated with the synthesis of avenalumins

In this chapter, it was examined if the formation of the cinnamic acid derivatives, the precursors

of avenalumins, could be activated in the incompatible oat-rust interactions. Also, the amount of the precursors detected in the infected leaves was compared to the amount of accumulated avena- lumins, to evaluate the participation of the compounds for the antifungal resistance.

MATERIALS AND METNODS

Plant,pathogen and inoculation The growth of oat plants and the inoculation with P coronata f.

sp avenue were done as previously described The oat cultivar Shokan 1 and the races 203 and

226 were used in this experiment Extractzon ojphenollc aczds Five grams of races 226- and 203-inoculated leaves and the uninocu- lated were used respectively for extraction with methanol The extract was filtered through a filter paper and then concentrated into a small volume under vacuum below 40°C. After eliminating undissolved materials by centrifugation, the extract was evaporated into dryness and the residue was solubilized in hot water Diethyl ether was added to the aqueous solution The aqueous phase was separated from the ether phase and extracted with ether after acidifing the aqueous solution to pH 2 0 with 1N HCl. The obtained ether phase was concentrated into a small volume and extracted with 5% NaOH solution The alkaline aqueous solution was acidified with IN HCI to pH 2 0 and re-extracted with ether Acidic compounds in the original extract were isolated in this ether phase. Analy szs and estzmation of phenollc aczds The acidic fraction was analyzed by high-pressure liquid chromatograph (HPLC), Tri-rotor 11, Japan Spectroscopic Co LTD and a gas chromatograph interfaced with a mass spectrometer (Hitachi CC-MS spectrometer M70) Phenolic acids which include the precursors of avenalumins were analyzed by HPLC with the CIS column a t pressure of 200 Kg/cm which gave a flow rate of 1 5 ml/min The solvent was methanol-water-acetic acid (40 : 80 : 5, V/V), which was modified after the report by Wulf and Negal [ 1391 Acetic acid was used in the solvent to suppress ionization of the acid group The standard curves of cinnamic acid, p-coumaric acid, ferulic acid and caffeic acid were prepared for qualitative and quantitative estimation of the compounds

The compounds in the acidic fraction were derivatized to trimethylsylylated compounds by N, 0 , -bis(trimethylsilyl)trifluoroacetamide and analyzed by GC-MS in the following conditions; col- umn OV-1 2% 1 m, column temperature 125 to 240°C, S°C/min

RESULTS

The enhanced accumulation of p-coumaric acid and ferulic acid, direct precursors of'avenalumins

I and I1 respectively, was confirmed in GC-MS analysis. As shown in Figure 29, the peaks of p-

coumaric acid and ferulic acid from race 226-inoculated leaves were higher than those from race

203-inoculated and the uninoculated when the same amount of each sample, equivalent to 40 mg

of' fiesh leaves, was used for analysis.. The peak indicated by the arrow at scanning number 210

shows the characteristic ions of'p-coumaric acid and the one marked by arrow a t scanning number

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2 1 9 x 3 0

2 4 9 x 6 0

B 2 9 5 x 3 0

3 0 8 X 3 . 0

3 2 3

3 3 8

T I C X O . 1

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

0 1 0 2 0 3 0 4 0

c Z 1 9 % 3 0

2 4 9 i 6 . 0

2 9 5 x 3 0

3 0 8 x 3 0

3 2 3

3 3 8

1 0 0 ZOO 3 0 0 4 0 0 5 0 0 6 0 0 S C A N NO

Fig. 29. Mass chromatograms of TMS derivatives of the acidic compounds from oat leaves (cv. Shokan 1) infected with Puccinia coronata f sp avenae races 226 and 203 Each sample equivalent to 40 mg fresh weight of leaves was supplied for Mass chromatography. A, B, C indicate uninocu- lated, race 203- and race 226-inoculated oat leaves (cv Shokan 1) respectively

310 shows the characteristic ions of f'erulic acid. A distinct peak specific to the incompatible interaction at scanning number 550 was considered to be a compound associated with avenalumin I, based on the characteristic ions.

In HPLC analysis, the errors of the standard deviations to the average values for the amount of phenolic acids were less than 3%.. The enhanced production of' the phenolic compounds was also indicated by HPLC-analysis, as found by GC-MS analysis (Fig. 30). In race 226-Shokan 1 leaves,

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Minutes

Fig 30 High-pressure liquid chromatography of the acidic compounds from oat leaves (cv Shokan 1) infected with Puccznla coronata f sp auenae races 226 and 203 The amount supplied for the chromatography was equivalent to 100mg fresh weight of leaves for the uninoculated and the race 203-inoculated, and 50 mg for the race226-inoculated Arrows, from left to wright, show the peaks corresponding to caffeic acid, p-coumar ic acid, ferulic acid and cinnamic acid re- spectivel y

Table 19 Amounts of phenolic acids and avenalumins I, 11,111 in oat leaves (cv. Shokan 1) infected with Puccinia coronata f sp avenae race 226

Time after inoculation (h) Compounds

36 42

Avenalumin I 183* 205 * Avenalumin I1

Avenalumin 111

Cinnamic acid tr tr

p-Coumar ic acid

Caff eic acid

Ferulic acid

* pg/g fresh wt tr =trace

the peaks corresponding to those phenolic acids were higher than those in race 203-inoculated a n d uninoculated leaves, even though the amount used for the analysis of the extract from race 226- inoculated leaves was a half of that used for estimation from the race 203-inoculated (Fig. 30).. The amounts of the precursors formed in the incompatible oat-rust interaction were roughly esti- mated by comparing with the peaks of their reference compounds.. Although the increased rate of

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the accumulation was found, the amounts detected were very low in comparison with those of avenalumins (Table 19); their maximum amounts would be estimated to be several pg per gram fresh weight of the race 226-inoculated leaves.

DISCUSSION

The possible involvement of aromatic compounds in disease resistance is covered in a number of reviews [33, 101, 1251 In the case of stem rust of wheat disease, Farkas and KirBly [33] reported that infected resistance varieties showed increased tissue concentration of phenols at a n earlier stage of disease development than did varieties regarded as susceptible Rohringer et a1 [99] and Fuchs et a1 [37] reported more active synthesis of certain aromatic compounds in infected wheat lines possessing the Sr 6 allele when compared to the corresponding susceptible near-isogenic line. However, Seever and Daly [ 1071 analyzed total, combined and bound phenols in leaves of healthy or inoculated near-isogenic lines of wheat carrying either the Sr 6 allele for resistance or the s r 6 allele imparting susceptibility to race 55 of P grarnlnls f sp trltcc!, and contrary to the previous reports, no significant difference in total phenolic compounds was found among healthy and inocu- lated, resistant and susceptible plants at any stage of disease development They concluded that, if aromatic compounds are involved in the expression of resistance controlled at this gene locus, such involvement cannot be demonstrated by techniques such as paper, thin-layer, or gas chro- matography, employed previously in studies of other host-parasite combinations

In the present study with crown rust of oat, however, the increase of the accumulation of the phenolic acids was detected in the incompatible interaction by the techniques of HPLC and GC- MS analysis. Although the amount of the compounds was very low, the promoted accumulation of the phenylpropanoids in the resistant tissues is considered to be linked with the production of avenalumins In addition, it has been shown in the previous Chapter IV that a n inhibitor of phen- ylalanine ammonia-lyase greatly reduced the production of avenalumins in the resistant tissues These results indicate the involvement of the shikimic route to synthesize the phytoalexins Also, acetyl CoA seems to be involved in the formation of 5-(p-hydroxypheny1)-2,4-pentadienoic acid, the precursor of avenalumin 111. According to Uritani's summary of the biosynthetic pathways of ipomeamar ones, the common character istic for the possible biosynthetic pathways is the involve- ment of acetyl CoA [ 1311 This seems to hold true for the production of avenalumin 111

The precursors of avenalumins I and 11, cinnamic acid and p-coumaric acid, showed about the same level of antifungal activity to crown rust as avenalumins, as described in Chapter 11. How- ever, the amounts of the precursors detected in the incompatible race-inoculated oat leaves were so small; thus, the result indicates that the largely-accumulated avenalumins in the infected leaves were major antifungal compounds and mainly responsible to the resistance of oat leaves to crown

r ust Since avenalumins are relatively simple compounds as phytoalexins, elucidation of the biosyn-

thetic and biodegradation pathways would be possible and desirable for understanding the mecha- nism of avenalumin accumulation in the incompatible oat-rust interaction.

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CHAPTER IX

GENERAL DISCUSSION

It was found in this study that oat plants could produce the phytoalexin, avenalumin, whose production was highly associated with the expression of resistance in oat leaves against the crown rust fungi Detailed evaluation of avenalumins in relation to resistance strongly indicates that avenalumins could be causal agents of the expression of genes for reistance against P. coronata infection The chemical structures of avenalumins (Fig 4) look very different from those of the previously-identified phytoalexins in other plant genera such as pterocarpans and terpenoids Avenalumins are water-soluble, nitrogen-containing phenolic derivatives and the first chemically- identified phytoalexins of cereal plants responding to obligate parasites

These facts suggest that the production of phytoalexin may be involved in the resistance mecha- nisms of Gramineous plants against parasitic fungi, though there have been some questions on the role of phytoalexin formation relating to resistance of Gramineous plants [23]. In this respect, the detection of avenalumins is very significant in regards to the generality of phytoalexin involve- ment in resistance to plant disease It would be interesting to look for whether a similar class of phytoalexin could be found in other cereal plants such as wheat and barley, since it has been generally known that plants within the same genus could produce phytoalexins whose chemical structures are usually similar [23, 661

Another noteworthy fact evidenced in this study is that avenalumins are produced in differential time and rate in response to the differential strength of various PC genes for resistance to P coronata f sp avenae This strongly suggests that the same mechanism of avenalumin production may be involved in the expression of the various resistance genes, and the distinguishing character of the various genes for resistance is rather attributed to differential recognition efficiency between oat cultivars and crown rust races

Recent works on the resistance mechanisms in gene-for-gene systems of some plant diseases have focused on the mechanisms that may determine specific recognition between the host plant and parasite interactions [27,28, 59,611 Although various kinds of theories on the recognition mech- anism could be considered, biochemical models for the recognition mechanisms should be accomo- dated with various genetical and biochemical events which have been found during the expression phase of resistance. Keen [ 591 postulated the "specific elicitor-specific receptor model", proposing that constitutive structural elements of the incompatible but not compatible pathogen race are recognized by specific receptor molecules in the incompatible host genotype According to Keen's interpretation, this model seems to elucidate most of the essential requirements for the biochemical models In fact, it was demonstrated that surface and/or extracellular glycoproteins from P megasperma f sp glycinea function as race specific defence elicitor [61, 1341 Keen and Legrand [61] obtained glycoproteins from isolated cell walls of several fungus races and observed that these gave race-specific phytoalexin elicitation in bioassays on soybean plants of various resistance genotypes Wade and Albersheim [ 1341 showed that the glycoprotein fraction from culture fluids

of P megasperma f sp glycznea races could protect soybean plants against infection by the compat-

ible fungus race, while no protection occurred by those from compatible races

On the other hand, the "specific suppressor model" has been proposed with P ~nfestans-potato

disease system Doke, Kuc and their colleagues 126, 271 isolated high molecular weight phyto-

alexin elicitor and low molecular weight glucan molecules from mycelial homogenates and

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germination fluids. They found that low molecular weight glucan molecules appeared to function a s race-specific suppressors of hypersensitivity and phytoalexin accumulation would be caused by the high molecular weight elicitor, unless the specific suppressor did not function.. It was therefore proposed that race-specific suppressors confer gene-for-gene specificty in the host-parasite system [26].. Oku et al.. showed the significance of' suppressor of phytoalexin production in infection of pea leaves by Mycosphaerella pinodes [91]. Ouchi et al. [94,95] reported the phenomenon referred as "aquired accessibility" and Ouchi and Oku [93] emphasized that the active suppression of host def'ence mechanism is a prerequisite f'or the establishment of pseudosymbiotic association between the host cell and parasite, a basis of' pathogenesis..

Although the previously-obtained wall materials from the germinated crown rust fhngus did not show the specific activity paralleled with the disease reactions, this does not exclude the possible involvement of specific recognition materials in the oat cultivars-crown rust interactions.. Further consideration of the methods of' isolation as well as extraction would be necessary to discuss the specific-receptor model in fhture.. However, the present study points out the important events in avenalumin production which have to be accomodated with the models of' recognition mechanisms in the interactions between oat cultivars and crown rust races. These facts are (1) there is no avenalumin accumulation during accomplishment of' susceptibility in any compatible combinations of' type 4, (2) there are intermediate types 0 to 2 in incompatible combinations, (3) the accumulation of avenalumins can be nonspecifically induced by various biotic and abiotic stresses.

In the elicitation of' avenalumin accumulation, it should be noted here that victorin which is a host-selective toxin essential fh the pathogenesis of Helminthospor ium victor~ae [85] could function as a n elicitor of' avenalumin accumulation only in the susceptible PC 2 oat genotype [79]. Since the PC 2 gene conferring toxin sensitivity and resistance to the crown rust fungus is considered to be either identical or closely linked [22], the preliminary results indicate that victorin may function by eliciting a plant def'ence mechanism by producing avenalumins ['79].. Also, it was observed that, unlike the crown rust fungus, H , victorihe is relatively insensitive to the oat phytoalexins in vitro. This may account for growth of H, victoriae in the susceptible oat leaves containing signifi- cant amounts of the phytoalexins.. These results imply that this disease system between victorin or H. victorihe and PC 2 oat line could have considerable significance in understanding, not only the nature of elicitation of' oat phytoalexin by victorin, but also a relationship between a host selective toxin and phytoalexin production. This will certainly be the next project to be studied..

The time schedule of the events in oat leaves, cv.. Shokan 1, in response to incompatible race 226 of' P. coronata f.. sp.. avenae has been summarized [ 1181. The changes of' RNA and protein metabolism of oat leaves following infection with compatible and incompatible races of P.. coronata f: sp. avenae have been extensively analyzed [ 117, 121, 122, 142, 1431. The results revealed that the activation of mRNA and protein synthesis by the plant is required f'or the resistance expression against incompatible rust fungi. Although the rapid accumulation of' phytoalexin avenalumins occurs after the activation of RNA and protein synthesis, the newly-synthesized RNA and protein have not been characterized yet in relation to the synthesis of avenalumins. As a possible biosyn- thetic pathway has been postulated (Fig. 29), the enhanced synthesis of phenylpropanoid precursors and involvement of' phenylalanine ammonia-lyase have been confirmed and thus support the

biosynthetic routes. Much effort should be made to analyze the enzyme that regulates the synthetic

route of avenalumins.

The role of avenalumin production in disease resistance should be extensively evaluated in other

fungal infections of' oat leaves.. Recently, Kidger and Carver [63] demonstrated extensive auto-

fluorescence in the epidermal cells of oat leaves responding incompatibly to E.. grumiizi:~ f'. sp. avenue.

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Since the present study suggests that avenalumins accumulate in the fluorescent epidermal cells of oat leaves invaded with fungal pathogens, it is most likely that the production of avenalumins, the phytoalexins of' oat, is involved in resistance expression of oat leaves against the powdery mildew infection.. On the other hand, a possibility of involvement of some other defence mechanisms in oat plants against parasite infections may not be ruled out Preliminary works revealed that

avenalumins could be produced by infection with saprophytic fungal pathogens. However, the

activation of the post-inhibitin, 26-DGAs, in oat leaves seem to be more effective for infection with these saprophytic fungal pathogens [I161 In case of infection by Pyricularia species, both the accumulation of avenalumins and the activation of 26-DGAs are most likely responsible for the restriction of fungal growth within oat leaves It is very unlikely, however, that avenalumins are involved in the induced resistance against Helmznthosporium species, because the pathogens are relatively insensitive to the avenalumins. Besides the phytoalexin production, the activation of 26- DGAs seems to cause the restriction of growth of intracellular hyphae of the Helmiizthosporium pathogens in oat leaves

Possible application of the basic research, as conducted in this study, to plant protection would always be desirable In the crown rust of oat, the use of resistant cultivars was one of the most successful means of controlling the disease, and it was found in this study that the molecular basis of observed resistance in the cultivars was due to the production of phytoalexin, avenalumins. The detection of avenalumins may be useful to devise screening techniques which enable the selection of the cultivars with the greatest ability to produce the antifungal compounds..

In facts, the idea of protecting plants against diseases by means of activating their natural resistance mechanisms has been discussed repeatedly [67, 102, 141, 1441. It has been pointed out

by Ayers et al. [5] that the elicitors of phytoalexin production may be potential biochemical candidates for the induction of protection.. Hoppe et al.. [51] isolated an elicitor fraction from cell walls of germinated spores of' U. phaseoli which after injection into bean leaves induced pro- duction of the phytoalexin phaseollin and resistance against the bean rust.. Also, the chemical means efficient in induced resistance were shown by Cartwright et al.. [ 141 .. A systemic fungicide, dichlor-dimethyl-cyclopropane carboxylic acid, increases the capacity of rice to synthesize phyto- alexins momilactones A and B in response to infection by P. oryzae and thereby seems to protect the plant against the rice blast. In the field, a type of induced resistance has been known as a so-called "cross protection" for the protection against subsequent inoculation as induced by pre- inoculation of' plants with nonpathogens or pathogens and as observed with fungi, bacteria, and viruses [67, 102, 1441 .. Recent reports by Kuc and his colleagues indicate that cucurbits can be immunized against viral, bacterial, and fungal diseases by infection with viruses, bacteria or fungi [67] .. It was demonstrated that lignification may have a role as a mechanism for the containment of' fungi in immunized plants [44].

As one of the possible applications, the use of'tissue culture techniques in the production of novel disease-resistant plants has been also thought for a long time after the demonstration that plants could be regenerated from cultured cells of tobacco or carrot [ 1361 . Especially in cases where a host-specific toxin is responsible for the pathogenecity of a fungal parasite, the induction and selection of toxin-resistance in vi'tro have been desirable [9, 12, 38, 39,40, 1231. Carlson [12]

demonstrated a selection of' tabacco cultures resistant to methionine sulfoximine from Pseudomonas

tabaci, the causal agent of' tobacco wildfire disease, and that the leaves of the plants regenerated

from the compound-resistant callus were insensitive to methionine sulfoximine or P. tabaci.

Gengenbach and Green [38] and Gengenbach et al.. [39] made a n important advance in the

selection of maize tissue cultures that are resistant to T-toxin of Drechslera maydis (Nisikado)

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Subram et Jain.. A high level of' resistance to T-toxin was selected in originally susceptible tissue cultures, originated from corns carrying Tms-cytoplasm, by the successive subculture of tissue on media containing T-toxin [ 38, 39,401 .. The regenerated plants resistant to the toxin were resistant to inf'ection with the pathogens.. Using the host specific toxin from Alternalih solani', the causal

agent of early blight disease of' potato, Matern et al. [76] also demonstrated the selection of toxin-insensitive and sensitive potato clones regenerated from single mesophyll protoplasts and enhanced resistance to the early blight disease.. Recently, more attention has been placed on the techniques fbr plant cell or tissue culture for exploiting the manipulation of plant genome a t the cellular level. It may be possible that a so-called biological or genetical engineering would be fruitful f'or plant breeding, such as promotion of' resistance, in the future..

The basic research on the defence mechanisms of' plants as described in this study may not be applicable immediately to actual protection of' oat plants in the fields. It is believed, however, that the studies of the molecular bases of' resistance and/or susceptibility in plants are basically important, not only for application of such bioengineering techniques to breed resistant plants, but also for addition of' continuous impact on the improvement of new divices f'or plant protection, in the never ending fight against diseases.

CHAPTER X

SUMMARY AND CONCLUSIONS

1. It was examined whether phytoalexin production is involved in resistance of oat (Avena satzva L..) against inf'ection of' Puccinia coronata f:, sp, avenae. The antifungal compounds accumu- lated only in the incompatible oat cultivar and crown rust race interactions.. Three major compounds were isolated and their antifhngal activites against oat crown rust were confirmed. They are regarded as phytoalexins and given the trivial names avenalumins I, I1 and 111.

2.. Avenalumins I, I1 and 111 were isolated and the chemical structures were elucidated. Avena- lumin I was 2-[2-(4-hydroxyphenyl)ethenyl]-6-hydroxy-4H-3,l-benzoxazin-4-one, avenalumin I1 was 2-[2-(3-methoxypheny1)enthenyll-4H-3, 1-benzoxazin-4-one. Avenalumin 111 was 2-[4-(4- hydroxyphenyl)-l,3-butadienyll-4H-3,l-benzoxazin-4-one.. Avenalumins are highly hydrophilic luminescent phenolic compounds. Avenalumins are the first nitrogen-containing phytoalexins found in plants and they are the first chemically-identified ones found in cereal plants in response to rust fbngi..

3.. Avenalumins I, I1 and 111 inhibited germination and germ-tube growth of the crown rust fungi to a similar extent. About 50% inhibition of germ-tube growth was obtained a t 200-250 pg/ml. No obvious difference in sensitivity was f'ound among crown rust races.. Synthesized avenalumins I, I1 and 111 were also antifungal at equal level to those of isolated avenalumins. The antifungal activity of' avenalumins was detected in the t.. 1.. c.. plate bioassay; the minimum amount detecting the activity was 5 pg per spot f'or each avenalumin..

4. The relationship between the degree of expression of resistance and the production of avenalumins was investigated in the interactions of 2 physiological races of the crown rust fungi

and 21 oat PC lines each carrying a difftrent single major gene for resistance. Avenalumins

accumulated in all incompatible interactions.. More rapid and abundant accumulation of' avena-

Iumins was f'ound in the more incompatible interactions where fungal growth was more rapidly

restricted.. Large accumulation of' avenalumins coincided with the time of' detection of retardation

of hyphal growth within leaf' tissues. Very little accumulation of avenalumins was found in any

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compatible interactions It is suggested that the production of avenalumins is regulated as a result of resistance gene expression, and in the expression of the various PC genes a single mechanism dssociated with the production of avenalumins could be involved in restricting fungal development

5. The amount of accumulated avenalumins was linearly correlated with infection rate through stomata (R= 0 98); the regression line was Y = 23X, if the amount of avenalumins was dependent variable Y and the infection rate was the dependent variable X No avenalumins were detected in areas adjacent to or apart from the infected area It was thus considered that avenalumins accumulate locally in infected stomatal sites In highly resistant interactions, the estimated con- centration of avenalumins in an infected stomatal site greatly exceeded the inhibitory level for growth of germ-tubes from the uredospores Avenalumins were able to diffuse into intercellular spaces of infected leaves It is suggested that the highly hydrophilic avenalumins could occur a t the infected micro-site in a form able to inhibit growth of intercellular hyphae of crown rust.

6 Cellular localization of avenalumins accumulated in oat leaves infected with either crown rust through stomata or rice blast fungus through the cuticular layer was demonstrated by micro- spectrophotometry using the fluorescence emission and u. v-absorption spectra characteristic of avenalumins Thin sections of incompatible rust-infected leaves revealed collapsed mesophyll cells which emitted autofluorescence and absorbed u v -light with spectral characteristics close to those of the avenalumins Epidermal cells accumulated quite high levels of avenalumins in response to cuticular infection by the facultative pathogen Spectrophotometry of the freshly stripped epidermis showed the presence of autofluorescent materials, with spectra characteristic of avenalumins, in infected and the directly adjacent cells U v -absorption spectra close to those of avenalumins were also detected in fluorescent cells Extraction of epidermal strips with methanol reduced fluorescence emission and u v -absorption in fluorescent cells by ca 40% of the original, thus suggesting that avenalumics could be partially eluted out of the fluorescent cells Epidermal cells which were injected with aqueous solution of avenalumin I became fluorescent. It is suggested that avenalumins accumulate locally in fluorescent mesophyll and epidermal cells formed in oat leaves at infection sites of fungal pathogens

7 The rapid accumulation of avenalumins in incompatible crown rust-infected oat leaves, cv, Shokan 1, which occurred at 20°C was greatly inhibited when infected plants were grown a t 25 to 35°C. At 30°C, avenalumin accumulation was almost totally inhibited and much greater hyphal growth accurred than at 20°C When infected plants were grown at 20°C for 36 hours and then transferred to 30°C, most of the previously-accumulated avenalumins disappeared rapidly within 48 hours and hyphal growth continued. However, no subsequent hyphal growth resulted when the plants were transferred to 30°C a t 72 hours after inoculation This suggests that resist- ance of oat against crown rust could be affected by altering the m vzvo concentration of avenalumins and the rate of avenalumin biosynthesis a t 20°C is higher than that for degradation; at 30°C, however, either the biosynthesis of avenalumin was prevented or the rate of biosynthesis was far less than that of biodegradation

8 Treatment of plants with a-aminooxyacetate, a potent competitive inhibitor of phenylalanine ammonia-lyase, greatly inhibited the rapid accumulation of avenalumins in normally incompatible

interactions This was correlated with an increase in susceptibility of the host plants Significant

inhibition of avenalumin accumulation was observed 48 hours after inoculation when inoculation

was followed by application of the inhibitor a t lOpM, but much less inhibition of hypersensitive

host cell collapse occurred in the treated leaves It is suggested that the large accumulation of

avenalumins is crucial for resistance expression in oat leaves against a n incompatible crown rust

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race, and phenylalanine ammonia-lyase is involved in biosynthesis of the avenalumins. 9 There are avenalumin-eliciting metabolites in the fungal walls of the germinated spores.

The wall-released materials stimulated the production of avenalumins which were the major antifungal compounds accumulated in oat leaves treated with the biotic elicitor. No biotic elicitor specific to the disease reaction in oat-rust interactions was found, however Slight stimulation of avenalumin accumulation was observed in oat leaves treated with M HgCI, The production of avenalumins in oat leaves treated with the elicitors was dependent on the light illumination.

10 The enhanced accumulation of p-coumaric acid and ferulic acid which are direct precursors of avenalumins I and I1 respectively was confirmed by spectral analysis with a gas-chromatography interfaced with a mass spectrometer and a high-pressure liquid chromatography. 'The amounts of the cinnamic acids were, however, much inferior to those of avenalumins accumulated in the incompatible race-infected oat leaves The results indicate that avenalumins are major com- pounds active against rust development in oat leaves, and the biosynthetic pathway of the phenolic acids are involved in the formation of avenalumins

11 Based on the critical phytopathological evaluations of avenalumins, it may be concluded that avenalumins I, I1 and 111 are the phytoalexins of oats and the direct causal agents of resistance

of oats against the crown rust fungi

ACKNOWLEDGEMENT

The author wishes to thank Dr.. Masaki Yamamoto, Professor of' Kyoto University, f'or critically reading this article and valuable suggestions.. The author is grateful to Dr.. Akinori Ueyama, Professor of' Kyoto University and Dr. Hiroshi Fukami, Professor of Kyoto University, f'or their critical review of' this article..

The author extends his sincere gratitude to Dr. Toshikazu Tani, Professor of Kagawa University, f'or critically reading this article and continuous advice and encouragement throughout this study.. I am indebted to Dr. Tamio Ueno and Dr.. Hiroshi Fukami, Pesticide Institute, College of Agricul- ture, Kyoto University, and Dr.. Hiroshi Irie, Department of' Pharmaceutical Science, Nagasaki University, for their cooperation in the elucidation of' chemical structures of avenalumins I, I1 and 111, the phytoalexins of oat.. The author appreciates Dr. M.. D. Simons, Iowa State University, U. S. A , , and Dr.. D. E. Harder, Agriculture Canada Research Station, Winnipeg, Canada, for kind- ly supplying the oat seeds of PC lines. Sincere thanks are also extended to Dr.. Jiko Shishiyama, Kyoto University, Dr.. J.. M. Daly, University of' Nebraska, Lincoln, U.. S. A,., and Dr.. N. T.. Keen, University of' California, Riverside, U. S. A, , for their critical suggestions for preparation of this article.

The author appreciates Dr. Hiroyuki Yamamoto and colleagues in Laboratory of Plant Pathol- ogy, Kagawa University, for their kind cooperation throughout this study.

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97 Rathmell, W G 1973 Phenolic compounds and phenylalanine ammonia lyase activity in relation t o phytoalexin biosynthesis in infected hypocotyls of Phaseolus vulgarzs Physiol Plant Pathol. 3: 259-

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100 Rohringer, R , W K Kim, D J Samborski and N K Howes 1977 Calcofluor : an optical brightner for fluorescence microscopy of fungal plant parasites in leaves Phytopathology 67: 808-810.

101. Rohringer, R and D J Samborski 1967 Aromatic compounds in the host-parasite interaction. Annu. Rev Phytopathol 5: 77-86

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103 Sato, K , Y Ishiguri, N Doke, K Tomiyama, F Yagihashi, A Mur ai, N Katsui and T Masamune 1978. Biosynthesis of the sesquiterpenoid phytoalexin rishitin from acetate via oxylubimin in potato. Phy- tochemistr y 17: 1901-1902

104 Sato, N , K Kitazawa and K Tomiyama 1971 The role of rishitin in localizing the invading hyphae of Phytophthora znfestans in infection sites at the cut surface of potato tuber Physiol Plant Pathol. 1: 289-295

105. Sato, N andK Tomiyama 1976 Relationship between inhibition of intercellular hyphal growth of Phytophthora znfestans and rishitin concentration in the infected potato cells Ann Phytopathol. Soc. Japan 43: 598-600

106 Schonbeck, F and E Schlosser 1976 Preformed substances as potential protectants. In Physiologi- cal Plant Pathology Ed by R Heitefuss and P H Williams pp 653-678 Springer-Verlag, Belt in

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108 Shiraishi, T., H Oku, M. Yamashita and S Ouchi 1978. Elicitor and suppressors of pisatin induction in spore germination fluid of pea pathogen, Mycosphaerellapznodes Ann Phytopathol Soc Japan 44: 659-665

109 Sirnons, M D 1970 Crown rust of oats and grasses Monograph No 5 Amer Phytopath. Soc. Hef-

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111 Staples, R C and G H. Toenniessen 1981 Plant disease control John Wiley & Sons, New York,

339 PP 112 Stoessl, A 1980 Phytoalexins-a biogenetic perspective Phytopathol Z 99: 251-272 113 Stoessl, A , J R Robinson, G L Rock and E W B Ward 1977 Metabolism of capsidiol by sweet

pepper tissue: some possible implications for phytoalexin studies Phytopathology 67: 64-66 114 Stoessl, A , 3 B Stothers and E W B Ward 1976 Sesquiterpenoid stress compounds of the solana-

ceae Phytochemistry 15: 855-872 115 Suzuki, H , K Oba and I Uritani 1974 Occurrence of 3-hydroxy-3-methylglutaryl CoA reductase in

sweet potato Agr Biol Chem 38: 2053-2055

116 Tani, T and S Mayama 1982 Evaluation of phytoalexins and preformed antifungal substances in relation to fungal infection In Plant Infection Ed by Y Asada, W R Bushnell, S Ouchi and C P Vance pp 301 314 Japan Scientific Societies Press, Tokyo and Springer-Verlag, Berlin

117 Tani, T and H Yamamoto 1978 Nucleic acid and protein synthesis in association with the crown rust resistance of oat leaves Physiol Plant Pathol 12: 215-223

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119 Tani, T , H Yamamoto, T Onoe and N Naito 1973 Effect of temperature on susceptibility of seed- ling oat leaves to crown rust Tech Bull Fac Agr Icagawa Univ 25: 53-59

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122 Tani, T , M Yoshikawa and N Naito 1973 Template activity of ribonucleic acid extracted from oat leaves infected by Puccznza coronafa Ann Phytopathol Soc Japan 39: 7-13

123 Thanutong, P , I Furusawa and M Yamamoto 1982 Degeneration of plants from protoplast and dis- ease resistance 2 On the tobacco clones resistant to toxins from Pseudomanas syrzngae pv tabacz and Alternarza alternata tobacco pathotype Ann Phytopathol Soc Japan 48: 362 (Abstract)

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126 Tomiyama, K 1980 Resistance of potato against Phytophthora tnfestans with a special reference to mechanisms of hyper sensitive reaction Plant Pr otection 34: 2-8

127 Tomiyama,K , T Sakuma, N Ishizaka, N Sato, N Katsui, M Takasugi and T Masamune 1968 A new antifungal substance isolated from resistant potato tuber tissue infected by pathogens Phytopa- thology 58: 115-116

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Japan 44: 91-92 (Abstract)

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130 Trivedi, N and A K Shinha 1978 Production of a fungitoxic substance in rice in response to Drech- sIera infection Trans Brit Mycol Soc 70: 57-60

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132 Vance, C P , R T Sherwood and T. K Kirk 1980 Lignification as a mechanism of disease resistance. Annu Rev Phytopathol 18: 259-288

OLIVE 香川大学学術情報リポジトリ

133 VanEtten, H D and S G Pueppke 1976 Isoflavonoid phytoalexins in biochemical aspects of plant parasite relationships Ed by J Friend and D R T hrelfall pp 207-289 Academic Press, London

134 Wade, M and P Albersheim 1979 Race-specific molecules that protect soybeans from Phytophthora megasperma var sojae Proc Natl Acad Sci USA 76: 4433-4437

135 Wahlroos, 0 and A I Virtanen 1959 The precursors of 6-methoxy-benzoxazolinone in maize and wheat plants, their isolation and some of their properties Acta Chemica Scandinavica 13: 1906-1908

136 Wareing, P F and I D J Ph~llips 1970 The control of growth and differentiation in plants Per gamon Press, Oxford, 303 pp

137 Wood, R K S 1982 Active defence mechanisms in plants Plenum Press, New York, 381 pp. 138 Wood, R K S and A Graniti 1975 Specificity in plant diseases Plenum Press, New York, 354 pp. 139 Wulf, L W and C W Nagel 1976 Analysis of phenolic acids and Ravonoids by high-pressure liquid

chromatography i Chromato 116: 271-279 140 Yamamoto, M 1965 Suscept-pathogen interrelationships in potato late blight Tagungsberichte 74

Biochem Probleme Krank Pflanze pp 175-199 Aschersleben 141 Yamamoto, M. 1974 Potato late blight, with special reference to the resistance of potatoes to the in-

vasion of Phytophthora infestans Rev Plant Protec Res 7: 45-56 142 Yamamoto, H, H Hokin and T Tani 1977 Peroxidase and polyphenol-oxydase in relation to the

crown rust resistance of oat leaves Phytopathol 2 91: 193-202 143 Yamamoto, H , T Tani and H Hokin 1976 Protein synthesis linked with resistance of oat leaves to

crown rust fungus Ann Phytopathol Soc Japan 42: 583-590 144 Yarwood, C E 1956 Cross protection with two rust fungi Phytopathology 46: 540-544 145 Yoshikawa, M , K Yamauchi and H Masago 1978 Glyceollin: its role in restricting fungal growth

in resistance soybean hypocotyls infected with Phytophthora megasperma var sojae Physiol Plant Pathol 12: 73-82

146 Yoshikawa, M ,I( Yamauchi and H Masago 1979 Biosynthesis and biodegradation by soybean hy- pocotyls infected with Phytophthora megasperma var sojae Physiol Plant Pathol 14: 157-169.

147 Zahringer, U, J Ebel, L J Mulheirn, R L Lyne and H Grisebach 1979 Induction of phytoalexin synthesis in soybean dimethylallylpyrophosphate: tr ihydr oxy-pterocar pan dimethylallyl transferase from elicitor-induced cotyledons FEBS Lett 101: 90-92

148. Zimmer, D E and J F Schaffer 1961 Relation of temperature to reaction type of Puccinza coronata on certain oat varieties Phytopathology 51: 203-203.

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－ 63一

エンバク冠さび病の抵抗性発現における

アベナルミンの役割

真 山 滋 志

エンバクいびβ〃α；αJ′pαL）晶辟とエンバク冠さび病菌（ア〝CC～〝お〟′♂〝αぬf∴spαグβ〝α）レース間における特異

的抵抗性横構の解明を目的として，抵抗性発現時にさび病菌の発育を阻著する直接的要匪＝．こついて研究し，品種

特異的な生体防御反応と連動してフィトアレキシン（アベナルミンと命名）が生成されることを発見した．アベ

ナルミンの検出，単離精製および化学構造決定，抵抗性における役割，および生成経路などについて－・連の検討

を行い，エンバク冠さび病における特異的抵抗性発現機構について考察したその結果を要約すると次のとおり

である．

1．ェンバク品種勝冠1号は，冠さび病菌レース226に抵抗性であるが，レース203に対しては雁病性である

この品種・レースの組合せを用いて抵抗性発現時に産生される抗菌性物質の検索を行ったい 抵抗性および雁病性

感染葉，また無接種葉中の熱メタノーリレ抽出液をセファデックスLH20－カラムクロマトグラフィーにより分画

した．．各画分を濃縮し，その－・定量をシリカゲル薄屑クロマトグラ■フィーによって分画後（展開溶媒クロロホル

ム：メタノ1－ル：水＝65：35：10，V／v），薄層坂上に直接冠さび病菌の夏胞子を塗布し，温室で16時間培養して，

同菌の発芽および発芽管伸長に対する抗菌活性を検定した．その結果，抵抗性薬において顕著に増加する3種の

抗菌性物質が検出された同物質は健全菜ではまったく認められず，慣病性菜ではきわめて微量検出されるにす

ぎなかった．同物質はアベナルミン（Avenalumin）I，I王，IIIと命名された

2」冠さび病菌レース226を接種した勝冠1号初生薬を大虫に熱メタノールで抽出し，減圧浪縮後に少量のメ

タノールに溶解してセフ7・デソクスLH20カラム（長さ35cm）を用いてメタノ・－ルで溶出分画したい えられた

アベナルミンを含む粗画分を濃縮し，再度セ■ファデックスLH20カラムクロ・マトグラフイ1－（長さ120cm）に

よってアベナリレミンⅠ，ⅠⅠ，IH，をそれぞれ含む画分をえた各画分をさらにシリカゲル蒋層クロマトグラフィー

で分画して，アベナルミンI，ⅠⅠ，ⅠⅠⅠを単離した

単離されたいずれのアベナリレミンも他の多くの■7イトアレキシンと異なって水易溶性であり，かつ，紫外線照

射下で螢光性の物質であったノ．紫外線，赤外，NMR，およびMSスペクトル分析の結果，アベナルミンⅠは［2－

［2－（4－hydr－OXyphenyl）ethenyl］－6－hydroxy－4H－3，l－benzoxazin－4－One，■アベナルミンI‡は2－［2－（3－methoxy－

phenyl）ethenyl］－4H－3，1rbenzoxazin－4－One，アベナルミンIIIは2一［4－（4山hydroxyphenyl）－1，3－butadienyl］

・－4H－3，トb¢nZOXaZin－4－Oneと同定されたこれらの化合物は化学合成によって確認されたアベナルミンは

含窒素化合物として従来のフィトアレキシンとは異なるユニークなものであり，ムギ類で明らかにされた最初の

フィトアレキシンである

31アベナルミンⅠの水溶液は200／ノg／mJでエンバク冠さび病菌レース203および226の夏胞子の発芽を抑制

し，発芽管伸長を約50％阻零した“アベナルミンノHおよぴⅠⅠⅠも同程度の抗菌活性を示した．また，同濃度で

コムギ黒さび病菌およびイネいもち病菌の発芽および発芽管伸長を約50％阻審した．肌・方，アベナリレミンⅠ，ⅠⅠ，

1Ilを展開したシリカゲル薄屑板上で，冠さび病菌の夏胞子を直接塗布して抗菌活性を検討したところ，いずれ

も5／ノg以上で明確な阻止円が形成された

4・エンバク冠さび病に対する抵抗性迫伝子を単一・に保有するエンバクタc（P〟Cぐj乃メαCク′ク〃α払）系統21種と冠

さび病菌レース203および226との組合せを用いて，抵抗性の強さとアベナルミン蓄積との相関性について検討し

た．抵抗性の強さは感染菌糸伸長と感染型で判定したところ，抵抗性発現の強い組合せほどアベナルミンの苔碍

はより早くかつ大益であり，また，感染菌糸伸長の停止期とアベナルミンの蓄積時期もー・致した．しかし，雁病

性ではアベナルミンははとんど蓄療しなかった．レース226およびレース203に対して，まったく逆の反応を示す

エンバク品種でほ，抵抗性発現時にのみアベナルミンの啓砧が認められた．以上の結果は，アベナルミンの生成

が冠さび病菌に対する抵抗性迫伝子の発現と密接に関連していることを示すとともに，個々の抵抗性迫伝子の発

OLIVE 香川大学学術情報リポジトリ

ー64－

現には，アベナリレミン生成を誘導する単一・の機構が作動していることを示唆するものである．

5．．アベナルミンが抵抗反応と相関して産成される抗菌性物質であっても，感染部位とは異なる組織中に蓄顧

されたものであれば抵抗反応に直接関与するとは考えられないそこで，アベナルミン蓄積の局在性について調

べた，アベナルミンはエンバグ菓の接種部位に苦境するが，隣接の非接種部位ではまったく蓄硫されない．また，

接種部位での蓄積量は冠さび菌の気孔感染率に依存して直線的に増加し，生薬1グラム当りのアベナルミン〃g

鼻（Y）と気孔感染率（X）との間にY＝23Xの相関が認められた．したがって，アベナルミンは感染部組織に局

在して欝潰するものと考えられるレー・ス226感染勝冠1号葉において，接種36時間後における気孔感染率と蓄

積丑間の相関係数から，1偶の感染気孔部のアベナルミン兢を算出し，それを侵入菌糸の存在する容硫あたりの

温度で表わすと，約1，8り0〃g／Cm3以上の高温度に苗窮していると推定された、、したがって，感染部位の組織内

で充分抗菌的な浪度で存在すると考えられる．

顕微分光測光故によって，アベナルミンの紫外線吸収および螢光放射の波長特性をモニターして，感染組織中

での局在部位を調べたその結果，抵抗性葉の気孔部に形成される螢光崩壊細胞（過敏感壊死細胞）およびその

隣接細胞にアベナルミンと歎似の紫外線吸収および螢光放射波長が認められ，病原菌の被侵入細胞およびその隣

接細胞におけるアベナルミンの蓄積が示唆された．

6感染巽中のアベナルミンゐ蓄街並を温度処理およびアベナルミンの合成1乱害剤によって制御して，感染菌

糸の伸長に対する影響を調べた勝冠1号葉は温度感受性であって，レース226に対して，200cでは抵抗性で多

量のアベナルミンが蓄積されるが，300C

でアベナリレミンを蓄積させた感染斐を300Cへ移行すると，アベナルミンは速やかに減少し，感染菌糸の伸長が

促進した一・方，・アユニルアラニン・アンモニア・リアーゼ（PAL）の阻磐剤であるatアミノオキシ酢酸（10～

100′′M）水溶液を感染葉の切断茎より吸収させると，アベナルミンの寄掛ま約80％阻誓され，感染菌糸伸長が促

進された．しかし，過敏感壊死細胞の阻審はわずかであったこれらの事実から，病原菌を直接阻著する因子と

してアベナルミンの苔潰が重要な役割を果すものと推定した．

7“エンバグ品種勝冠1号薬にレース226およびレース203を接種後，経時的にアベナリレミンの定量を行ったと

ころ，抵抗性および雁病性茹とも病原菌が気孔を通過する接種10時間ごろからわずかに検出され，接種24時間後

まで少蒐ながら増加するしかし，接種28時間以後になると抵抗性菓で急激に増加するが，催病性では逆に減少

して検出できなくなるこのように，感染過程における蓄積の様相には接種後24時間こ1ろを境として，さび病菌

が気孔を通過後，柴肉細胞に侵入するまでの期間と柴肉細胞への侵入彼の期間の二相性が認められた螢光崩壊

細胞は第一・相瑚には形成されず，第二相期で抵抗性葉で形成されたこれらの様相はアベナルミンの蓄硫誘導磯

構の解明の手がかりとして重要であると思われる，

8さび病菌の菌体細胞壁抽出物質を・エンバク葉に塗布しても，主要な抗菌性物質としてアベナルミンの蓄撥

が認められた同壁成分のアベナルミン誘導活性は本来のエンバグ品種－レースの組合せに非特異的であったが，

アベナルミンの蓄積は壁成分の濃度および処理時間に依存したり また，アベナルミンの同処理敷こおける蓄積は

光照明下で顕著であった フィトアレキシンの非生物的エリシターとして知られる水銀，銅イオン処理によって

もアベナルミンほ蓄潰誘導されたが，その蓄積藁は少丑であった

9アベナリレミンの化学構造から，アベナリレミンの生合成過程には，シキミ酸経由のフェニルプロパノイド化

合物とアントラニル酸とが結合して生成されることが示唆される… 抵抗性感染葉においてアベナルミンⅠおよび

ⅠⅠの前駆物質であるダークマル酸，フェルラ酸の蓄積促進を確認した．また，α－アミノオキシ酢酸処理の結果から，

PALが同生成経路に関与することが示唆されたしかし，これら前駆物質の蓄私立はアベナルミンの蓄私立に

比べて，きわめて少なく，感染葉に生成される主要な抗菌性物男がアベナルミンであると確認された．．

以上の研究結果を総合して，エンバク冠さび病における特異的抵抗性発現機構において，フィトアレキシン・

アベナルミンの生成苗砧が主要な役割を演ずるものと結論づけた

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