Phytoalexin accumulation, phenylalanine ammonia lyase activity and ethylene biosynthesis in fosetyl-Al treated resistant and susceptible tobacco cultivars infected with Phytophthora nicotianae var. nicotianae

Physiological and Molecular Plant Pathology (1990) 37, 207-219

207

Phytoalexin accumulation, phenylalanine ammonia lyase activity and ethylene biosynthesis in fosetyl-Al treated resistant and susceptible tobacco cultivars infected with

Phytophthora nicotianae var . nicotianae G . S . NEMESTOTHY

and D . I .

GUEST

School of Botany, University of Melbourne, Parkrille, Victoria 3052, Australia (Accepted for publication July 1990)

NC 2326, a cultivar of tobacco resistant to race 0 of the black shank pathogen Phytophthora nicotianae var. nicotianae, responds to stem inoculation by rapidly accumulating sesquiterpenoid phytoalexins and activating phenylalanine ammonia lyase activity at the infection front . In cv . Hicks, a near-isogenic susceptible cultivar, both responses are slower . Pretreatments of leaf discs with propylene oxide, which kills the cells, mevinolin, a specific inhibitor of sesquiterpenoid biosynthesis, or the non-specific amino-transferase inhibitor, aminooxyacetic acid (AOA), inhibit post-infection phytoalexin accumulation in both cultivars, and induce susceptibility in cv . NC 2326 . Aminohydrazinophenylpropionic acid (AHPP), a specific inhibitor of phenylalanine ammonia lyase activity and aminoethoxyvinylglycine (AVG), an inhibitor ofethylene biosynthesis, do not affect the susceptibility of either cultivar. Plants of the cv. Hicks are protected from infection by the systemic phosphonate plant protectant, fosetyl-Al . Sesquiterpenoid phytoalexins, lignin and ethylene accumulate, and phenylalanine ammonia lyase activity increases more rapidly in fosetyl-Al-treated Hicks stems than in untreated stems . Propylene oxide, mevinolin and AOA inhibit sesquiterpenoid phytoalexin accumulation and the effectiveness of fosetyl-Al in cv. Hicks . Fosetyl-Al does not enhance sesquiterpenoid phytoalexin biosynthesis in cv . NC 2326, and only marginally reduces pathogen growth in the initial stage of infection, before resistance is expressed . Mevinolin and AOA do not induce total susceptibility in fosetyl-Al-treated NC 2326, indicating that factors other than sesquiterpenoid phytoalexins are also involved in the mode of action of fosetyl-Al in this cultivar.

INTRODUCTION

Plants respond to infection by producing physical and chemical barriers which function as wall reinforcements, antibiotics or lytic enzymes . In every detailed comparative study of these responses, the differences between resistance and susceptibility are quantitative rather than qualitative . Although susceptible plants possess the machinery necessary for resistance, it is not activated in sufficient magnitude or speed to restrict the infection [18] . t To whom correspondence should be addressed . Abbreviations used in text : ACC, 1-aminocyclopropane-l-carboxylic acid ; AHPP, L-2-hydrazino-3phenylpropionic acid ; AOA, aminooxyacetic acid ; AVG, aminoethoxyvinylglycine ; AOPP, L-2-aminooxy3-phenylpropionic acid ; MES, 2- (N-morpholino) ethane sulphonic acid ; PAL, L-phenylalanine ammonia lyase ; sed, standard error of the difference between two means . 0885-5765/90/090207+13 $03 .00/0

© 1990 Academic Press Limited

208 G . S . Nemestothy and D . I . Guest Phosphonates such as fosetyl-Al (aluminium tris-[o-ethyl phosphonate] ; Aliette [Rhone-Poulenc Rural Australia Ltd]), control diseases caused by some Peronosporales, particularly the downy mildew pathogens and Phytophthora species [1] . Despite the ability of fosetyl-Al, or its degradation product phosphonic acid to control these diseases, it has, compared to other systemic fungicides, little direct activity on mycelial growth of the pathogen at sub-millimolar concentrations such as those found in protected plant tissues [3, 8-10, 24, 26] . Fosetyl-Al is hydrolysed in the plant to the phosphonate ion [8, 9] . Evidence that phosphonate inhibits mycelial growth of Phytophthora spp . under phosphate-limiting conditions in vitro has been used to support the hypothesis that fosetyl-Al has a direct fungicidal mode of action [8, 9] . However, phosphonate concentrations in planta only reach ED50 levels, determined under similar phosphate conditions in vitro, after application rates at least several times those needed to prevent disease [9, 24, 26] . Phosphonate may be concentrated in hyphae by active uptake in vitro, and possibly in planta, but even at high concentrations it is fungistatic rather than fungitoxic [4, 9 ; B . R . Grant, University of Melbourne, pers . comm .) . The strongest evidence for a direct fungicidal mode of action of phosphonates is based on studies of induced mutants of Phytophthora capsici with five- to ten-fold reduced sensitivities to phosphonates, both in vitro and in vivo [8] . Until the biochemical change underlying the reduced sensitivity of these mutants is known, their relevance to an understanding of the mode of action of phosphonates will remain unclear . These mutants may simply avoid sub-lethal physiological stress from exposure to phosphonates . This mechanism could be anticipated by the hypothesis proposing an indirect mode of action, because the unstressed mutants would not trigger the hosts defence responses [11-14, 24-26] . The indirect mode of action hypothesis predicts that phosphonates present in plants at concentrations insufficient to prevent mycelial growth induce physiological stress in the pathogen, causing it to elicit lethal host responses [13] . The timing and magnitude of host defence responses is affected by phosphonates . Phosphonates alter the respiratory response of infected plants [14], and stimulate cytoplasmic and phytoalexin defences of the host [3, 11, 12, 24, 25], mycorrhizal infections, carbohydrate accumulation and root leakage [17] . An inhibitor of aminotransferase and phenylalanine ammonia lyase activities, aminooxyacetic acid (AOA), reduces the efficacy of phosphonates against Phytophthora spp . on tomato and cowpea [3, 25] . The authors concluded that the action of phosphonates in these hosts requires phenylpropanoid biosynthesis, including, in cowpea, isoflavonoid phytoalexin biosynthesis . An alternative hypothesis, based on uptake assays of tritiated phosphonate by AOA-treated mycelium, is that AOA inhibits the uptake of phosphonate [8] . However, Bompeix et al. found that under the experimental conditions used, significant dissociation of the 3H-label from the phosphonate molecule may have occurred, making it uncertain as to what the label was attached to when its uptake was inhibited [2] . Similar experiments with more specific metabolic inhibitors are required . In this report we examine the effects of fosetyl-Al on three responses associated with disease resistance in plants : sesquiterpenoid phytoalexin accumulation, increases in phenylalanine ammonia lyase (PAL ; EC 4 .3 .1 .5) activity, lignin deposition and ethylene production . Sesquiterpenoid phytoalexins accumulate in tobacco in response to infection by a wide range of pathogens and elicitor treatments, although their role

Mode of action of fosetyl-Al in tobacco cultivars

209

in race-specific resistance has not been studied . Lignin, synthesized from the phenylpropanoid pathway, may be involved in the expression of resistance in some host-parasite interactions, including tobacco [20] . Ethylene biosynthesis appears to coincide with phenylpropanoid phytoalexin accumulation in soybeans, but is not otherwise implicated in resistance [22] . We compare these three responses in the nearisogenic cultivars of tobacco, Hicks and NC 2326 . NC 2326 has a gene for resistance to race 0 of the pathogen derived from Nicotiana plumbaginifolia [23] . In addition, we report the effects of a range of biosynthetic inhibitors on resistance and on the activity of fosetyl-Al : mevinolin, a specific inhibitor of HMG-CoA reductase and the acetatemevalonate pathway ; AHPP, a specific inhibitor of PAL and phenylpropanoid biosynthesis ; AOA, a non-specific inhibitor of aminotransferases ; AVG, a specific inhibitor of ethylene biosynthesis ; and propylene oxide, a volatile sterilant which kills cells with minimum disturbance to cell structure . Using these inhibitors, we aimed to identify which metabolic pathways are important to resistance, and to the mode of action of phosphonates .

MATERIALS AND METHODS

Inoculum Phytophthora nicotianae var . nicotianae B . de Haan (DAR 35088) was obtained from Mr J . Walker, Biological and Chemical Research Institute, New South Wales Department of Agriculture, Rydalmere, N .S .W . A second isolate, M 3049, was obtained from Mr G . Johnson, Department of Primary Industry, Mareeba Queensland . Both isolates are race 0, i .e ., they are virulent on cultivar Hicks and avirulent on cultivar NC 2326 . They were maintained on oatmeal agar at 28 ° C ; zoospores were produced as previously described [12] . The ED,, of fosetyl-Al (Technical grade, 98 % active ingredient ; kindly supplied by Rhone-Poulenc Rural Pty Ltd) against each of these isolates was determined on a defined mineral medium containing 1 . 33 mm phosphate [12], and on Difco Corn Meal Agar, which contains 0 . 4 mm phosphate [4] . Bioassay Ideally the effects of fosetyl-Al and inhibitors on pathogen growth in vivo would be assayed on inoculated tobacco stems, as used for the PAL and phytoalexin assays . We found this to be impractical because the leading edge of the lesion is internal, and thus not visible without splitting the stem . Leaves are naturally infected by the black shank pathogen [19], and lesion diameters on inoculated leaves are easy to measure without destroying the tissue . We developed a leaf disc bioassay to quantify the effects of fosetylAl and inhibitors on susceptibility . Four centimetre discs were taken from young, dark green leaves and rinsed thoroughly under running water . Propylene oxide-treated discs were exposed to 1 ml of propylene oxide in a one litre bell jar for 1 h before inoculation and incubation . A 3 mm hole was made in the centre of each disc, and the discs incubated on a bed of glass beads in a 4. 5 cm Petri dish containing 1 mg 1 - ' benzimidazole in 5 ml of 0 . 04 M MES (pH 6 . 5) . Other discs were treated with 100 .tg ml -1 fosetyl-Al in the same incubation medium, with or without AOA (Sigma ; 100 JIM), AHPP (Cambridge Research Biochemicals ; 100 pm), AVG (Serva Fein-

210

G . S . Nemestothy and D . I . Guest

biochemica ; 30 pm) or mevinolin (Sigma ; 10 gm) . A 4 mm mycelial plug of the pathogen was placed over the central hole, and diameter of the resulting necrotic lesion on three replicate discs was measured daily, in repeat experiments . The rate of lesion extension in Hicks was linear until the margin of the disc was reached .3-4 days after inoculation (data not shown) . Lesions expanded slowly on NC 2326 leaf discs for the first 3 days, but stopped soon afterwards . Leaf discs sham-inoculated with oatmeal agar plugs failed to develop lesions . Assay for phytoalexin production

Six to eight week-old tobacco plants were, where indicated, watered with fosetyl-Al (10 mg a .i ./pot) 7 days before inoculation . Inhibitors and inoculum were applied by a cut stem technique . Stems were decapitated just below the apex, and inoculated with 50 pd drops of either zoospores (approximately 500) or water . In the experiment using enzyme inhibitors, 50 pl drops of water, 10 pM mevinolin, 100 µM AOA or AHPP or 30 pM AVG were placed on the cut surface . Six hours later 500 zoospores in 50 pl drops were placed on the same surface, and the inoculated plants were incubated at 30 °C/ 20 °C in 12 h day/night cycles . At the times indicated after wounding or inoculation, 4 mm sections (0 . 05-0 . 10 g) from the necrotic front of individual stems were soaked in cold 50 % ethanol, homogenized and extracted with ethyl acetate as previously described [11] . The ethyl acetate extracts were dried to a film under partial vacuum and redissolved in 1 ml g - ' fresh wt . methanol containing 100 pg ml - ' of myristic acid methyl ester as the internal standard . Replicate 1 pl samples were injected on column into a 25 m x 0. 32 mm BP-1 capillary column . The Hewlett Packard 5890 gas chromatograph was equipped with a flame ionization detector coupled to a Hewlett Packard 3396A integrator . Helium carrier gas flow was regulated at 1 ml min -1 . Column temperature was programmed to 100 ° C (0. 5 min), increasing by 20 ° C min - ' to 180 ° C, then 10 °C min' to 320 ° C . Purified phytuberol, phytuberin, and rishitin samples were kindly donated by Professor J . Kuc, Department of Plant Pathology, University of Kentucky ; rishitin by Dr L . C . Davidse, Department of Phytopathology, Wageningen Agricultural University ; and capsidiol by Dr E . W . B . Ward, Agriculture Canada Research Centre, London, Ontario . Their identities were confirmed by GC-MS (Dr R . H . Dunstan, School of Biochemistry, University of Melbourne) . Phenylalanine ammonia lyase activity In the experiment on the dynamics of PAL activation following wounding or inoculation in both cultivars, stems were inoculated, with 50 p1 drops of either zoospores (approximately 500) or water, then incubated as described above . The enzyme inhibitors used in these experiments were 50 pd drops of water, 30 pM AVG, 100 pm AOA or AHPP . At the times indicated after wounding or inoculation, 4 mm sections (0. 05-0. 10 g) from the necrotic front of individual stems were homogenized in liquid nitrogen in the presence of 0 . 05 % w/w Polyclar AL, then suspended in 10 ml g -1 fresh weight of 1 mm dithiothreitol in 0 . 1 M sodium borate buffer, pH 8 . 8 . The suspension was centrifuged (10 min, 10000 g, 4 ° C) and 50 pl of the supernatant was added to 1 ml of substrate (1 MM L-phenylalanine in 0. 1 M sodium borate buffer) and incubated at 30 ° C for 1 h, with shaking every 15 min . The amount of trans-cinnamic

Mode of action of fosetyl-Al in tobacco cultivars

211

acid formed as the product of the PAL-catalysed reaction was calculated from the differences in absorbance at 290 nm between duplicate extracts with and without substrate [16] . Total protein was estimated using the 'Biorad' dye-binding assay [5] . Nine replicates of each treatment were assayed in duplicate in repeat experiments . Lignin accumulation in inoculated Hicks stem tissues before and 24 h after inoculation was estimated by the ligninthioglycolic acid method . Four millimetre-long sections (0.05-0 . 10 g) from below the wounded or inoculated site were extracted in methanol and NaOH, neutralized with HCI, then extracted with thioglycolic acid and HCI as described by Hammerschmidt [15] . The data are presented as the average difference in absorbance at 280 nm g -1 fresh wt ., of extracts from replicate stems of cv . Hicks before and 24 h after wounding or inoculation . Assay for ethylene production Fosetyl-Al (10 mg/plant) was applied as a root drench in 100 ml water 24 h before each experiment . Young, dark green, fully expanded leaves from 6-8 weeks old glasshouse grown plants were washed with distilled water, surface sterilized for 10 s in 70 % ethanol, then rinsed several times in sterile distilled water . The adaxial surface of each leaf was lightly dusted with carborundum powder (Union Carbide, 220 grid) and rubbed lightly with sterile cotton gauze soaked in 2- (N-morpholino) ethane sulphonic acid (MES) buffer (0 . 3 M, pH 6 . 0) . After rinsing, 1 cm discs were cut from the leaf lamina and floated, abaxial surface downwards, on 0. 5 ml MES buffer containing 100 tM ACC (Sigma), and where indicated, 30 µM AVG, in 20 ml vials . After 4 h incubation, 1 ml zoospore suspension (10 5 ml -1 ) or sterile distilled water was added, the vials were sealed, and incubated at 30 °C in the dark . One millilitre samples of air taken from three replicate sealed vials at 0, 2, 8 and 16 h after inoculation were assayed for ethylene content by gas chromatography . Duplicate 2 gl samples were injected into a Hewlett Packard 5830A gas chromatograph fitted with a 0 . 25 x 244 cm stainless steel Porapak `T' (80-100 mesh) column and a flame ionization detector . Nitrogen carrier gas flow was 45 ml min - ' . Oven, injector and detector temperatures were 100, 110 and 120 °C respectively . RESULTS ED50

The ED 50 s of fosetyl-Al on Hewitt's agar and CMA were 260 and 141 Pg ml - ' respectively for DAR35088, and 320 and 162 pg ml - ' for M3049 . Phytoalexin production The phytoalexins capsidiol, phytuberin, phytuberol, and rishitin accumulated in both cultivars of tobacco following stem inoculation with zoospores of Phytophthora nicotianae var . nicotianae (Table I and Fig . 1) . Small amounts (total sesquiterpenoid concentrations less than 5 .tg g -1 fresh wt.) also accumulated in treated and untreated wounded, uninoculated stems of both cultivars (data not shown) . There were significant differences between the cultivars in the timing and amount of phytoalexins accumulated following inoculation : in NC 2326 maximum total phytoalexin levels (135 .5 gg g- ' fresh wt .) accumulated 24 h after inoculation, whereas the total levels in Hicks never



212

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Mode of action of fosetyl-Al in tobacco cultivars

213

FABLE 1 .

Effect offosetyl-Al (10 mg/plant) and inhibitors on total sesquiterpenoid phytoalexin accumulation in tobacco stems (mg g- ' fresh weight) 24 h after inoculation with zoospores of Phytophthora nicotianae var .

nicotianae

Treatment

cv . Hicks +fosetyl-Al

cv . NC 2326

cv . NC 2326 +fosetyl-Al

50

107

134

114

9 46 66 65 6

8 48 78 101

6 52 108 120

8 60 121 115

cv . Hicks

Control inhibitor : Mevinolin AOA AHPP AVG SED (6 replicates) =

TABLE

2

Lesion diameters (mm) on fosetyl-Al (100 mm)- and inhibitor-treated tobacco leaf discs 60 h after inoculation with Phytophthora nicotianae var. nicotianae

Treatment

cv . Hicks +fosetyl-Al

cv . NC 2326

cv. NC 2326 +fosetyl-AI

27

12

16

4

28 29 22 22 25

25 24 14 11 25

35 29 18 15 32

12 14 5 4 16

cv . Hicks

Control inhibitor : Mevinolin AOA AHPP AVG Propylene oxide SED (9 replicates) =

2

exceeded 55 . 0 gg g - ' fresh wt ., even after 48 h (Fig . lE) . The difference is mostly due to the higher levels of capsidiol and phytuberin in NC 2326 (Figs IA and B) . The pattern of phytoalexin accumulation in stems of fosetyl-Al treated Hicks was similar to that in NC 2326 (Table 1 and Figs IA-E) . Most of the increase over untreated Hicks was due to the major phytoalexin, capsidiol, although levels of the other phytoalexins also increased (Figs 1B-D) . Fosetyl-Al pre-treatment marginally depressed phytoalexin levels in NC 2326 24h after inoculation, but the rate and pattern of accumulation were basically unchanged . Lesion diameters on NC 2326 and fosetyl-Al-treated Hicks leaf discs were significantly smaller than those on untreated Hicks (Table 2) . Lesions on fosetyl-Al-treated NC 2326 leaf discs were even smaller than on untreated discs . FIG . 1 . Dynamics of sesquiterpenoid phytoalexin accumulation in tobacco stems following inoculation with zoospores of Phytophthora nicotianae var . nicotianae ; cv . Hicks ( D-), cv . Hicks+fosetyl-Al (10 mg/plant) (- •- ), cv . NC 2326 (-N-) and cv. NC 2326+fosetyl-Al (10 mg/plant) (-0-) . A. Capsidiol ; B . Phytuberin ; C . Rishitin ; D . Phytuberol ; E . Total sesquiterpenoid phytoalexins . Each point represents the mean of three replicates, including in Fig. I E, ±SE .

214

G . S . Nemestothy and D . I . Guest Mevinolin completely inhibited phytoalexin accumulation 24 h after inoculation in all treatments (Table 1) . AOA reduced phytoalexin accumulation in all treatment/ cultivar combinations to the basal levels in stems of untreated, inoculated Hicks . Both inhibitors, as well as propylene oxide, which kills the leaf cells, increased the susceptibility of both fosetyl-Al-treated and untreated leaf discs of both cultivars (Table 2) . The protective effect of fosetyl-Al on cv . Hicks, was completely lost following treatment with AOA, mevinolin or propylene oxide . In cv . NC 2326, mevinolin, AOA and propylene oxide induced levels of susceptibility similar to that in cv . Hicks, and reversed the additional protective effect of fosetyl-Al : lesion diameters in untreated NC 2326 leaf discs are greater than in fosetyl-Al-treated discs, but the same as in mevinolin, AOA or propylene oxide-treated, fosetyl-Al-treated discs . AHPP partially inhibited the fosetyl-Al-induced increase in phytoalexin accumulation in cv . Hicks, but not in any other cultivar/treatment combination, without apparently affecting the susceptibility of leaf discs (Tables 1 and 2) . AVG did not affect phytoalexin accumulation or leaf disc susceptibility in any cultivar/treatment combination . Phenylalanine ammonia lyase activity and lignin deposition

PAL activity peaked 24 h after wounding or inoculation in cv . NC 2326, and after 36 h in cv . Hicks (Figs 2A and B) . PAL activity in both cultivars increased after inoculation, however the increase was much greater in the resistant cultivar, NC 2326 (Table 3 and Fig . 2B) . Fosetyl-Al caused a 47 % increase in PAL activity and induced 12 (A) l0 8 6

0

10

20 30 Time after inoculation (h )

40

50

FIC . 2 . The effect of fosetyl-Al on the dynamics of phenylalanine ammonia lyase activity in stems of tobacco following (A) wounding ; and (B) inoculation with zoospores of Phytophihora nicotianae var . nicotianae ; cv . Hicks (-p-), cv . Hicks+ fosetyl-Al (10 mg/plant) (- •- ), cv . NC 2326 (-/-), cv . NC 2326 +fosetyl-Al (10 mg/plant) (-O-) . Each point represents the mean of nine replicates +SE .

21 5

Mode of action of fosetyl-Al in tobacco cultivars TABLE 3 Effect offosetyl-Al on phenylalanine ammonia lyase activity in tobacco stems (µkat g- ' protein) 24 h after inoculation with zoospores of Phytophthora nicotianae var. nicotianae

Treatment

cv . Hicks

cv . Hicks +fosetyl-Al

cv. NC 2326

cv . NC 2326 +fosetyl-Al

6. 2

9.2

10 . 1

11 . 5

5. 0 4. 5 6. 0 1 .0

6 .6 2 .6 7.7

7.5 9.0 11 . 2

6. 8 9. 6 11 . 6

Control inhibitor : AOA AHPP AVG SED (6 replicates) =

TABLE 4 Effect of fosetyl-Al on the relative rates of lignin deposition from 0 to 24 h after wounding or inoculation with zoospores of Phytophthora nicotianae var. nicotianae in stems of tobacco cv. Hicks'

Wounded Inoculated

Untreated

Fosetyl-Al

-0 . 76 + 0 .80 -0 . 44+ 1 .06

0. 05 + 0 . 15 2. 96+ 1 . 29

'Relative rates of lignin deposition, ±SE, were estimated from the differences between the average A480 9-1 fresh wt . values of thioglycolic acid extracts of three replicate stem sections at 0 and 24 h, i .e. A 280 (24 h) - A280 (0 h) .

lignin deposition in first 24 h after inoculation of the susceptible cultivar, Hicks (Table 4) . No lignin deposition was measured in wounded or inoculated, untreated Hicks stems in the first 24 h, although significant lignin deposition was detected from 2448 h (data not shown) . PAL activity was not greatly affected by fosetyl-Al treatment of NC 2326, and lignin deposition was not assayed (Fig . 2B and Table 3) . AOA and AHPP reduced the amount of PAL activity extracted 24 h after inoculation in both cultivars, whether fosetyl-Al-treated or not (Table 3) . AVG had no effect on PAL activity . The effect of these inhibitors on lignin deposition was not assayed . Ethylene production Wounding stimulated ethylene production after an initial lag of 8 h in both cultivars (Fig . 3A) . Fosetyl-Al increased wound ethylene production in NC 2326 at 8 h, and in Hicks at 16 h after wounding . Inoculation induced more rapid ethylene production in fosetyl-Al-treated and untreated- NC 2326 and in fosetyl-Al-treated Hicks than in untreated Hicks (Fig . 3B) . AVG strongly inhibited ethylene production in all treatments of both cultivars (data not shown) . AVG had no effect on infection or colonisation of leaf discs in any cultivar/treatment combination (Table 2) .

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G . S . Nemestothy and D . I . Guest zoo

(A)

Jr Fic . 3 . The effect of fosetyl-Al on ethylene accumulation in excised tobacco leaf discs following (A) wounding ; and (B) inoculation with zoospores of Phytophthora nicotianae var . nicotianae ; cv . Hicks (-p-) , cv . Hicks + fosetyl-Al (10 mg/plant) (- •- ), cv . NC 2326 (-M-), cv. NC 2326 + fosetyl-Al (10 mg/plant) (- O - ) . Each point represents the mean of three replicates ±SE .

DISCUSSION We have shown that a resistant and a susceptible cultivar of tobacco differ in the magnitude and timing of phytoalexin, PAL and ethylene responses following infection by Phytophthora nicotianae var . nicotianae . Each of these responses began earlier and proceeded at a faster rate in the resistant cultivar . Inhibition of sesquiterpenoid phytoalexin biosynthesis by the specific inhibitor of acetate-mevalonate biosynthesis, mevinolin [6], and the non-specific inhibitor of aromatic biosynthesis, AOA [22], induced susceptibility in a leaf disc bioassay of the resistant cultivar . These results provide strong circumstantial evidence that sesquiterpenoid phytoalexin biosynthesis is involved in the expression of race-specific resistance of tobacco to this pathogen . Specific inhibition of PAL by AHPP did not affect the susceptibility or resistance of leaf discs, suggesting at most a minor role for phenylpropanoids, such as lignin, in the early stages of resistance of tobacco to this pathogen . Although we did not directly measure the effects of AOA or AHPP on lignin accumulation, both inhibit PAL, a key enzyme in their biosynthesis, and AOA inhibits lignin accumulation in infected potato tuber discs [15] . Thus the major effect of AOA was probably due to the inhibition of phytoalexin accumulation, as it has the same effect on lesion diameter as mevinolin . Lignin was not found to be an important factor in the hypersensitive response of tobacco to TMV [20] . By contrast, phenylpropanoids play an important role in the non-host resistance of potatoes [15] and in the race-specific resistance of some legumes to pathogenic Phytophthora spp [21] . Isoflavonoid phytoalexin biosynthesis was inhibited,

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and susceptibility was induced, in the normally incompatible interaction between races of Phytophthora megasperma f. sp. glycinea and soybean, by the related PAL inhibitor, L2-aminooxy-3-phenylpropionic acid (AOPP) [21] . Like Massala et al . [20], who also used AOPP, we failed to observe the phenomenon of superinduction of PAL following the application of AHPP in tobacco plants . We interpret the inhibition of extractable PAL activity by AHPP and AOA as being due to either an effect on the production of the enzyme, or to the tight binding of the inhibitors to the active site of the enzyme [22] . We have also demonstrated that, coinciding with its ability to protect a susceptible cultivar against the black shank pathogen, the phosphonate compound, fosetyl-Al, enhances sesquiterpenoid phytoalexin accumulation, PAL activity, lignin deposition and ethylene biosynthesis following inoculation . Two pieces of evidence suggest a causal link between enhanced host defences and disease protection . Firstly, mevinolin and AOA inhibit sesquiterpenoid phytoalexin accumulation and reduce the efficacy of fosetyl-Al in the susceptible cultivar. AOA would also reduce lignin deposition [151 . Secondly, at the rates applied and at the phosphate concentrations present, the measured concentration offosetyl-Al or its toxophore, phosphonate, at the infection site is only partially inhibitory to mycelial growth in vitro [26] . Our results support the hypothesis that in this and some other host-parasite interactions, enhanced speed and magnitude of phytoalexin accumulation is involved, along with a direct obstruction of normal growth of the pathogen, in the complex mode of action of phosphonates [3, 11, 12, 14, 24-26] . The relative importance of the "direct" and "indirect" components of the mode of action varies with the characteristics of the particular host-parasite pair studied, the application rates and possibly environmental factors . Our hypothesis is supported by the evidence that fosetyl-Al had no additive effect on the resistance, or of the phytoalexin, PAL or ethylene responses of, the resistant cultivar, NC 2326 . When phytoalexin biosynthesis was blocked in the resistant cultivar, lesion growth was still partially inhibited in fosetyl-Al treated leaf discs, indicating that factors other than phytoalexins are involved in the resistant response in this cultivar. There is unlikely to be a significant direct effect of fungal growth, because there was no residual inhibition of lesion growth in fosetyl-Al-treated Hicks leaf discs following treatment with mevinolin . A primary effect of phosphonates on the pathogen may be to interfere with its ability to avoid phytoalexin elicitation [13], perhaps through alterations to watersoluble cell wall components of Phytophthora [7] . Many other elements of the resistant response may be triggered by a phosphonate-affected pathogen in a resistant host . By disturbing the pathogens recognition-avoidance mechanisms, fosetyl-Al could enable the activation of an idle capacity in the susceptible cultivar, supporting the hypothesis that all plants have the potential to express resistance [18] . The specific PAL inhibitor, AHPP, caused a slight but significant and consistent reduction in the fosetyl-Al-induced increase in phytoalexin accumulation in cv . Hicks . We observed this reduction in three repeat experiments . PAL is not directly involved in sesquiterpenoid biosynthesis, and AHPP had no effect on phytoalexin accumulation in the resistant cv . NC 2326 . This minor effect is specific for fosetyl-Al treatment, and suggests linkage between PAL activity and phytoalexin elicitation in tobacco following treatment . We have observed the same effect using another specific PAL inhibitor, AOPP (unpublished results) . A possible explanation may be that lignin deposition

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G . S . Nemestothy and D . I . Guest

synergistically complements the efficiency of the phytoalexin response . AOA, although not as effective as mevinolin in inhibiting phytoalexin accumulation, was equally effective in reversing genetic resistance and the effect of fosetyl-Al . The specific ethylene biosynthesis inhibitor, AVG, did not inhibit phytoalexin accumulation in tobacco . Ethylene is not involved in the control of phytoalexin accumulation or the expression of resistance in soybeans [22] . Ethylene production is primarily a wound-induced response, although the magnitude of this response is affected by inoculation, and the host genotype . AVG did not affect the response of fosetyl-Al-treated leaf discs to infection . Increased biosynthesis of ethylene therefore, is not involved in the mode of action of fosetyl-Al . We have shown that the phosphonate compound, fosetyl-Al, enhances responses normally associated with stress disease resistance . The suppression of sesquiterpenoid phytoalexin biosynthesis, and to a lesser degree, lignin deposition, reduces the activity of fosetyl-Al . We propose that rapid phytoalexin accumulation, and possibly lignin deposition, are involved in the mode of action of fosetyl-Al against this pathogen in tobacco, in combination with and following the disturbance of the pathogen by fosetylAl or its toxophore, phosphonic acid . Identification of the precise site(s) of metabolic disturbance caused by phosphonates may provide some insight into the regulation of the host-parasite interaction . Thanks to Dr R . H . Dunstan, Russell Grimwade School of Biochemistry, University of Melbourne, for GC-MS analyses of the sesquiterpenoids, to Judy Carrigan, Robyn Gross, Rodd Jones and Zophia Felton for their valued assistance, and to Drs B . R . Grant, M . D . Coffey and B . J . Howlett for their comments on the manuscript . Statistical analyses were performed by Dr R . Jarrett, Statistical Consulting Centre, University of Melbourne, using the Genstat 5 statistical package . This work was supported by the Australian Research Grants Scheme, the Tobacco Research Trust Fund and the Commonwealth Tertiary Education Commission .

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