Factors affecting accumulation of pisatin by pea leaves

Physiological Plunt Pathology (1976) 9, 285-297

Factors affecting T.

J.

ROBINSON

accumulation

of pisatin by pea leaves

and R. K. S. WOOD

Botany Department, Imperial College, London SW?, U.K. (Acceptedfor publication August 1976)

Disks from pea leaves were floated on solutions of various substances; pisatin levels in the solutions and in the disks were measured after incubation. Filtrates from cultures of Ascochyta ptii and Mycosphaerella pinodes which increased accumulation were no more effective than were uninoculated media; filtrates from cultures of Penkillium expansum were ineffective. Increased accumulation by media depended almost entirely on their sugar content and probably was not caused by bacteria or fungi in solutions that greatly increased accumulation. Cupric chloride solution in the dark increased accumulation much less than expected, but in the dark, it was much more effective in the presence of sucrose. In the light cupric chloride alone had about the same effect as cupric chloride and sucrose in the dark, and sucrose had no additional effect. Similar results were obtained with mercuric chloride and sodium selenite. Apart from sucrose, galactose, glucose and raffiose also greatly increased accumulation of pisatin in disks treated with cupric chloride in the dark; arabinose, galacturonic acid, gluconic acid, dulcitol, mannitol, mannose, rhamnose and xylose were much less effective. In the absence of cupric chloride, galactose, glucose and raflinose increased accumulation in the dark to about the same degree as did sucrose. Acetate, cinnamate, phenylalanine, tyrosine and glycine had little effect on accumulation in the dark, with and without cupric chloride. Amounts of pisatin in solutions below disks were 1.5 to 2.9 times those in disks. Solutions that greatly increased accumulation when applied to the surface of disks were ineffective when iniiltrated into disks. Solutions of sucrose and cupric chloride did not increase accumulation when sprayed on young plants.

INTRODUCTION

Characteristically, leaf spots caused by pathogens reach a certain size, usually small relative to the leaf, and then get no bigger although surrounded by tissue ostensibly little different from that in which they first became established and then grew. Leaf spots of pea caused by Ascochytapisi and Myco.s$haerellapinodes have been studied on the basis that lesions would be limited if they or adjacent uninfected tissue came to contain fungitoxic substances in concentrations sufficient to prevent growth of the pathogens ; evidence which did implicate the phytoalexin pisatin in this way has been obtained [II]. It was decided, therefore, to study how growth of the pathogens in pea leaves could lead to accumulation of pisatin in and around the lesions. And because synthesis of phytoalexins is commonly supposed to be one of the many responses to damage caused by pathogens and other agents, we studied accumulation of pisatin in pea leaves caused by substances in cell-free filtrates from cultures of the two pathogens. Surprisingly, these filtrates were no more effective than were uninoculated media. This led to an investigation of culture media as stimulators of accumulation of pisatin and of the effects of other factors especially in relation to the results of earlier work along similar lines [1, 6, 9, 18, 201. The results of our study are summarized below.

288 MATERIALS

7. J. Robinson and R. K. S. Wood AND

METHODS

Plants Pea seeds (cv. Onward) were placed in a solution of sodium hypochlorite (2% available chlorine) for 10 min, washed, soaked overnight in water, then sown 3 cm deep in Vermiculite kept moist with Long Ashton nutrient solution [12] modified by containing iron as the ethylenediamine-tetra-acetic acid ferric monosodium salt at a final concentration of 55 meq/l. Plants were grown in a glasshouse at 18 to 28 “C with additional illumination for 16 h/day from fluorescent tubes and used at about the 5 leaf-pair stage, 20 to 24 days after sowing seed. Fungi Isolates of Ascochytapisi and Mycosphaerellapinodes were from infected pea plants [IO]. Spores were obtained from 7 to 14 day cultures grown at 24 “C on neutral V8 juice agar (V8 juice (Campbell’s Soups Ltd) 1 vol., water 4 vol., excess CaCO,) ; they were washed before suspending in water to required concentrations. Chemicals All were Analytical Reagent grade unless stated otherwise. Petroleum spirit was redistilled before use; water was glass distilled. Vitamin-free casamino acids were supplied by Difco Laboratories. Culture media These were as follows (g/l) : (a) Nutrient broth: sucrose 30, NaNO, KsSO, 0.35, FeS04.7H,0 0.01.

2.0, KCl 0.5, Mg glycerophosphate

(b) Sucrose nitrate : sucrose 30, NaNO, Z-0, KH,POI l-0, MgSOI.7H,0 KCl 0.5, FeSOJ.7H,0 O-01, ZnSO,.7H,O 0.01, CuS04.5H,0 O-01. (c) Glucose ammonium tartrate: 1.0, MgSO,.7H,O O-5.

glucose 12.5, ammonium

tartrate

0.5, O-5,

1.0, KH,PO,

(d) Sucrose casamino acids: sucrose 15.0, casamino acids 4.6, KHsPO, 1.0, MgSO,. 7H,O O-5, FeSO,.7H,O O-003, ZnSO,.7H,O O-0004, CuS0,.5H,O 0*0004, MnS0,.4H,O o-0004, Na,MoOl.ZHzO o-0005. Media were sterilized by autoclaving otherwise.

at 15 lbf/in2 for 15 min unless stated

Culture jltrates Still cultures were grown at 25 “C in 300 ml bottles in 35 ml medium inoculated with 1 ml water containing c. lo6 to 107 spores. Shake cultures were grown at 25 “C in 250 or 500 ml conical flasks with 75 or 100 ml medium inoculated with 2 or 3 ml water containing c. 2 or 3 x lo6 to 10’ spores; 250 ml flasks were shaken on an orbital shaker (150 rev/min) and 500 ml flasks were shaken on a reciprocating platform ( 150 cycles/min) . Fluid from cultures was made cell free by centrifuging and then sterilized by membrane filtration.

Accumulation

of pisatin by pea leaves

287

Inoculation and examination of leajets and disks

Detached leaflets were gently rubbed to make them more easily wetted and placed top surface upwards in transparent polystyrene boxes (3 x 11.7 x 17.5 cm) on polyvinylchloride grids above the surface of c. 100 ml water per box with cut ends of petioles just submerged. Drops (5 ~1) each with c. 3000 spores were placed at the centre of each half leaflet which were incubated in a growth cabinet at c. 20 “C and illuminated, for 16 h/day, or kept in the dark. Disks (c. 9 mm diameter) from leaflets were inoculated and treated in the same way. After incubation leaf tissue was cleared in hot lactophenol, stained in cotton blue in lactophenol for a few minutes and then differentiated in hot lactophenol. Stimulation

of accumulation of pisatin

Healthy, fully expanded leaflets were cut and gently rubbed between thumb and forefinger to increase wettability. Disks were cut with a metal cork borer from the leaves supported on paper tissues. The disks were bulked by being floated for c. 1 h on a large volume of sterile water. Test solutions were sterilized by heat or by membrane filtration when heat might have been damaging. Twenty-five drops, each c. 0.15 ml, were spaced on the bottom of a 9 cm diameter plastic Petri dish and one disk, upper surface downwards, was placed on each drop. Lids carrying wet tissue were replaced and the dishes incubated in the same ways as inoculated tissues. There were usually 75 or 100 disks for each treatment. After incubation the liquid beneath disks was collected and added to 2 x 5 ml water in which disks and dishes were washed. This liquid, to be called diffusate, was used soon after collection, or stored at 4 “C for short periods or at - 20 “C for long periods. Extraction and assay of pisatin

Pisatin was extracted from diffusates by partitioning 4 times with equal volumes of petroleum spirit (b.p. 40 to 60 “C) later removed by evaporation under reduced pressure at 40 to 45 “C. Undue exposure to light was avoided because pisatin is degraded by U.V. light. Dry extracts were dissolved in ethanol and an absorption spectrum was obtained. If it were characteristic for pisatin, concentrations were calculated from absorbance at the absorption maximum of 309 nm. Samples were discarded on the rare occasions when the spectrum was atypical and particularly when the absorbance ratio at peaks of 309 and 286 nm was not close to 1.47, the value for pure pisatin [4, 51. Almost always, by far the most important compound in extracts was pisatin with peaks of absorption at 309, 286, 280 and 213 mn and with the characteristic double peak of anhydropisatin at 358 and 339 nm after acidification. Solubility and other properties were also characteristic of pisatin [16, 171.

Disks were finely ground in 5 to 10 ml ethanol, the extract was collected and the residue washed with 6 x 5 ml ethanol. Total extract was centrifuged at 2500 g for 15 min, and ethanol removed under reduced pressure at 45 “C. The residue was shaken with 2 x 50 ml water and glass beads before 20 ml of the combined extract was partitioned with petroleum spirit which was then assayed for pisatin as described

288

T. J. Robinson

and R. K. S. Wood

above. Unless stated otherwise pisatin is expressed as pg/lOO disks. The mean diameter of disks (samples of 100 taken at intervals over 3 years) was 9.3 mm which gives an area of c. 68 cm2 for 100 disks from which pisatin/unit leaf area can be calculated. The fresh weight of 100 disks was 0.7 to 1.3 g so that pg/lOO disks is near f*g/g fresh weight tissue. Treatments usually contained 75 or 100 disks. They were either replicated 3 times in one experiment or included in separate experiments with suitable controls. Means for replicates of treatments given below are close to results for individual replicates unless otherwise stated. Throughout this research substantially different amounts of pisatin were often obtained from tissue treated similarly but in different experiments. Experiments were, therefore, repeated a number of times to confirm key results. RESULTS

Effect of culture jiltrates

on accumulation of pisatin

Ascochyta pisi and Mycosphaerella @nodes were grown in shake culture for 15 days at

25 “C in 100 ml of the following: (a) shoots of 21-day-old plants comminuted in water (1 g fresh weight/l 0 ml water) ; (b) as (a) +5 g sucrose/l00 ml; (c) as (a) -I- 5 g starch/100 ml. The fungi grew well on these media. Disks were placed on water, on uninoculated media and on cell-free filtrates from cultures. Only uninoculated medium (b) which contained sucrose caused greater accumulation of pisatin than did controls with water. The fungi also grew well in still culture on sucrose-nitrate medium. The results for cultures of different ages are summarized in Table 1. TABLE

1

Stimulation of accumulation of pisatin by sucrose-nitrate medium and culture jiltrates

Treatment

Pisatiu (yg/lOO disks) Age (days) of culture 5 12 Expt 1 Expt 2 Expt 1 Expt 2 48 268 557

31 3::

Water A. pisi culture filtrate Uninoculated medium

26 169 289

Water M. pinodes culture filtrate Uninoculated medium

22 325 314

24 160 217

Water P. expansum culture filtrate Uninoculated medium

44 81 428

< 15 86 320

19 Expt I

35 477 a3

37

Expt 2 117 383 39

64 239 224 -

A. pisi 5 day culture filtrates were less effective than was uninoculated medium, and filtrates from 12 and 19 day cultures were no more active than water. Filtrates from M. pinodes cultures differed from those of A. @isi cultures in being more active at

Accumulation

of pisatin by pea leaves

289

5 days and in remaining active at 12 and 19 days, but none was more effective than uninoculated medium. P. expansumwas used because Bailey [I] stated that in culture it produced substances that increased accumulation of pisatin, but in our experiments filtrates fi-om cultures were no more active than water in this respect. Diluted V8 juice also stimulated accumulation which was not surprising in view of its complexity and reports that accumulation is increased by coconut milk and many other substances of natural origin [2, 71. However, sucrose-nitrate medium, and glucose-ammonium tartrate medium which also was effective, do not contain such substances and further work showed that the sucrose or glucose in these media were as active as the complete media. These results were unexpected. A possible explanation of them is that accumulation of pisatin by disks was increased not by the media or by sugars but by microorganisms which grew in the liquid beneath the disks. Certainly after 2 days sucrosenitrate medium beneath disks usually contained many bacteria and occasionally the leaf appeared slightly damaged, but there was little or no growth of bacteria in glucose-ammonium tartrate medium or in solutions of the sugars, and disks appeared undamaged. Also, nutrient broth in which bacteria grew profusely did not increase accumulation of pisatin above that of controls with water. Also, V8 juice diluted with water and at pH 5.0, unsuitable for rapid growth of most bacteria, was much more effective than V8 juice at pH 7-O which is suitable for bacterial growth. Growth of fungi was less readily detected and it was insufficient to be readily visible by light microscopy. Also, the failure of filtrates from cultures of A. phi, A4. pinodes and P. expansumto increase accumulation above that caused by the medium alone suggests that growth of fungi on disks would not explain effects of the media and the sugars. Attempts to eliminate effects of micro-organisms by treating disks with solutions of sodium hypochlorite were unsuccessful because these treatments damaged the disks and because effects of this damage and of decreasing numbers of microorganisms could not be separated. However, it was found that treatment with hypochlorite prevented accumulation caused by solutions of cupric chloride in which, presumably, micro-organisms play no part. Further experiments with sucrose-nitrate and glucose-ammonium tartrate media, with each of their components, and with the media less each of their components showed that the activity of glucose-ammonium tartrate medium in causing accumulation of pisatin was attributable wholly to glucose, and that most of the activity of sucrose-nitrate medium depended on sucrose though a small part could be attributed to Fe, Cu and Zn. Bacteria grew in some of the solutions but growth was quite unrelated to the accumulation of pisatin in diffusates. In total the evidence suggested strongly that the two synthetic media primarily acted directly in causing an accumulation of pisatin and at most only secondarily by stimulating growth of bacteria, and that sucrose and glucose were the active components in the media. Stimulation of accumulation of pisatin by metal compounds One of the experiments on stimulation of accumulation by synthetic media included treatment of disks with CuCl, (10-s M) reported to be an effective stimulator of

290

T. J. Robinson and R. K. S. Wood

synthesis of pisatin [IS]. Again, surprisingly, it was no more effective than water. This experiment was repeated with a wide range of concentrations of CuCl,, HgCl, and NasSeOs, also reported earlier to be stimulators of synthesis [18]. HgCl, was little more effective than water. In two experiments NasSeO, at the optimum concentration of 3 x 10e5 M gave c. 160 Erg pisatin/lOO disks but this has to be set against unusually high values of c. 100 for water controls in these experiments. In two of three experiments, &Cl, at 1 x 10-l to 1 x IOF M was no more effective than water. In a third experiment, at the optimum of 3 x IO-* M, diffusates contained 110 pg pisatin/lOO disks compared with 33 for water controls. In spite of some anomalies it was clear that CuCI,, HgCI, and NasSeO, were far less effective with disks of leaf tissue than would be expected from earlier reports for pods and were not nearly so active at optimum concentrations as sucrose and glucose. CuCI, was now tested with sucrose with the results given in Table 2. TABLE

2

Stimulation of accumulation of pisatin by CuCI, and sucrose Treatment

Pisatin (pg/lOO disks)

Water Sucrose (30 g/l) (A) CuCI, (3 x 1O-4 M) (B) Sucrose and copper (A + B)

37 144 165 429

Although the figures for CuCl, were considerably higher and those for sucrose were lower than usual the additive effect was of interest so it was studied further. In earlier work with CuCl,, leaf disks were incubated in the dark [I], as, presumably, were pods used in the earliest experiments [5]. Accordingly disks were incubated in the dark and in the light. The results (Table 3) showed clearly that CuCl, was much more effective in the light than in the dark and that the effect of light could be simulated by adding sucrose to disks kept in the dark. TABLE

3

Stimulation of accumulation of pisatin in dark and light Pisatin (Kg/ 100 disks) Light

Dark Treatment Water Sucrose (30 g/l) (A) CuCl, (3 x IO-” M) (B) Sucrose and copper (A-t B)

Expt 1

Expt 2

Expt 1

Expt 2

38 128 90 373

23 140 64 338

47 112 396 394

32 85 338 329

The effect of light and sucrose on stimulation by CuCI, was confirmed many times and was studied in more detail in further experiments, representative results of which are given in Table 4. Accumulation of pisatin was highest at G. 3 x lo-* M CuCl,; this optimum was similar in dark and light, with and without sucrose.

Accumulation

291

of pisatin by pea leaves 4

TABLE

Eflects of sucrose,CuC12and light on accumulationof pisatin Pisatin (Kg/100 disks) Treatment

Dark

Copper (3 x 1O-4 M) 111 303 443 449

No copper 105 149

(M)

Light

Sucrose (30 g/l, 0.088 M) 196 184 426 353

No sucrose 88 114 128 303

CUcl, (M) 0 1 x 10-k Sucrose g/l 0 30 (O-088)

Light

Dark

Accumulation of pisatin was linearly related to log concentration sucrose up to 0*088 to 0.175 M, the highest values tested, in dark and light, with and without copper. Without sucrose, light greatly increased accumulation caused by CuCl,. With optimum sucrose (c. 30 g/l, 0.088 M), light had little effect but did increase accumulation at concentrations below the optimum. Accumulation of pisatin caused by copper was greatly increased in the dark by sucrose which also had some but much less effect in the light. Stimulation of accumulation by sugars, mannitol and metal compounds

Na,SeO,, HgCl, and CuCI, were tested with sucrose, glucose and mannitol in dark and light. Representative results given in Table 5 show that in interaction with light and dark and with sugars, HgCI, and NasSeO, had effects similar to those caused by CuCl,, and that glucose acts in much the same way as sucrose especially in greatly increasing accumulation of pisatin caused by the metal compounds in the dark compared to light. In contrast mannitol did not increase accumulation in the dark with or without metal compounds but in the absence of metals it was active in the light. TABLE

5

Effects of sugars, mannitol and metals in pisatin synthesis Treatment

Pisatin (pg/lOO disks) Nil

Metal Compound

Sucrose Glucose (30 g/l, 0,088 M)

Mannitol

0 0

Dark Light

53 15

316 196

257 248

35 163

c&l, (3 X lo-* M) CuCl, (3 x 10-b XI)

Dark Light

93 351

324 342

502 549

76 376

Dark Light

2::

327 391

450 408

Dark Light

114 316

408 491

397 514

Na,SeO, Na,SeO, HgCl, HgCl,

(1 (1

(3 x 10-s M)

(3 x lk5 X

lO-5

X lo-‘M)

M)

M)

129 316

292

T. J. Robinson

and R. K. S. Wood

Further experiments (Table 6) studied the effects of a variety of sugars and related compounds with and without CuCl, in the dark in which the sucrose/metal compound interaction was most pronounced. Solutions of galacturonic and gluconic acid were adjusted to pH 7 with NaOH to avoid the damage caused by solutions at lower pH. TABLE 6 Effects

of CuC12, sugars and

Treatment

(0.1 M)

related compounds on accumulation of pisatin Pisatin (pg/ 100 disks) No CuCl, CuCl, (3 x 10-Q M)

Water Arabinose Galacturonic acid Gluconic acid Dulcitol Galactose Glucose Mannose Marmitol Raffinose Rhamnose Xylose

35 44 36 2 206 136 88 41 164 48 42

105 (70)” 110 64 70 105 480 545 168 133 581 135 154

Results are means of three experiments done in two series. a One result probably anomalously high; figure in parentheses gives mean of two results which were similar, 61 and 79 yg/lOO disks.

Glucose, galactose and rafhnose stimulated accumulation of pisatin in the absence of CuCl,. Other substances with the possible exception of mannose had little or much less effect. The same three sugars also caused very substantial increases in accumulation in the presence of CuCl, and again other substances were ineffective. Precursorsof pisatin and related compounds It is possible that less pisatin accumulates in response to CuCl, in the dark because of shortage of precursors present in higher concentrations in light. This possibility TABLE 7 Effect of various metaboliks on accumulation of pisatin Treatment

(0.01 M)

Water Acetate Cinnamate Phenylalamne Tyrosine Glycine Sucrose

Pisatin (pg/lOO disks) No GuCl, CuCl, (3 x 104 Id) 25 < 15 <15 13 10 22 158

66 76 <15 57 44 37 260

Results are means for three experiments in which there was close agreement between replicates.

Accumulation

of pisatin by pea leaves

293

studied by using acetate, cinnamate and phenylalanine which are probably precursors of pisatin. Tyrosine, structurally similar to phenylalanine, and glycine were also tested. Solutions of acids were adjusted to pH 7-O with NaOH before use. The results (Table 7) suggest that the precursors have no effect on accumulation of pisatin with or without CuCl s; tyrosine and glycine were also ineffective. In contrast, the effect of sucrose although not so striking as in certain other experiments was still pronounced. Cinnamate with and without CuCl, severely damaged the disks so it should have been tested at lower concentrations that were not damaging. The other compounds caused no macroscopically visible damage but these too need to be tested at lower concentrations. Also, in further work it would be desirable to measure uptake of test substances by the disks.

was

Pisatin in dzfiiates

and tissue

Pisatin in diffusates as defined above depends on the amounts that accumulate in disks and on the amounts that diffuse into the solution beneath disks. The latter were investigated for disks in the dark and in the light, on water and on different solutions, with the results shown in Table 8. TABLET Pisatin in dffusates and in disks Pisatin (pg/lOO disks) DifFusates Disks

Treatment Water, dark Water, light

45 56

Sucrose, dark Sucrose, light

190 149

CuCl,, dark CuCl,, light Sucrose, C&l,, dark Sucrose, CuCl,, light

Ratio

29 22 66 99

1.6 2.5

117 360 .

60 237

2.0 l-5

554 447

285 248

1.9 l-8

Meall

2.0

2.9 1.5

The results were somewhat variable with diffusates containing l-5 to 2.9 times as much pisatin as disks, but they suggest that diffusion of pisatin from disks is not critical in determining amounts in diffusates. They also raise the question of how a substance with so low a solubility as pisatin (c. 30 pg/ml) moves so readily from tissues into ambient fluids, and emphasize the high concentrations to which pisatin may accumulate in leaf tissue, bearing in mind that the figures in Table 8 are pg/ 100 disks with a fresh weight c. 1 a0 g, i.e. pg/g fresh weight. Synthesis of @satin in water-soaked disks caused by M. pinodes remain larger in detached leaves floating on soaked. It was of interest, therefore, did not become water-soaked when Lesions

small in leaves attached to plants but get much water [IO]. These lesions rapidly become waterto study accumulation of pisatin in disks which floated on solutions and in disks infiltrated with

294

T. J. Robinson and R. K. S. Wood

solutions. A further reason for these experiments was the supposition that substances can cause pisatin to accumulate in disks floating on solutions only after they have passed into the disks. It might be expected, therefore, that accumulation would be more rapid if solutions were brought into contact with many more cells by infiltration. Disks were infiltrated under reduced pressure with water or with various solutions before incubation for 48 h in the usual way. Infiltrated disks remained water-soaked during incubation. Results (Table 9) showed that infiltration decreased greatly or prevented the accumulation of pisatin that occurred in disks that had not been infiltrated. TABLE 9

Effect of inaltration on accumulationof pisatin Infiltration

Incubation

None Water None Water None Water

Cl&I,” CuCl, Sucroseb-nitrate medium Sucrose-nitrate medium CuCls and sucrose CuCl, and sucrose

(3 X lk4 b Sucrose (30 g/l).

’ cU’&

Pisatin (pg/ 100 disks) Dark Light

on solution

131 26 160 15 175 15

350 52 131 15 327 85

M).

Synthesis of pisatin in leaves on plants

Various copper compounds have been among the most widely used of protectant fungicides and are still important. Accumulation of pisatin in leaf disks is increased It seemed possible, therefore, that application of copper by solutions of CM&. fungicides to leaves might increase accumulation of pisatin to levels that might supplement the action of the fungicides. This was studied by applying 2.5 g copper oxychloride/l to run-off from young pea plants (three to four pairs of leaflets). Other plants were sprayed in the same way with water, or with a solution of sucrose (30 g/l) because this also increased accumulation of pisatin by disks from leaves. Plants were kept in a glasshouse at 18 to 28 “C in high humidity for 2 days. Pisatin was then extracted from samples of plants. Results (Table 10) showed no significant difference in levels of pisatin in plants sprayed with water, a suspension of copper oxychloride or with sucrose. But it was TABLE 10

Pi-satinin wholeplants Pisatin (pg/g fresh weight) Expt 1 Expt 2

TreatmenC Water Copper oxychloride Sucrose (30 g/l) a Sprayed to run-off.

(2 g/l)

11.0 3.5 6.8

5.5 6.3 9.5

Accumulation

of pisatin by pea leaves

of interest that apparently weight.

295

healthy leaves contained 5.5 to Il.0 pg pisatin/g fresh

DISCUSSION

The work reported above was based on the use of disks cut from pea leaves floated on solutions of various substances. This is an artificial system as indeed are most systems that have been used to study accumulation of phytoalexins. Clearly it is rash to extrapolate from results obtained by the use of such systems, as others have warned [14, 151, but their use in practice is more or less inevitable and can be justified if it gives interesting results which then are interpreted with caution. The advantages of leaf disks are that they are of tissue of a type which is attacked by leaf spot fungi and that they allow relatively large-scale experiments in which the unit of reacting tissue is a large number of disks taken at random from a population of plants grown under the same conditions. A disadvantage is the difficulty of eliminating contaminating micro-organisms other than by the use of substances which are likely to affect the processes under study. But there are ways in which this disadvantage can be circumvented. Possibly more important is the fact that a disk from a pea leaf rapidly becomes physiologically different from an attached leaf This can be partly offset by keeping the experiment as short as possible and by choosing species with leaves that probably deteriorate slowly under conditions similar to those used in the work. Another objection is that phytoalexins may be synthesized in response to mechanical damage and that tissues damaged in cutting of disks may contribute materially to the phytoalexin that accumulates in the disks. However, experiments reported elsewhere [I91 showed that this “edge” effect was not an important factor in our work. Lastly, there is the difficulty of knowing how much of the substances in solutions on which disks of leaves are incubated moves into cells inside disks, a problem common to most other techniques used to study accumulation of phytoalexins. The results given in this paper must be interpreted in the light of the limitations summarized above. In other papers “induction” and words with the same root refer to processes which lead to increases in amounts of phytoalexin. Its use does not always imply control at the genetic level in the sense of Jacob & Monod [13] though some workers have postulated controls along these lines. Thus, Hadwiger & Schwochau Hypothesis” based, inter alia, on reports that [9] have proposed an “Induction accumulation of pisatin is associated with increases in protein synthesis and of a certain fraction of rapidly labelled RNA. They propose that induction involves a pisatin operon with an operator site and a polycistronic structural gene for the enzymes of the pisatin pathway. Metabolites stimulate synthesis of pisatin by inhibiting a regulator gene thus preventing synthesis of a substance which otherwise would have repressed the pisatin gene. It is not easy to see how this hypothesis would explain the range of interactions with light and CuCl, or the wide range of substances now known to stimulate accumulation of pisatin. Here it may be relevant that accumulation of pisatin is usually associated with increased activity of phenylalanine-ammonia lyase (PAL) [SJ, that the activity of PAL is affected by light and that in the light activity can be stimulated 24

296

T. J. Robinson and R. K. S. Wood

by various substances including sucrose [3]. Our results for sugar/CuCls/light interactions may be explained by assuming two controls in the synthesis of pisatin. Irrespective of this there remain the facts that accumulation is stimulated by sucrose and other sugars at concentrations that can hardly be regarded as injurious and that more than one substance can interact in the process of stimulation. It is of interest that solutions of sucrose and CuCl, that stimulate accumulation of pisatin when applied to the surfaces of disks of pea leaves were ineffective when infiltrated into leaves. The possibility that effects of infiltration may explain in part the rapid spread of lesions caused by M. pinodes which become water-soaked is partly offset by the fact that under the same conditions lesions caused by A. pisi remain limited. There is also the discrepancy between the increased accumulation caused by solutions of CuCl, and sucrose applied to leaf disks and the failure of these solutions to cause accumulation when applied as sprays to leaves attached to plants. The research described in this paper was supported by the Agricultural Council to whom the authors express their gratitude.

Research

REFERENCES 1. BAILEY, J. A. (1969). Effects of antimetabolites on production of the phytoalexin pisatin. PhyfoCherniS6~8, 1393-1395. 2. BAILEY, J. A. (1970). Pisatin production by tissue cultures of Pisum sativum. Journal of General Microbiolopy 61,409-415. 3. CREASY, L. L. (1968). The increase in phenylalanine ammonia lyase activity in strawberry leaf disks and its correlation with flavonoid synthesis. Phytochemistry7,441~446. 4. CRLIICKSHANK, I. A. M. BE PERRIN, D. R. (1960). Isolation of a phytoalexin from Pisum sativum L. Naature 187, 7!%-800. 5. CRUICKSHANK, I. A. M. & PERRIN, D. R. (1961). Studies on phytoalexins. III. The isolation, assay and general properties of a phytoalexin from Pisum satiuum L. Australian Journal of Biological Science14, 336-348. 6. CRUICKSHANK, I. A. M. & PERRIN, D. R. (1963). Studies on phytoalexins. VI. Pisatin: the effect of some factors on its formation in Pisum sat&m L. and the significance of pisatin in disease resistance. AustralianJournal of Biological Science16, 111-128. 7. HADWIGER, L. A. (1966). The biosynthesis of pisatin. Phytochemistry5,523-525. 8. HADWIGER, L. A. (1967) Changes in phenylalanine metabolism associated with pisatin production. Phytopathology57, 1258-1259. 9. HAD’WIGER, L. A. & SCHWOCHAU, M. E. (1969). Host resistance responses-an induction hypothesis. Phytopathology59, 223-227. 10. HEATH, M. C. & WOOD, R. K. S. (1969). Leaf spots induced by Ascochytapisi and Mycosphaerella pinodes. Annals of Botany 33, 657-670. 11. HEATH, M. C. & WOOD, R. K. S. (1971). Role of inhibitors of fungal growth in the limitation of leaf spots caused by AscochytapG and Mycosphaerellapinodes. Annals of Botany 35, 475-491. 12. HEWITT, E. J. (1952). Sand and water culture methods used in the study of plant nutrition. Technical CommunicationCommonwealthBureau of Horticulture and Plantation Crops No. 22. 13. JACOB, F. & MONOD, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. 3ournal of Molecular Biology 3, 318-356. 14. KEEN, N. T. & HORSCH, R. (1972). Hydroxyphaseollm production by various soybean tissues: a warning against the use of “unnatural” host parasite systems. Phytopafhologv62, 439-442. 15. Ku&, J. (1972). Phytoalexins. Annual Review of Phyfopathology 10, 207-232. 16. PERRIN, D. R. & BOTTOMLEY, W. (1961). Pisatin: an antifungal substance from Pisum sativumL. Nature 191, 76-77. 17. PERRIN, D. R. & BOTTOMLEY, W. (1962). Studies on pisatin. V. The structure of pisatin from Pisum sativum. Journal of the American Chemical Society84, 1919-1922. 18. PERRIN, D. R. & CRUICKSHANK, I. A. M. (1965). Studies on phytoalexins. VII. Chemical stimulation of pisatin formation in Pisum sativum L. Australian journal of Biological Science18, 803-816.

Accumulation of pisatin by pea leaves

297

19. ROBINSON, T. J. (1974). Pisatin and limitation of pea leaf spots. Ph.D. Thesis, University of London. 20. SCHWOCHAU, M. E. & HADWIGER, L. A. (1968). Stimulation of pisatin production in Pisum s&urn by actinomycin D and other compounds. Archives of BiocFRmist7yand Biophysics 126, 731-733.