Use of plant cell cultures to study production and phytotoxicity of Alternaria solani toxin(s)

PhysiologiGal

Plant Pathology

(1982)

21, 295-309

Use of plant cell cultures to study production and phytotoxicity of Alternaria solani toxin(s) AVTAR

and

K. HANDA, RAY M. HASEGAWA

A.

MARY

BRESSAN,

L. PARK

PAUL

DGpcrrtm-ent of Horticulture, (Acctpkdfor

publication

Purdue

University,

West Lafayette,

Indiana

47907,

U.S.A.

June 1982)

The effect of toxin preparations from culture filtrates of Altemaria solani (TPA) on the growth of cell suspensions of A. so&G host and non-host plants was examined. Cells of host species were more sensitive to TPA than cells of non-host species. Both the inoculum density and the stage of growth of cells used for inoculum affected the tolerance of the cells to TPA. The inhibitory effects of TPA on cell growth are reproducible when the cells are obtained from a defined stage of growth and are inoculated at a predetermined inoculum density. Thus cultured cells provide an accurate assay for quantification of TPA phytotoxicity. This standardized growth assay was used to measure changes in production of phytotoxic activity of TPA during the growth of A. solani cultures. Results indicated that maximum production of extracellular phytotoxic activity occurred in the early log phase of fungal growth. The growth assay was used also to measure changes in tolerance of potato cells to TPA after selection. By repeated exposure to increasing amounts of TPA, potato cells became successively more tolerant to TPA. At least a 4-fold increase in tolerance was observed.

INTRODUCTION

solani (Ell. and G. Martin), the causal organism of early blight disease of tomato and potato, causes significant economic losses of these crops. Alternaric acid was the first phytotoxic compound identified from culture filtrates of A. solani [il. Since then numerous other toxic compounds have been isolated from culture filtrates of various Alternaria species [16]. Although most of these compounds, including alternaric acid, are toxic to a wide range of plant species [S, 7, IS], compounds have been isolated from culture filtrates of A. mali, A. kikuchiana and A. alternata which have host specific toxicity and appear to be responsible largely for pathogenicity [14, 19, 291 of these organisms. The production of host specific toxins by A. solani has been reported only recently [23]. It has been reported that A. solani secretes 2 lipid-like compounds into culture medium and that both are required for the elicitation of necrotic and chlorotic symptoms typically associated with the disease [23]. Although plant tissue cultures have been used for several practical applications in plant pathology [see II, 201, they have been used rarely to study the phytotoxic compounds produced by plant pathogens. Recently, plant protoplasts have been used to quantitate phytotoxic compounds from culture filtrates of Phyt&ithora citrophthora [3]. The aim of the present study was to investigate whether cell suspensions of plant species known to be hosts or non-hosts for A. solani can be used to Alternaria

This research was supported by Purdue Improvement Funds. Journal Paper No. 8827 0048-4059/82/060295+ 15 $03.00/O

University Agriculture Experiment Station Purdue University Agriculture Experiment @ 1982 Academic

Press Inc.

(London)

Program Station. Limited

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study the production of host specific toxin(s) by A. solani. The results presented here show that toxin preparations from culture filtrates of A. solak (TPA) were more toxic to cell suspensions of plant species which are hosts for A. sol& lhar. :u cell suspensions of plant species which are not hosts for this pathogen. Results also show that cell suspensions of host species can be used to quantitate in a reproducible manner the amount of extracellular phytotoxic activity of TPA produced by A. solani during its growth in culture. Furthermore: we have obtained cell lines of potato which exhibit enhanced tolerance for growth t.o TPA. MATERIALS AND METHODS Plant cell and callus cultures

Potato cell suspensions were derived from callus initiated from tuber tissue of Solanum cell suspensions of potato, approximately 1 g portions of 5 to &week-old friable callus were transferred to 25 ml of medium (Table 1) in 125 ml Erlenmeyer flasks. Procedures for the initiation and maintenance of cell suspensions of tomato, &ycopersicon esculentum mill. cv. VFNT Cherry, have been described previously [43. Carrot (Caucus carota L.) callus was obtained from Professor J. M. Widholm, University of Illinois, Urbana, Illinois and cell suspensions were initiated and grown in carrot medium (Table 1). Callus of Phaseolm wrightii (a wild species from Arizona) was initiated from hypocotyl explants in the medium used for tomato cells (Table 1). To initiate cell suspensions, 1 g portions of 6-week-old friable callus were transferred to 25 ml of bean medium (Table 1) in 125 ml Erlenmeyer flasks. Stock cell suspensions were maintained by transferring about 1.6 g of cells from stationary phase cultures into 1 litre Erlenmeyer flasks containing 200 ml of medium. tubevosum L. cv. Superior. To initiate

'FABLE Culture

media for plant

Potato Supplement ___Sucrose Casein hydrolysate Myoinositol Folic acid Biotin Glycine Nicotinic acid Pyridoxin-HCI Thiamin-HCl 2,4-Dichlorophenoxyacetic Indoleacetic acid Kinetin

PH

(mg l--l) --

1 cell cultwes

Tom-to

Carrot

medium

medium

medium

25 000-O 1000~0

30 000.0 -

30 000,o

Bean medium ---x

100.0 0.5 0.05 2.0

0.5 0.5 0.5 3.0

acid -

0.2 5.7SO.l

1004 -^-

roo.0 0.5 2.0 0.5 0.5 0.5 0.4

0.5 0.5 1.0 0.5 5.0 0.3 &O&O.1

5.OiO.l

30 oai-0 -

1004l -0.5 0.5 1.0 2-O _-

l,O 57-&0.1

Callus and cell suspension cultures of all plant species were maintained on medium containing Murashige and Skoog salts [25’] and the additional supplements indicated. Ali media were sterilized by autoclaving for 15 min at 121 “C. Media were soliied (if needed) with Bacto agar, 10 81-1 and the pH of all media was adjusted with 0.1 N NaOH or HCl prior to autoclaving.

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Callus and cell cultures were maintained at 26 “C under 16 h daily illumination of 1500 lx (Cool White fluorescent lamps). Cell suspension cultures were grown on either gyratory or reciprocating shakers (80 to 100 cycles mm-r). Toxin @reparation Altemaria solani (Eli. and G. Martin) was obtained from Professor James Shepard, Kansas State University andmaintained on a mediumof potato broth [36] containing 10 g 1-r dextrose (PD) solidified with 15 g l-1 Bacto agar (Difco Labs, Detroit, Michigan, U.S.A.). The pathogenicity of A. solani was confirmed by inoculating potato (Solarium tuberosum L. cv. Superior) plants in the greenhouse with mycelial and spore suspensions. Spores of A. solani were prepared according to the procedure of Shahin & Shepard [32]. To prepare large quantities of A. solani toxin (TPA), the fungus was grown in 500 ml Florence flasks, each containing 150 ml of PD broth. Flasks were inoculated with 2 mycelial plugs (1 cm diameter) cut from leading edge of A. solani colonies grown on PD agar. After 12 to 16 days, the fungus was harvested by filtration through 2 layers of cheesecloth and the filtrate was lyophilized. The residue was extracted with methanol, filtered through Whatman No. 1 filter paper and the filtrate evaporated in vacuuoat 40 “C. The residue obtained was redissolved in distilled water (1 /I 0th of the original volume of the culture filtrate) and the pH was adjusted to 5.5 with 0.1 N NaOH or HCl. For controls, uninoculated PD broth was lyophilized, the methanol soluble material was resuspended in I/lOth of the original volume of distilled water and the pH was adjusted to 5.5. All preparations were sterilized by filtration through Millipore GSW membrane filters before adding to autoclaved media. Assay of phytotoxic activity of TPA using potato cell suspensions To assessthe phytotoxicity of TPA, potato cells obtained from cultures in the stationary phase of growth were inoculated at a cell density of O-2 g cells per 25 ml culture medium into 125 ml Erlenmeyer flasks containing varying amounts of TPA. The amount of TPA required to inhibit the increase in the fresh weight of the potato cells by 50% (LDsO) compared to controls was determined. This amount of TPA was equivalent to one LD,, unit of TPA. The total number of LD,, units present in the original volume of TPA was calculated. Unless otherwise stated, the amounts of TPA used in experiments are given as LD,, units. Based on this assay procedure, filter sterilized TPA preparation did not lose phytotoxicity for at least 2 months at room temperature. Phytotoxicity of A. solani toxin preparations To determine the response to TPA of plant species used in this study individual leaflets of potato, tomato and bean were excised from plants growing in the greenhouse and were placed into Petri plates containing 15 ml of various dilutions of TPA. Amounts of TPA ranged between 0.4 and 18 LDso units. Leaflets were incubated in a constant temperature room (26f 1 “C) under illumination of 1500 lx [Cool White fluorescent lamps) and were examined daily for the development of early blight symptoms.

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TPA production during the growth of A. solani To study the phytotoxic activity of TPA produced during the growth of A. so21ani. 100 Florence flasks (500 ml) each containing 150 ml of PD broth were inoculated as described above. At different time intervals the filtrate from 6 to 10 flasks -was harvested, lyophilized and extracted as described above. The toxicity of these preparations to potato cell suspensions was measured by determining the LD,, of each preparation as described. The LD,, units present per gram fresh weight of mycelia was then calculated for each stage of fungal growth. Analysis of TPA

inhibition of cell growth during the plant cell growth cycle Cells in stationary phase of growth were harvested under aseptic conditions on fritted glass filters. Four litre flasks containing 2 1 of medium were inoculated with these cells at a density of 8 g 1-l and were incubated on gyrator-y shakers. At various intervals following inoculation, cells were collected by filtration under sterile conditions and were used to inoculate 125 ml Erlenmeyer flasks containing 25 ml medium with various amounts of TPA. Unless otherwise mentioned, cells were harvested after 17 days and the fresh weight gain was determined as described previously [4]. Growth assayof cells The growth of cells was determined by measuring fresh and dry weight gain as described previously [4]. Ail data points represent the mean of measurements on at least 2 separate cultures. RESULTS Exposure of leaves of dzrerent plant species to TPA

Plate 1 shows the effect of TPA on potato, tomato and bean leaves after 72 h exposure. In response to large amounts of TPA (>8 LD,, units), leaves of both potato and tomato turned yellow and developed areas of necrosis within 3 to 4 days. With iesser amounts of TPA (between 3 to 8 LD,, units) tomato and potato leaves developed necrotic lesions with chlorotic borders all over the leaf surface. The least amount of TPA required to cause the development of visible symptoms was 3 LD,, units, The time required for symptoms to develop appeared to be proportional to the amount of TPA present in various treatments. Bean leaves showed little sensitivity to TPA. The bean leaves developed a few necrotic lesions without any chlorotic borders only at the highest concentration of TPA tested (16 LD,, units) (Plate 1). Similar results were obtained when 100 pi of different dilutions of TPA (between O-4 and 16 LD,, units per 25 ml agar) contained in agar plugs (6 mm diameter, 3.5 mm thick) were applied to single puncture wounds on the leaflet blades of potato, tomato and bean. Under these conditions, the minimum amount of TPA required for potato and tomato leaflets to exhibit areas of necrosis surrounded by a chlorotic border was lower (O-8 ID,, units) than that required when leaflets were floated in a solution containing the TPA (3 LD,, units). When leaves were exposed to TPA by using the agar plugs, bean leaves were cmnpletely insensitive to TPA at all concentrations tested.

PLATE 1. (Left to right) Reaction of leaflets of tomato (Lycopersicm esculentum Mill. CY. VFNT tubemum L. cv. Superior), and bean (Phaseolus &g&ii) to TPA. Leaflets were incubated in the presence in individual Petri plates. Other procedural details are the same as described in Materials and Methods.

Cherry), potato of 8 LD,, units

(Solmum of TPA

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Efect of TPA on cell suspensioncultures of A. solani host and non-host plant species Figure 1 shows the comparison of growth of cell suspensions of various plant species in the presence of varying amounts ofTPA. Cell suspensions of potato and tomato,

0

0.2 Aitwourio

04 toxin

06 (ml

0.8 per 25

ml medium)

Fro. 1. Effect of increasing concentrations of TPA on the relative growth of cell suspensions of tomato (Lcopcrsic4n esculentum Mill. cv. VFNT Cherry; 0), potato (S4&mm tubemum L. cv. Superior; ), carrot (Daucus camta cv. Danvers; A) and bean (Phnscolus wrighlz’i; 0). In all experiments, O-2 g of cells (in stationary phase of growth) were inoculated into 25 ml of medium containing varying concentrations of TPA in 125 ml Erlenmeyer flasks. Cells were collected on a Whatman No. 4 filter paper in a Buchner funnel with an aspirator after 17 days of growth. The cells were removed from the filter paper and the fresh weight was then determined [4] (- - - -) LDss.

l

the plants of which are natural hosts of A. solani, showed greater inhibition of fresh weight gain in response to increasing dosages of TPA than did cell suspensions of plant species which are non-hosts, carrot and bean. The amount of TPA required to inhibit fresh weight gain by 50% (LD6,,) was the same (0.12 ml per 25 ml culture medium) for both potato and tomato. Cell suspensions of carrot and bean showed sensitivity only at much higher concentrations of the same TPA. The LD,, for carrot and bean cells was 0.52 ml and 0.72 ml TPA per 25 ml culture medium respectively. In fact with bean cells, a 2*5-fold stimulation of fresh weight gain was observed at a toxin concentration which completely inhibited the growth of potato and tomato cells. These results indicate a species specific response to TPA used in the present study as compared to the more general toxic effect of Altemaria toxin(s) reported by other investigators [IS, 7, 161. Kinetics of growth inhibition by TPA No significant differences in the growth rate or in the final cell densities at stationary phase were observed for potato cells grown in the presence or absence of TPA (Fig. 2). However, a substantial increase in the lag period (time required to measure

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an increase in fresh weight) was observed with increasing concentrations of TPA in the medium. Cells growing in medium with l-25 LD,, and 2.5 LD,, units of TPA exhibited lag periods of 16 and 24 days, respectively, compared to a lag period of 4 to 6 days for control cultures (Fig. 2). In subsequent experiments the phytotoxic activity of TPA was determined by harvesting the cultures immediately after the control cultures had reached the stationary phase of growth.

Days oftet

inoeoloticn

FIG. 2. Kinetics of growth inhibition by TPA. Potato cells in stationary phase of growth were collected and resuspended into fresh potato medium without TPA to a density of 0.2 g ml-‘. This suspension was then inoculated into potato medium (200 ml in 1 litre Erlenmeyer flasks) at 8 g fresh wt of cells 1-r in the presence and absence of I.25 and 2.5 LD,, units TPA respectively. At indicated time intervals the fresh weight densities of the cultures were determined as described in Fig. I. 0, in the absence of TPA; a and 0 in the presence of 1.25 and 2.5 LD,, units of TPA respectively.

Effect of inoculum density on the growth of potato cell suspensionsin the presenceaud absme of TPA The effect of inoculum density on the growth of potato cells in the presence and absence of TPA was tested with cells in the stationary growth phase, More than 90% inhibition of growth (gain in fresh or dry weights) of cells was observed in the presence of TPA concentration equivalent to 2 LD,, units when the initial inoculum density was less than or equal to 0.2 g cells per 25 ml culture medium (Fig. 3). However, with inoculum densities greater than 0.2 g cells per 25 ml culture medium, the growth inhibition due to the presence of TPA in medium decreased markedly with increasing cell densities. No significant inhibition in cell growth due to the presence of TPA was observed at inoculum densities greater than 0.35 g of cells per 25 ml-* culture medium (Fig. 3). In subsequent experiments an inoculum density of 0.2 g cells per 25 ml culture medium was used to determine the phytotoxic activity of TPA. Changes in TPA inhibition of cell growth during a tin&e growth cycle of sttqkhn a&res Cells were taken from different stages of the growth cycle and their sensitivity to TPA was examined. Figure 4 shows the response of tomato cells of Werent ages (different days following inoculation) to increasing concentrations of TPA. The amount of TPA required to inhibit the increase in fresh weight of tomato e&s by 50% varied greatly depending upon the age of the cells which were expo~ecl to the

Production

and toxicity

of Alternaria

lnoculum

solani toxins

density

(g per 25 ml medium)

FIO. 3. Effect of inoculum density on growth of potato cells in the presence and absence of TPA. Potato cells from stationary phase were resuspended in fresh medium and then inoculated at the indicated cell density in 25 ml of potato medium in 125 ml Erlenmeyer flasks in the absence (0) and presence (0) of 2 LD,, units of TPA. After 17 days cells were collected on a Whatman No. 4 filter paper and the fresh I-) and dry . (-, - -) weights ’ were determined as described in Mate&& and Methods. .

Alfernuria

toxin

(ml per 25 ml medium)

FIG. 4. Effect of increasing concentrations of TPA on the relative growth of tomato cell suspension as a function of the growth cycle stage. Cells taken after various days following inoculation were subcultured into medium containing increasing concentrations of TPA. The cells were allowed to grow for 17 days at which time fresh weights were determined. All inoculum densities were 0.2 g fresh wt per 25 ml of culture medium. The figure shows the fresh weight gains in media containing various amounts of TPA as a percentage of fresh Also shown is the volume of toxin required to inhibit weight gain in the medium without toxin. 50% gain in fresh weight at each stage of growth cycle.

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toxin. The sensitivity of tomato cells was lowest just before the cells entered exponential growth and increased during all other stages of growth (Fig. 5). The LD,, of’ stationary phase cells was between 0.16 to 0.2 ml TPA per 25 ml culture medium. whereas the LD,, when the cells were at minimum sensitivity was 0.4 ml TPA per 25 ml culture medium (Figs 4 and 5).

0

16 8 12 Days after inoarlation

4

FIG. 5. Amounts of TPA of growth cycle stage. Other

required details

20

24

’

to inhibit growth of tomato cells by 500/b as a function are as described in Fig. 4. 0, Fresh weight; 0, LD,,.

A reverse trend in sensitivity to TPA was observed with potato cell suspensions which exhibited minimum sensitivity in the stationary phase (Fig. 6). The LD,, of stationary phase cultures was 0.48 ml TPA per 25 ml culture medium as compared with 0.16 ml TPA per 25 ml culture medium for cells from early exponential growth phase. The relative sensitivity to TPA decreased rapidly as cells entered the rapid growth phase and reached the minimum sensitivity (LD,,z0*46 ml TPA per 25 ml culture medium) around mid log phase of growth (Fig. 6).

-z 5.I B E 4-z

0

4

8 Days after

12 16 inoculation

20

FIG. 6. Amounts of TPA required to inhibit growth of potato cells by 50% as a function of growth cycle stage. Other details are as described in Fig. 4 except that potato cdfs WCC used instead of tomato cells. 0, Fresh weight; 0, LDbo.

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To determine if non-host species also show growth cycle dependent changes in sensitivity to TPA, bean cells taken from different stages of the growth cycle were examined for their sensitivity to the same A. solani toxin preparation (Fig. 7). The pattern of relative sensitivity to TPA at different stages of growth of bean cells was similar to that of tomato cells. In both cases cells showed minimum sensitivity to TPA just before they entered the exponential growth phase. However, concentrations of TPA required to inhibit the increase in fresh weight of bean cells by 50% were 4 to 8-fold higher than those required to inhibit tomato or potato cell growth by 50%.

FIG. 7. Amounts of TPA of growth cycle stage. Other used instead of tomato cells.

Days after inoculation required to inhibit growth of bean cells by 50% as a function details are as described in Fig. 4 except that bean cells were 0, Fresh weight; 0, LD,,.

An assayof TPA phytotoxicity bmed on the growth inhibition of cellsin srcspenrion The inhibitory effects of TPA on cell growth are reproducible when the cells are obtained from a defined stage of growth and are inoculated at a predetermined inoculum density. This reproducible growth inhibition by TPA can be used as an assay to quantitate the phytotoxicity of TPA. The use of cell suspensions from plant species which are hosts or non-hosts to A. solani may allow the determination of species specific phytotoxicity of different toxin preparations (Fig. 1). Potato cells appeared to be the best cells for the quantitation of phytotoxicity of TPA since the LD,, of potato cells remains virtually unchanged during the late exponential and stationary phase of growth (Fig. 6). Thus, the differential response of cells at different stages of the growth cycle to TPA can be eliminated easily by using cells from these growth phases. Tomato cells also can be used to determine the amounts of phytotoxic activity present in TPA. However, changes in the response of tomato cells to TPA during the cell growth cycle (Fig. 5) make it more difficult to eliminate the effects of growth cycle on the assay procedure. Production of phytotoxic activity during the growth of A. solani Figure 8 shows that when A. solani mycelia are grown in PD broth, the maximum phytotoxic activity (LD,, units g-l fresh wt) occurred in the early log phase of

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growth. Although there was rapid growth of mycelia 12 days after inoculation, and the mycelial fresh weight increased from 15 to 40 g 1-l in the next 4 days, there was no increase in total phytotoxic activity of the fungal filtrate during that period. The amount of extracellular phytotoxic activity per gram fresh weight of mycelia declined from a maximum of about 65 LD,, units to about 5 LD,, units g-l fresh weight of fungus even though the fungus was growing rapidly during this time (Fig. 81, These results suggest that the amount of extracellular phytotoxic activity is regulated during the growth of A. soiani.

Days after

iroxlatii

FIG. 8. Growth curve of A. soluni in potato dextrose broth and the phytotoxic activity (total LD,, units) of culture filtrate obtained after various periods of growth. Ako shown is the depletion of glucose from the medium. Phytotoxic activity was determkd with potato cell cultures as described in the text. 0, Fresh weight; A, toxin, 0, glucose.

Selectionof potato cell su.$ensionswith enhancedtolerance to A. solani toxin preparations As seen in Fig. 2, potato cells eventually (after a prolonged lag period) grow in the presence of TPA. Once a cell population has grown in the presence of TPA the cells are apparently changed (either by selection, adaptation or both) such that their growth is not as sensitive to the same toxin preparation. Figure 9 shows that the tolerance of cells to TPA is increased as the cells are grown in the presence of TPA. This tolerance of potato cells to TPA can be substantially increased by growing these cells in a medium containing higher amounts of TPA. As shown in Fig. 9(b), the LD,, of potato cells increased about 5-fold as the tolerant cells obtained from one cycle were transferred sequentially to medium containing increasing amounts of TPA. DlSCUSSiON Recently there has been considerable interest in selecting cultivan resistant to pathogens which produce pathogenic toxins using in vitro techniques El, 2, 5, 8, 9, 11, 13, 18, 23, 381. However, to apply selection pressure in a reprodneible manner it is essential to be able to determine quantitatively the phytotoxic activity present in a toxin preparation. Several bioassays have been used to detect the

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toxins (a)

0 Alfwnur~

200 toxin

IpI

Selected

400 per 25 ml

60 0 medium)

cell lines

Fro. 9. (a) Relative growth (after 17 days) of unselected (Ss) and selected cell populations (S,, S,, S,, S,) of potato inoculated into medium containing varying concentrations of TPA. Eighty microlitres of TPA per 25 ml of culture medium was equivalent to one LD,, unit. S,, S,, S,, S, represent the cell populations which were selected to grow in the presence of 2, 3, 4, and 5 LDss units of TPA respectively. To select S, potato cells, Ss cells were inoculated in the presence of 2 LD,, units of TPA. After an extended lag period (Fig. 2), these cells (S,) which eventually grew up in the presence of 2 LDss units of TPA were maintained in the presence of 2 LD,, units of TPA. S, cells were then inoculated in the presence of 3 LDss units of TPA and those cells (S,) which grew up were maintained in 3 LDss units of TPA. Similarly Ss and S, cell populations were selected from Ss and S, cells in the presence of 4 LD,, and 5 LDss units of TPA respectively. S, and S, cells were maintained routinely in the presence of 4 LD,, and 5 LD,, units of TPA. Other details are as in Fig 1. (b) Relative resistance of selected cell lines of potato to TPA indicated as the LD,,, in ~1, of same TPA per 25 ml of culture medium.

production of host specific and non-specific toxins by various pathogens. In most cases assays based on the induction of typical disease symptoms such as chlorosis, necrotic lesions, chlorotic halos, etc., have been used to detect the production of host or species-specific toxin(s) by pathogens [24, 31, 37, 39, 40-J. Assays based on physiological effects such as stimulation of respiration or changes in membrane permeability have also been used to quantitate the phytotoxicity of culture filtrates and mycelial extracts of fungal pathogens [ZZ, 39, 401. Some early researchers used the degree of root and shoot growth inhibition as a measure of toxin activity present in extracts [24, 301. These assays have been used to obtain meaningful results in several hostpathogen interaction studies. However, based on these assays, it is difficult to predict the concentration of toxin required to achieve selection of toxin resistant cells and plants using in vitro techniques. The experiments reported here show that cell suspensions of plant species which are hosts of A. solani can be used successfully to determine the amounts of species-specific toxicity present in TPA. Our results show that A. solani toxin(s) was 4 to 8-fold more toxic to cell suspensions of plant species susceptible to A. solani (potato and tomato) than to cell

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suspensions of non-host plant species (Figs 1, 4, 5, 6 and 7). Since Alternaria spp. are known to produce several phytotoxic compounds [S, 7, 161, the observed inhibitory effects at higher levels of TPA on cell suspensions of non-host species may bc due to the presence of other non-specific phytotoxic compounds in TPA. Although it is difficult to generalize from a single experimental system, the above results offer an attractive possibility of using plant cell suspensions to distinguish and quantitate the production of host specific and general toxic compounds by various pathogens. Plant tissue culture techniques have many potential advantages for the study of host-pathogen interactions. Most experiments with cell suspensions can be conducted in well defined and controlled chemical and physical environmcnt~. However, many factors can affect the determination of phytotoxic activity of a toxin or any other compound when tissue culture systems are used for such determinations [28, 42, 43]. Our data demonstrate clearly that initial inoculum density (Fig. 2) and the stage of cell growth (Figs 5, 6 and 7) greatly influence the susceptibility of cells to a given TPA. During growth, cells in suspension undergo developmental changes including changes in several cellular activities [IO, 12, 1.5, 21, 26, 27, 33, 34, 35, 411. Depending on the mode of action of a phytotoxin, these changes can have significant effects on the response of the host cells to phytotoxins. Furthermore? it is clear from our data (Figs 5, 6 and 7) that the effect of cell growth stage on the tolerance of the host cells to the toxin cannot be generalized from the response of the cell suspensions of one plant species. Even though plants of both potato and tomato are severely affected by A. solani, the potato cell suspensions showed maximum sensitivity to TPA at a growth phase where the tomato cell suspensions were most tolerant. The cell cultures of bean (a non-host to A. solani) also showed the cell growth stage dependent effect on the sensitivity of the cells to TPA. Ef&cts of inoculum density and the physiological stage of growth on sensitivity to phytotoxins have been observed in callus cultures also [42]. The inhibition of cell growth in culture by phytotoxic compounds can be explained in many ways. In general, the presence of phytotoxic compounds in the medium affects the growth of cells either by increasing the lag period (time period before cells begin to grow) or by reducing the growth rate. In some cases, the inhibition of cell growth is due to the killing of cells which lowers the inoculum density and increases the lag period which is generally thought to be the mechanism involved in cell selection. Before cell suspensions can be used to measure phytotoxicity-, the pattern of observed cell growth inhibition should be determined. Without such information, measurements of relative growth may be misleading if cells exposed to toxin are allowed to grow after growth of control cell cultures has ceased. The influence of these possible mechanisms on the determination of phytotoxicity by tissue culture techniques has been discussed [42, 431. In the present study, TPA inhibited the growth of cells by increasing the lag period (Fig, 2). However, other phytotoxic compounds may inhibit the cell growth by reducing the growth rate. We cannot tell from our results whether the enhanced ability of potato cells to grow in the presence of TPA (Fig. 9) is the result of a true selection of variant cells within the normal population, or the result of adaptation of cells to the TPA imposed stress, or a combination of these, i.e. selection of adapted cells. It should be pointed out, however, that it is unlikely that selection of variant cells could occur in only

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16 days (Fig. 2) unless the variants are already present at a high frequency, If it is assumed that the observed growth pattern of TPA-tolerant cells (Fig. 2) is the result of the growth of resistant variants within the unselected population such variants would have to occur at a frequency of about I.15 and O*O7o/ofor TPA concentrations of 1.25 and 2.5 LD,, units respectively. This is assuming a lag period of 0 days. An increasing lag period requires even higher frequencies of variants. For example, a lag period of 4.5 days (equivalent to that of the unselected population) would require these variants to occur at a frequency of 4.75 and 0.3% for TPA concentrations of I-25 and 2.5 LD,, units respectively. Based on this analysis of Fig. 2, it seems more likely that adaptation of the cells rather than the growth of a small fraction of variant cells is responsible for the isolation of cell lines with enhanced tolerance to TPA. At present we do not know if this enhanced tolerance to TPA is stable in the absence of TPA. However, stability of tolerance characters in the absence of stress agents has been reported for other selective agents using in vitro systems [I, 2, 5, 8, 9, 13, 23, 38) while other reports indicate failure to maintain the resistance in the absence of the stress agent [4, 9, 17, 381. At present little is known about the production of toxin(s) in A. solani. We have used growth inhibition of potato cell suspension cultures as an assay to determine the production of phytotoxic activity during the growth of A. solani. Our results show that A. solani produces maximum amounts of the extracellular phytotoxic activity during the early log phase of its growth. Our results are different from those reported by Matern et al. [23]. These authors had reported that toxin production peaked at 8 weeks following inoculation, coincident with maximum mycelial growth, when A. solani was grown in a modified Czapek-Dox medium. Toxin production peaked at 35 days following inoculation when grown in a medium developed by Lukens & Sisler [23]. Since we have used a medium (PD broth) different from that used by Matern et al. [23], it is not possible to make a direct comparison between the present study and that reported by Matern et al. [23]. However, it is possible that production of the maximum toxin during the growth of A. solani is influenced by the chemical composition of medium used for its growth. It is apparent from these results that the production of extracellular toxin by A. solani, during its growth, is regulated. A further understanding of mechanism(s) of regulation of toxin synthesis in A. solani may provide significant information concerning the host pathogen interactions and subsequent development of early blight in potato and tomato.

We are thankful to Drs L. D. Dunkle and R. L. Nicholson for suggesting improvements in the manuscript and to Dr R. L. Nicholson for technical advice. REFERENCES 1. BEHNICE,

M.

(1979).

Selection

of potato callus for &stance to culture filtrates of F%yftophthma of resistant plants. Theoretical and Applied Genetics55,67-71. Selection of diphaploid potato callus for resistance to the culture filtrate of

infestam and regeneration 2. BEHNKE,

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