Evidence for the involvement of ascochitine in phoma leafspot-wilt disease of Clematis

Physiological

and Molecular

Plant Pathology

(1994)

333

45, 333-348

Evidence for the involvement of ascochitine leafspot-wilt disease of Clematis

in phoma

G. R.

COLE*

SMITH*$,

*Department New Zealand (Accepted

of Plant

for publication

M. H. G.

MuNROt,

B. A.

and Microbial

Sciences and t Deparlment

FINERAN*

and A. L. J.

of Chemislry,

lJniversi&

of Canterbury,

Christchurch

I,

June 1994)

Ascochitine, a phytotoxic metabolite, was purified from cultures ofPhoma clematidina and identified by ‘H and r3C nuclear magnetic resonance spectroscopy and electron impact and chemical ionization mass spectroscopy. This toxin-induced electrolyte leakage from leaf discs of Clematis cultivars that were susceptible to fungal infection, while there was no significant electrolyte leakage from leaf discs of the cultivar most resistant to fungal infection. The level of ascochitine production in vitro by P. clemafidina isolates was related to isolate virulence. The fungal isolates could be characterized into two groups: (1) high virulence, high ascochitine. production, and (2) low virulence, low ascochitine production. Ascochitine was isolated from P. clemafidinn-infected leaf discs, indicating the toxin is produced in ho. Leaf tissues exposed to ascochitine solutions showed black flecking in proportion to the concentration of ascochitine. Scanning electron and light microscopy of infected leaves indicated that the fungal hyphae were well behind the necrotic zone in leaf spots, while transmission electron and light microscopy suggested that mitochondria and chloroplasts were the major organelles affected by ascochitine, although extensive cellular damage was evident. These observations suggest that ascochitine may be involved in the pathogenesis of P. clemnlidina against Clemafis, by killing plant cells prior to hyphae ramification through the necrotic tissue.

INTRODUCTION

Phomaclematidina(Thtim.) Boerema is the causal agent of leafspot and wilt of Clematis L. hybrids in New Zealand [26,27]. Clematis hybrids are ornamental climbers or shrubs, usually cultivated with emphasis on the colour and size of the flow,er which is comprised of sepalsrather than petals. The pathogen, originally identified as Ascochyta clematidinaThiim by Gloyer [12, 131 in the U.S.A., was transferred to the genus Phoma by Boerema in 1979 [4] on the basisof new definitions of the two genera [5]. Leafspot and wilt are both symptoms of the disease;wilting is usually the result of nodal rotting or stem girdling following hyphal extension into- the node f!rom the infected leaf [12, 13,271. Phytotoxins have been implicated in disease and symptom expression produced by a number of Phomaand Ascochytapathogens including A. fabae [22], A. pisi [2], A. imperfecta,P. herbarum and P. medicaginis[28]. The toxin ascochitine has been $ To whom correspondence should be addressed Experiment Stations, P.O. Box 86, Indooroopilly, 0885-5765/94/l

10333 + 16 008.00/O

at: David North Plant Q4068, Australia.

Research

0

Centre,

1994 Academic

Bureau

of Sugar

Press Limited

G. R. Smith et al. - 334 isolated from culture filtrates of A. f@ze [22] and A. pisi [2], and from pea leaves infected with Mycosphaerellapinodes ( = A. pinodes) or A. pisi [18]. Ascochitine (C,,H,,O,) is an ortho quinonemethide (azaphilone subgroup) and shows the chemical behaviour of this classof compounds [28]. It is selectively toxic to higher plants, germinating seeds,bacteria and fungi [22]. Brown necrotic lesions on Viciu faba coleoptiles were induced by 10 pg ml-’ ascochitine solutions [22], while Lepoivre [17] demonstrated that the susceptibility ofPisum sativum L. cultivars to fungal infection was related to the amount of electrolyte leakage from leaf tissue in&bated in 100 pg ml-’ ascochitine solution. Clematis hybrids display differential susceptibility to infection by P. clematidina [27]. Here we report on studies to determine if ascochitine is involved in development of diseaseand the pathogenicity of P. clematidina infecting Clematis.

MATERIALS AND METHODS Fungal isolates and Clematis cultivars Five P. clematidina isolates MT, HD, RC, EM and LB and five Clematis

cultivars Montana, Huldine, Rouge Cardinal, Ernest Markham and Lady Betty Balfour were as previously described [27].

Leaf disc pathogenici&

assay

Discs (16 mm diameter) were cut from washed, freshly harvested Clematis leaves, and floated abaxial side up on distilled water in Petri dishes maintained in a 25 “C (16 h) day, 15 “C (8 h) night cycle. Illumination was approx. 300 pE me2s-l. Spore suspensionsprepared as previously described [27] at 1 x 10’ conidia ml-’ were used to inoculate leaf discs which had been wounded by abrading 1 mm2 of the leaf surface with a sterile dissecting needle, as P. clematidina is a wound pathogen [27].

Toxin production

and isolation

The liquid medium for toxin production was essentially that of Oku & Nakanishi [22] except that @5g 1-l Difco yeast extract replaced the defined quantities of thiamine, pyridoxine, riboflavin and pantothenate. Samples of 250 ml medium in 500 ml ‘Erlenmeyer flasks were inoculated with two 5 mm discs cut from the growing edge of a 7-day old potato dextrose agar [PDA (Difco)] culture, and grown at 25 “C in the dark at 50 r min-l in a Gallenkamp INR-200 orbital shaking incubator. The toxin was purified from the culture filtrate as described by Lepoivre [17] using the method of Hald & Krough [14]. This involved filtering the contents of the flask (250 ml),‘acidifying the filtrate with 1N HCl to pH 3.0 and extracting with two 50 ml samples of chloroform. After phase separation the toxin was extracted from the chloroform with two 125 ml samplesof 0.4% (w/w) Na,CO,. The aqueous phase was then adjusted with 1N HCl to pH 3.0 and extracted with 150 ml of chloroform. The solvent was evaporated in vacua, the toxin crystals were redissolved in a minimum volume of methanol and then air-dried and weighed. Toxin was also extracted from infected leaf discs by finely grinding leaves in a minimum volume of distilled water,

Ascochitine

involvement

in phoma

leafspot

of Clematis

335

making the volume to 50 ml with distilled water, and isolating as above in proportion to the volumes involved. Toxin

identa$cation

Toxin sampleswere analysed by TLC on non-activated silica gel 1B (Baker-Flex) in either cyclohexane : chloroform : glacial acetic acid (7 : 2 : 1 by volume), or acetone :ethyl acetate: distilled water (5 : 5 : 2 by volume) [17]. The toxin was identified as ascochitine by U.V. spectroscopy in spectroscopic grade methanol, infra-red spectroscopy in fused KBr and Nujol, ‘H and i3C nuclear magnetic resonance (nmr) spectroscopy in CDCl, using a Varian XL-300, and by low resolution electron impact and chemical ionization massspectrometry using a Kratos MS80RFA massspectrometer. Toxin

assay

An electrolyte leakage assay was developed. Purified ascochitine was dissolved in acetone and diluted with freshly autoclaved water to a final solvent concentration of 5% (v/v). Initial studies indicated that ascochitine was soluble in ethanol, 2methoxyethanol and acetone, but only remained soluble after dilution with water when initially dissolved in acetone. A dilution seriesin 5 y0 (v/v) acetone was therefore prepared. The range of toxin concentrations that could be initially assayedwas limited by the low solubility of ascochitine. Leaf discs (16’mm diameter) were cut from mature Clematis leaves, randomized and placed adaxial side down in solution and submerged’ with an inverted Pasteur pipette. Each replicate contained approx. 170 mg of leaf tissue. Leaf discs were infiltrated by reducing the air pressure to about 2 cm Hg for two 5-min periods. The infiltrating solution was then replaced with 10 ml of fresh bathing solution and the vials were incubated at 25 “C in the dark and the conductivity of the bathing solution was measured in micro-Siemens (pS) (S = A V-’ or m-2 kg-’ s3A2) with a dip-type cell containing two platinum electrodes connected to a Triac conductivity meter. The cell constant was measured with a 0.01 N KC1 solution [I]. Five parameters of the assay were determined with leaf discs from the leafspot-wilt susceptible Clematis cv. Lady Betty Balfour. These were: (1) solvent effect; (2) effect of time of incubation on leaf tissueresponse; (3) tissueresponseto toxin concentrations; (4) assaysaturation concentration; (5) assayminimum concentration. Two parameters were determined with leaf discs from the wilt-resistant cv. Montana; (6) effect of leaf tissue weight; and (7) to establish if the assay would differentiate wilt-susceptible (Lady Betty Balfour) from wilt-resistant (Montana) tissue. The five Clenzatiscultivars were then assayed for sensitivity to ascochitine with the developed method.

Microscopy

Specimens for light microscopy were fixed in formalin-aceto-alcohol, dehydrated in a tertiary butyl alcohol series, and infiltrated and embedded in Paraplast. Sections were cut on a Jung microtome. Sections were stained with Azur II (50:50 Azur blue : methylene blue), or Pianeze III, [25] .. Samples for scanning or transmission electron microscopy were fixed, stained and examined as previously described [27]..

G. R. Smith et al.

336

Statistical analysis Experimental data were compared using one- or two-way analysis of variance, F-test or linear regressionanalysis. P values < 005 were considered significant.

RESULTS Leaf %sc pathogenici&

The size of the lesionswhich developed on the Clematis leaf discsfollowing inoculation with spore suspensionsindicated that fungal isolate MT was lessvirulent against all Clematis cultivars than the other four isolates (P < 001 ), and that all five fungal isolates were less aggressive when inoculated onto Montana leaf discs (P < O-01) compared with the other cultivars (Table 1). Isolates were ranked in order of most to least aggressive as follows HD > LB = RC > EM > MT based on overall aggressivenessagainst all cultivars, while comparison of overall lesion size on the cultivars gave a cultivar ranking from least to most resistant to leafspot as Huldine = Lady Betty Balfour = Ernest Markham < Rouge Cardinal < Montana.

TABLE Diamcfn.

of leafsspot lesion (mm)

1

on clematis leaf disGsinoculatedwith Phoma clematidina 7 days incubation

isolates after

Cultivar Fungal isolate

Lady Betty Balfour

LB MT EM HD RC Overall

11.9 22 68 13.2 8.6 85a

Montana

Ernest Markham

Huldine

Rouge Cardinal

3.9 2.2 67 5.7 3.8 45c

9.4 2.1 7.8 12.4 8.3 80a

86 4-7 63 140 9.9 8.6a

7.6 2.7 5.0 99 87 67b

Overall 8.3 a 2.8d 6.5 c ll.Oa 7-9 b

Data from 25 replicates of each combination were analysed by two-way ANOVA, and the means compared by Duncan’s New Multiple Range Test. The overall coefficient of variation ofthe experiment was 22.2%. The overall mean of each interaction is indicated for the isolates in the last column, and for the cultivars in the last row. The overall column indicates the relative virulence of each fungal isolate, e.g. isolate HD most virulent, MT least virulent. The overall row indicates the relative resistance of each cultivar to infection, e.g. Montana most resistant. Means followed by the same letter either &thin the last column, or within the last row are not significantly different at the P = 001 level.

Ascochitine production

Ascochitine was isolated from the culture filtrates of the five P. clematidina isolates. Initial results indicated that ascochitine production reached a maximum after 11 days in culture. Comparison of ascochitine production by the five isolates after 11 days indicated significant differences (P < 0.05) in the amount of toxin produced by the fungal isolates (Table 2). Isolates EM, LB and HD produced the most ascochitine,

Ascochitine

involvement

in phoma

leafspot

337

of Clematis

followed by RC and then minimal production by isolate MT. Ascochitine was also isolated from leaf discsinfected with P. clematidina [0*63 mg (SE 0.06) toxin per 1 g leaf tissue fresh wt], but not from uninfected leaf discs. TABLE In vitro

ascochitineproduction

(mg toxin

2

per 70 ml culkrejillrale)

by Phoma

clematidina

isolates afler

II days

LB Toxin

yield

190a

EM 191 a

HD

RC

MT

1%2a

163b

2.2c

Data from three replicates were analysed by one-way ANOVA. was &7 %. Means followed by the same letter are not significantly level by Duncan’s New Multiple Range Test.

The coefficient of variation different at the P = 005

Identijcation

The purified toxin had R, values of 0.53 in acetone-ethyl acetate-water and 0.25 in cyclohexane-chloroform-acetic acid on non-activated silica gel lB, and showed a yellow colour under white light and yellow-green colour under 365 nm monochromatic U.V. light, properties reported by Lepoivre [17] for ascochitine. The toxin had a carboxylic acid nature as evidenced by partitioning into the acidified phase during purification, decolourized potassium permanganate in acetone, was negative to Fehling’s solution, and reacted positively with ferric chloride, all of which are characteristics of ascochitine [22]. In addition the toxin displayed three absorption maxima (224, 289 and 420 nm) in methanol, corresponding well with the reported alcoholic solution maxima of 220, 286 and 415 nm reported for ascochitine [22]. The infra-red transmission spectra in Nujol and fused KBr were comparable with those obtained by Oku & Nakanishi [23] and Lepoivre [17]. The compound was positively identified as ascochitine by analysis of the ‘H and r3C nmr spectra which were identical to published data [S, 211, and confirmed by low resolution electron impact and chemical ionization mass spectra (M+ 276; MH+ 277: C,,Hi60, requires M+ 276, MH+ 277, respectively). Bioassay *

Electrolyte leakage induced by 10 l.tg ml-’ ascochitine was independent of the solvent concentrations assayed (no significant difference between the means at P = 0.05). This indicated, that there was no significant statistical interaction between ascochitine and acetone (Table 3), although the mean net responsesdo suggestsome synergistic effect. The variation in this experiment was large so that while an interaction cannot be statistically demonstrated, it is possiblethat someof the effect attributed to ascochitine was due to acetone or the toxin-solvent interaction. The net responseof leaf tissue to ascochitine (grossresponse-solvent effect) was used to compare the effect of ascochitine on the leaf tissue. The time-response curve of the susceptible tissue (Fig. 1) indicated that 20 h was an appropriate time to prepare the toxin concentration-response curve

G. R. Smith

338

et a/.

as it was near the top of the linear responsecurve of the tissue to the toxin. The toxin concentration responsecurve at 20 h was approximately sigmoidal, while the assay response saturated at 15 /tg ml-’ ascochitine. Regression line analysis of log,, transformed net solution conductivity at ascochitine concentrations of 0, 5, 10 and 15 pg ml-’ gave r = 0.85 (P < O*OOl) indicating that the electrolyte leakage response correlated with the toxin concentration over this range. Ascochitine concentrations of 1 and 2.5 pg ml-l induced electrolyte leakage wi,$h responsesfitting the dose-response curve, but could not be statistically differentiated from the solvent effect (Table 4). A regressionline through theselog,, transformed points had r = 0.92 (P < O-001), again indicating that, although these responsescould not be statistically differentiated, the tissue was responding to the ascochitine. The minimum ascochitine concentration necessary to induce statistically differentiable endogenous electrolyte leakage was therefore defined at 5 pg ml-’ for this assay. TAECLE~

Effect

ofacetone concentration

on ckctrolyle leakagefrom Clematis leaf discs co. Lady Betp Balfour in o 10 pg ml-’ ascochifine solution Mean

Solvent (% v/v) 5 10

response

@S) net (pS)

Mean

Control 75.8 104.8

Toxin

response

141.6 212.0

leakage from

TABLE 4 Lady Betty Boffour leaf d&s solutions

Toxin 0 Response

mean

($3)

Standard

658 107.2

72.9 a

7&3a

the net mean

responses.

The

after 20 h exposure to dilute arcochitine

concentration 1

error

1079 23.54

Data from five replicates were analysed by an F-test between difference between the means was not significant (P > 905).

Induced electro&e

incubated

25 91.6a

(pg ml-‘) 5

10

1270b

2164~

Data from five replicate experiments analysed by one-way ANOVA, and means compared by Duncan’s New Multiple Range Test. The coefficient of variation of the experiment was 16.9 %. Means followed by the same letter are not significantly different at the P = 0.05 level.

The weight of leaf tissue used in the electrolyte leakage assay, rather than the number of leaf discs, was found to be important. Five leaf discs of Montana have an average total weight of 270 mg, while the average total weight of five Lady Betty Balfour was 180 mg. When exposed to a 5 pg ml-’ ascochitine solution five Montana leaf discsleak significantly more electrolyte than five Lady Betty Balfour leaf discs (P < O-05). However, there was no significant leakage of electrolyte from three Montana leaf discs (average total weight 160 mg) (P > 0.05). Therefore all cultivars were

Ascochitine

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leafspot

339

of Clematis

260 -

4ll

240220 tis I

mo-

P :? s

HO160-

%

14nr

0

4

8

12 Time

FIG. I. Effect of ascochitinc concentration Clemdis cv. Lady Betty Balfour leaf discs. (+), IO (0) and 5 (A).

16 (h)

20

and time on net conductivity Concentration of ascochitine

24

28

of solutions bathing (pg ml-‘) 25 (O), 15

A”” 90-

70-

1

tz60s

:,.

3 i40- 50-

0 I -10 L$YB&Y

Huldine

RwF Cardinal

Ernest Markham

‘1 Montana

Clematis cultivar FIG. 2. Net solution using

conductivity of bathing five Clematis cultivars.

solutions

after

20 h incubation

in 5 pg ml-’

ascochitine

G. R. Smith

340

et a/.

compared using sensitivity to a 5 p.g ml-’ ascochitine solution based on the average total weight of the leaf discs, rather than the number of leaf discs. Electrolyte leakage was induced from leaf discs of the five Clematis cultivars by this concentration of ascochitine (Fig. 2), but the assaywas not sensitive enough to differentiate statistically between the responsesof cvs Rouge Cardinal, Huldine and Ernest Markham. Black flecking (small scattered black areas on the surface) of leaf discs exposed to ascochitine solutions, but not to solvent control solutions was also observed, with the degree of flecking appearing to be proportional to the toxin concentration. Black flecks were evident on leaf tissue of Lady Betty Balfour incubated in 1 or 2.5 pg ml-’ ascochitine, while the flecking was more apparent on tissue incubated in a 5 pg ml-’ solution. Leaf tissue from the cv. Montana did not show any black flecking when incubated in 5 pg ml-l ascochitine although some flecking was evident on tissues incubated in 7.5 pg ml-’ ascochitine solutions. The black colour of the tissue exposed to ascochitine was identical to the outer zone of leafspots on attached leaves (Fig. 3) or inoculated leaf discs (Fig. 4).

FIG. 3. Leafspot symptom of Phoma clmntidinn infection on cv. Lady Betty Balfour 9 days after inoculation. The leafspot has two major zones: an inner brown area surrounded by an outer black zone. Microscopy

Light microscope examination revealed alterations in the cellular organization of leaf cellsof cv. Lady Betty Balfour exposed to 5 pg ml-l ascochitine for 20 h. The cytoplasm appeared compressedand individual organelles such as the chloroplasts could not be distinguished (Fig. 5). The only change induced by the solvent was enlarged starch

Ascochitine

involvement

in phoma

leafspot

of Clematis

FIG. 4. Leafspot symptom of Phoma clernafidina infection on cv. Lady Betty Balfour leaf discs 7 days after inoculation. The outer black zone of the in uiuo leafspot is also evident on infected in uim~ leaf discs in both adaxial (top) and abaxial (bottom) views.

FIG. 5. Effect of ascochitine on cv. Lady Betty Balfour leaf cells as viewed by light microscopy of leaf sections. The cytoplasm of cells exposed to 5 pg ml-’ ascochitine for 20 h is relatively homogenous so that individual organelles such as chloroplasts and mitochondria can not be identified compared with leafcells from the solvent control (Fig. 5) (4 pm thick transverse section, Spurr’s resin, Azur II staining) x 600.

341

G. R. Smith et al. 342 grains within the chloroplasts ; otherwise the cellular organization and distribution appeared identical to that from a fresh leaf (Fig. 6). Extensive changes in cellular membrane systemswere evident when cells incubated in the toxin were examined by transmissionelectron microscopy. The organelle-limiting membranes lacked structural detail and were electron dense (Fig. 7) compared to solvent control tissues(Fig. 8). The internal structures of the mitochondria (cristae and stroma) were not discernible. Intact thylakoids were apparent inside the indistinct chloroplast-limiting membrane, but the stoma1 matrix had become very granular, suggesting a lossof integrity. The cytoplasm appeared coagulated with very coarseparticles. Both the plasmalemma and tonoplast appeared undamaged; this was also evident in cells from a natural leafspot (data not shown).

FIG. 6. The effect of 5 y0 acetone (solvent control) on cv. Lady Betty Balfour leaf cells as viewed by light microscopy. Enlarged starch grains in the chloroplasts is the only visible effect of exposure to 5% (v/v) acetone for 20 h. The chloroplasts were clearly visible and cellular organization appeared no different from cells in leaf discs incubated in water (4 pm thick transverse section, Spurr’s resin, Azur II staining) x 600.

Examination of freeze-fractured infected leaves by scanning electron microscopy revealed a well-defined lesion edge with leaf cells on either side clearly different (Fig. 9). The cytoplasm of cells within the leafspot was coagulated, and considerable quantities of a mucilaginous compound were evident especially in the intercellular spaces.The leaf spot margin was also clearly defined in tissuesstained with Pianeze III, and examined with a light microscope. The cytoplasm was collapsed or coagulated in cells within the leafspot, whilst unaffected cells at the leafspot margin

Ascochitine

involvement

in phoma

leafspot

of Clematis

343

FIG. 7. Effect of ascochitine on organelles of cv. Lady Betty Balfour leaf cells as viewed by transmission electron microscopy. In cells exposed to 5 pg ml-’ ascochitine for 20 h the granal matrix (gm) and ground plasm appear coagulated, and the chloroplast-limiting membrane is indistinct and unresolvable (a). Thylakoid stacks (th) are visible but appear to be degraded (b). The limiting membranes of the nucleus (nu) (arrowed) and mitochondria (mt) can not be clearly resolved (c). Bars equal @5 m.

appeared like other leaf cells. Hyphae were not found at the margin of any of the lesions examined: the leading hyphae were always observed a minimum of four or five plant cell diameters behind the margin edge. DISCUSSION

The phytotoxic metabolite, ascochitine, purified from cultures of P. clematidinaand from P. clematidina-infected Clematis leaves was identified by ‘H and 13C nmr spectroscopy, and electron impact and chemical ionization mass spectroscopy. Ascochitine appears to be involved in the infection of Clematisby P. clematidina.The level of ascochitine production in vitro by P. clematidinaisolates was related to isolate virulence as fungal isolates could be characterized into two groups : (1) high virulence, high ascochitine production; and (2) low virulence, low ascochitine production. Cultivar susceptibility to Phomainfection and ascochitine-induced electrolyte leakage from leaf discs also appeared related, although only the most and least sensitive cultivars were clearly differentiated by the assay. Ascochitine was produced in vivo and could be isolated from infected leaves. The toxin caused structural changes to cells similar to those observed in the cells of infected leaves, and the toxin solution induced a blackening of tissue similar to the colour of the outer zone of leafspots.

G. R. Smith

344

FIG. 8. The effect of 5 y0 acetone (solvent control) on cv. by transmission electron microscopy. The enlarged starch to incubation in 5 y0 (v/v) acetone for 20 h. The thylakoid limiting membrane are easily resolvable (a) and the mitochondrial-limiting membrane and internal organization the solvent (c). Bars equal @5 f.tm.

et a/.

Lady Betty Balfour leaf cells as viewed grains are the only visible change due stacks, plasmalemma and chloroplastground plasm is distinct (b). The appear unaffected by exposure to

The role played by any metabolite in diseaseis difficult to establish and prove, mostly due to the limited types of responsesof plant cells and tissuesto injury or challenge [9-I]]. In this study, several criteria were investigated to establish if ascochitine was involved in the pathogenesis of Clematisby P. clematidina.Ascochitine solutions blackened exposed tissuein proportion to concentration, so that the leaf discs exposed to toxin solutions appeared very similar in colour to the outer zone of leafspots. However, symptom production is inconclusive as a primary line of evidence [30], as it is recognized that plant cells have a limited number of responsesto toxicants [9-111. The effect of the acetone solvent in this study cannot be discounted. While there was no solvent effect that could be described statistically, the difference in responseof the cells exposed to 5 and 10o/oacetone solutions suggesteda reaction to the solvent. Some enlargement of starch grains in cells exposed to ascochitine was also evident. However, the tissueblackening doessupport the hypothesis that ascochitine plays a role in fungal virulence and cultivar susceptibility when considered as part of the total evidence. Ascochitine solutions have been previously reported to induce brown necrotic lesions on Vicia faba coleoptiles at similar concentrations (10 l,tg ml-‘) to those used in this study [22]. In the physiological complexity of the host-parasite interaction, it is difficult to distinguish causesfrom effects [30], and most criteria used to evaluate the pathological significance of toxins (e.g. symptom production in responseto treatment

Ascochitine

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leafspot

345

of Clematis

FIG. 9. Edge of the leafspot on cv. Lady Betty Balfour as viewed in freeze-fractured by scanning electron microscopy. (a) The edge of the leafspot lesion (le) was sharply the appearance of cells (arrow pair). Cells within the lesion (dt) appeared covered with (mu), while unaffected cells (ht) to the right appear normal. (b) Ramifying hyphae found within the lesion, but were never observed at the leafspot edge. Bars equal 50

specimens defined by mucilage (hy) were urn.

with toxin, presence of the toxin in the infected plant, correlation of toxin production with virulence) are logical but in practice are inconclusive as primary lines of evidence [30]. Satisfying two or more criteria increasesconfidence that the suspectedbiochemical has a role in development or expression of the diseasecomplex. While it is unlikely that any single metabolite will be found to be solely responsible for all aspects of a plant disease1291,each potential toxin must be considered on its own merits [24]. There was a relationship between virulence of the fungal isolate and ascochitine production, although the results did not differentiate the fungal isolates beyond two groups, namely LB, EM, HD, RC (high virulence, high ascochitine production) and MT (low virulence, low ascochitine production). Likewise, a relationship was found between cultivar susceptibility to Phoma infection and ascochitine-induced electrolyte leakage, although the electrolyte leakage assay did not differentiate the intermediate responses. The two extremes of sensitivity to the toxin (Lady Betty Balfour, highly sensitive and Montana, relatively insensitive) were clearly differentiated from the immediate responses (Rouge Cardinal, Huldine and Ernest Markham), while the leaf disc virulence assay was used to rank the cultivars from least to most resistant as Huldine = Lady Betty Balfour = Ernest Markham < Rouge Cardinal < Montana. The weight of leaf tissue, rather than the number of leaf discs, exposed to ascochitine was a critical factor in using the electrolyte leakage assay to differentiate between cultivar responses to ascochitine. This result may be related to that reported by

G. R. Smith et a/. 346 Damann et al. [7] who found that large leaf sampleslost electrolytes faster than small samples,and suggeststhat the weight of leaf tissueused in electrolyte leakage assaysis important, especially to allow differentiation of toxin sensitive from toxin insensitive tissueat a fixed toxin concentration. Ascochitine has previously been implicated in the &ease processof other Phomaand Ascochytaspecies,e.g. [li’, 221 and an electrolyte leakage assaydeveloped to measure the responseof pea leaf tissue to concentrations of ascochitine showed that resistant pea cultivars yere less affected by the toxin than varieties susceptible to A. pti infection [17]. Lepoivre [17] also established that the pathogenicity of A. pisi strains was correlated with their ability to produce ascochitine in vitro, and that ascochitine could be extracted from pea leaves infected by A. pisi or M. pi&es [la]. In this study, ascochitine was isolated from infected Clematisleaf discs, indicating that toxin production occurs in vivo and is not an artifact of in vitro culture. Furthermore, fungal hyphae were never observed at the well-defined leafspot edge, suggesting that the structural changes observed may be the result of some metabolic reaction in advance of the ramifying hyphae. The total evidence suggests that ascochitine is a phytotoxin and kills the plant cells prior to hyphae ramification through the necrotic tissues. Microscopic examination of tissues exposed to ascochitine revealed changes in cytoplasmic consistency, organelle distribution and internal structure, and appearance of the limiting membranes. While alterations to structure can occur long after physiological effects and fixation artifacts are of concern and may increase in already weakened membrane systems [1.5], these observations suggest that chloroplasts and mitochondria are the sites of ascochitine action. Chloroplasts are often sensitive indicators of exogenous chemical action, as shown for example by the effect of polychlorinated biphenyls on the ultrastructure of algal and fern cells [19,2&j. Another toxin in the azaphilone subgroup of the quinonemethides, citrinin [28], also affects membrane permeability [3] and inhibits algal respiration and photosynthesis [8]. These reported physiological effects of the quinonemethides and the ultrastructural evidence presented here suggestthat the mode of action of ascochitine may be against the limiting membranes of these two organelles. We have presented pathological, biochemical and ultrastructural evidence to suggest that ascochitine is involved in the infection of Clematisby P. clematidina.In previous studies with ascochitine, only one or two lines of evidence were investigated to support the hypothesis that ascochitine was involved in the development of disease,e.g. [l&18]. However, sensitivity of Clematiscultivars to ascochitine and in vitro pathogenicity tests do not relate closely to the observed field resistance. Huldine is generally regarded as wilt resistant, but leaks electrolytes in responseto ascochitine, and is susceptible in leaf disc infection assays.This is becausewhole plants display another defence mechanism in which the leaves senesceto prevent fungal infection of the node via the petiole [27]. Montana displays both biochemical and. physical resistance to infection by P. clematidina;the rate of fungal colonization in the leaf is considerably slower (biochemical resistance to ascochitine), and as the leafspot nears the petiole, the leaf abscisesto prevent infection of the node via the petiole (physical resistance). In many of the varieties bred for flower colour and size, both of these resistance mechanismsappear to be greatly reduced or lost, resulting in rapid invasion of the node by the fungus and ultimately wilting of the plant. Huldine would appear to be an intermediate: the

Ascochitine

involvement

in phoma

leafspot

of Clematis

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biochemical resistance is reduced, but the effective mechanism of leaf senescence operates to confer effective field resistance. Therefore, the interpretation of laboratory results must, in this case, be reconciled with observations of development of the disease in whole plants. We gratefully acknowledge a postgraduate scholarship from the New Zealand University Grants Committee, and the help of technical staff of the Departments of Plant and Microbial Sciences and Chemistry, University of Canterbury.

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