Biological activity of purified host-specific pathotoxin produced by Bipolaris (Helminthosporium)...

Physiologicnl Plant Pathology (1980) 16, 227-239

Biological activity of purified host-specific pathotoxin produced by Bipolaris (Helminthosporium) maydis, race T f

GARY PAYNE,: H. W. KNOCHE, Y. KONO§ and J. M. DALY

Laboratory of Apicultural Biochemistry, University of Nebraska, Lincoln, NE 68583, U.S.A.

(Acceptedfm publication October 1979)

A purified, chemically characterized toxin from Helmintkosporium maydis, race T, was examined for its host-specific effect on Texas male sterile (T) cytoplasm corn, This preparation consists of several nearly identical linear polyketols and it is shown that each component has the same specific toxicity for oxidation by mitochondria from susceptible corn. The stability of the dry preparation and knowledge of its mol. wts enabled a quantitative comparison to the dosage required to affect ion balance, dark CO, fixation, coleoptile elongation, and mitochondrial oxidation. Unexpectedly, the toxin was active at concentrations between 5 and 50 ng ml-i (6.5 x 10 --O to 6.5 x 10 -8 M) on all these processes in T corn, despite potentially different barriers to toxin penetration. N cytoplasm corn was not affected at concentrations 1000 times greater. These results are discussed in relation to possible modes of action. The quantitative specific effects of this preparation are compared to data on other race T toxin preparations for which chemical structures have been postulated.

INTRODUCTION In common with other host-specific [24] or host-selective pathotoxins [27], the toxin produced by Bipolaris (Helminthos@orium) maydis, race T, affects a variety of physiological and biochemical processes in susceptible plants. So far described are host-specific effects on ion leakage [2, II, 13, 161, mitochondrial oxidation [4, 9, 10, 12, 18, 211, root growth inhibition [2, 6, 141, stomata1 closure and transpiration [I], dark CO, fixation [6] and photosynthesis [I, 61. Although all the effects may ultimately be related to a single primary site of action, for example, mitochondrial oxidation [12, 181, there are dissenting [2, 201 and precautionary views [7].

At first glance, it would appear that useful criteria in identifying a primary site would be (a) the minimum time necessary to observe an effect and (b) the amount of toxin required. Arntzen and co-workers [Z], for example, have argued that because inhibition of root growth apparently occurs within minutes, while respiratory increases are observed only after hours, interference with plasma membrane function may be more important than interference with mitochondrial oxidation.

There are limitations to the use of either of the above criteria. Each of the above processes must be measured by different physical methods which differ intrinsically in their sensitivity and their ability to determine rapid changes. Furthermore, these

tPublished as Paper No. 5832, Journal Series, Nebraska Agricultural Experiment Station. Supported in part by Cooperative States Research Service Special Grant 216-15-22.

$Present address: Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27650, U.S.A.

$Present address: Institute of Physical and Chemical Research Wako-Shi, Saitama 351, Japan. 0048-4059/80/020227+ 13 $02.00/O @ 1980 Academic Press Inc. (London) Limited

228 G. Payne, H. W. Knoche, Y. Kono and J. M. Daly

assays require tissues with potentially dissimilar properties, such as rate of penetration by toxicant. As important, perhaps, is the fact that comparisons must be made of published data from several laboratories, and obtained with unstandardized toxin preparations varying in purity. Often, results are expressed only in terms of dilutions of crude culture filtrates.

Recently, Kono & Daly [17] reported that race T toxin consists of a mixture of C,, to C,, polyketols, each with apparently identical host-selectivity and toxicity (approximately IO-* to 10TB M). In this paper, we provide more detail on the activity of individual components of race T toxin. The possible structure of the principal component (C&HssOrs, 768 daltons) is illustrated below. Availability of stable purified toxin, and knowledge of its molecular weight, enabled us to quantitatively compare the sensitivity of several diverse systems affected by toxin.

POsIible structure

Bond 1 rm~ 1 toxin

In addition, two laboratories [3, 151 have postulated toxin structures at variance with that of Kono and Daly [17]. The data to be presented show that race T toxin prepared by our methods is intrinsically more toxic than the preparations for which other structures have been postulated, thus supporting the physical and chemical evidence for the purity of the preparations upon which the evidence for the structure shown above was obtained.

MATERIALS AND METHODS Toxin preparations

Toxin was isolated as described elsewhere [ 171. Because of limited solubiiity in water, stock methanol solutions were prepared at toxin concentrations of 1 to 5 mg ml-1 by transferring O-5 to 2 mg of toxin to Pierce Reactivials. After adding the appropriate volume of either pure or aqueous methanol (33%), the Reactivials were sealed with serum stoppers and heated to 40 to 60 “C to dissolve toxin completely. Upon cooling, a volume of the stock solution was withdrawn by microliter syringe, without breaking the seal of the Reactivial, and added to distilled water to give working solutions of 5 to 10 ng ml-‘, of which only ~1 amounts were used experimentally.

For most processes studied, the final concentrations of toxin to which tissue was exposed usually were less than 100 ng ml-l, resulting in a IO4 minimum dilution of the methanol in stock solutions. Nevertheless, potential complications, if any, due to the presence of methanol were checked with controls supplied with methanol at concentrations equal to or up to 10 fold greater than those arising from toxin stock solutions. No effects of methanol were observed at such concentrations.

Plant materials Corn (w64A Tms and W64N) was sown in Perlite, watered with distilled water and maintained in the dark at 28 “C. Coleoptiles and root segments were prepared from seedlings 4 days from sowing.

Host-specific pathotoxin from Bipolaris may&s

Coleoptile growth

229

4 m m coleoptile segments were cut starting 2 m m behind the tip and the interior seedling leaves then removed. Eighteen segments were placed in 4 ml of solution contained in glass vials 65 m m x 16 m m inside diameter. After capping with a serum stopper, the vials were continually rotated in the dark, along the long axis of the vial, in order to aerate and to minimise coleoptile curvature. The period of growth at room temperature (21 to 24 “C) was 17 h.

Ion leakage from roots In preliminary experiments, 1 cm root tips and a 1 cm segment immediately behind this were compared, but most data were obtained for the second segment. Unless indicated otherwise, segments were aerated for 4 h by bubbling compressed air through 300 to 400 ml of solution containing excised segments. To 3 ml of solution contained in small plastic dishes (6 m m deep by 38 m m diameter) were added generally 30 segments. The total fresh wt of 30 segments corresponded to approximately 200 and 250 mg for 1 cm root tips and second segments, respectively. Light was excluded by placing individual dishes under inverted aluminium weighing pans, removed only during the 10 to 20 s required for conductivity readings. We have no evidence that toxin binds to plastic but as a precaution new dishes were used for each run.

Conductivity (pmhos, pS) of bathing solutions was determined by withdrawing solution into a Radiometer (Copenhagen) conductivity cell (capacity 1 ml) through a syringe needle with the aid of a rubber bulb attached to the open end of the cell. The needle prevented entry of tissue pieces as the bathing solutions were being mixed by several rinses of the conductivity cell before measurement. After determining conductivity in time course studies, the solution in the cell was returned to the plastic dishes.

Dark CO 2 fixation Handling of leaf discs and assay procedure was as previously described [S].

Mitochondrial oxidation Mitochondria were isolated from 5 day-old etiolated shoots by the usual differential centrifugation procedures. The results will be described elsewhere [22]. After final pelleting through a cushion of O-6 M sucrose the mitochondria were suspended in O-4 M

sucrose (0.1 ml for each 10 g fresh wt corn shoots). Generally 0.07 to O-10 ml (approximately 0.5 mg of protein) was added to 3 ml of assay medium consisting of 200 m M sucrose, 20 mu KCl, 2 m M MgCl,, 1 mg ml-r bovine serum albumin, 20 m M TES [Jv-tris (hydroxy-methyl) methyl-2-aminoethane sulfonic acid] and 5 m M KH,PO, adjusted to pH 7.5. For studies where exogenous NADH was used, the concentration of NADH (1 mM) was in slight excess of the amount of substrate required by mitochondria to reduce oxygen tension to zero in the electrode vessel. When malate was the substrate, a concentration of 60 m M was added to give maxi- mum rates. A limiting amount of ADP (50 JIM for NADH and 100 pM for malate) was used to establish state 3 [8] rates before and after toxin treatment. When mitochondria were oxidizing exogenous NADH, the effect of toxin was measured as an


TABLEI

Effect of toxin on ion leakage andgrowth of I m m coleoptilesegments of Tms corn in thepresence of IAA

Coleoptile growth

Cont. IAA (M)

Toxin8 (ng W

, Final

Conductivity length Increase Inhibition (LunW (-4 (-1 of growth

0 0 188 6.8 2.8 - 10-d i.5 147 9.3 5.3 0 10-h 153 7.8 3.8 27 lo-’ 5 161 7.4 10-a 50 174 6.9

2”:; 36 45

lo-’ 500 288 5.8 1.8 66

a No effects on N corn at 50 pg ml --1.

increase in the state 4 rate within 2 min after addition of toxin. When malate was used as substrate, toxin was added in state 3 and the rate of the subsequent state 3 was compared with that of nontoxin treated control.

Except for studies on mitochondria, each datum given is the average of at least two replicates. Each experiment was repeated at least once.

RESULTS During the course of the work, several different preparations of purified toxin were tested on each process. All showed the same effectiveness.

Growth inhibition In the experiment of Table 1, as little as 2.5 ng toxin ml-l, over a 17 h period, inhibi- ted IAA-stimulated growth of Tms corn coleoptiles appreciably. No effects were observed on N-corn at up to 50 pg ml- l. Higher concentrations were not tested because of potential problems with solubility in aqueous media. The limited elongation obtained in the absence of IAA made evaluation of toxin effects difficult at the lower toxin concentrations. Also shown in Table 1 is concentration dependent stimulation of ion leakage, which correlates well with the growth inhibition if the conductance of the 10S4 IAA controls is used as reference. The data presently available do not permit conclusions about the relationship between these phenomena.

A number of variables (IAA concentration, presence or absence of sucrose, pH, aeration, salts, buffers) were examined for their effects on toxin inhibition of coleoptile growth. Consistent inhibition by toxin was observed at 5 ng ml-l, but the magni- tude (10 to 40%) appeared to depend upon the extent of coleoptile growth in the controls.

Ion leakage Private communications with several laboratories had indicated difficulties in detect- ing a rapid induction of ion leakage by toxin treated TMS corn tissue. Most success- ful attempts apparently have required a 2 to 4 h pre-incubation of roots with relatively unpurified toxin preparations, perhaps because the presence of ions in the preparations precluded early detection of low rates of induced leakage.

Host-specific pathotoxin from Bipolaris maydis

TABLE 2

231

Changes in conductivity (pmhos) of distilled water bathing segments cut between 2 and 3 ems behind tips of Tms and N corn. Data in parenthesis are for Jirst I cm root tip segments at 23 h. Segments

rinsed but not aerated.

I Time in toxin solutions (h)

A Toxin (ng ml-‘) 1.5 2.0 2.5 3.0 5.5’ 6.0” 23 1 cm

0 5

50 500

0 5

50 500

73 70 77 86

69 74 76 68

Tms corn 88 92 97 98

N corn 80 79 88 86

123 90 142 189 151 269 (510) 156 320 WO)

101 150 (220) 109 110 -* 103 158 (148) 92 115 (146)

(t No differences in pH of solutions at these times. b Not measured.

Purified preparations of race T toxin do not contribute to the conductance of distilled water and, in preliminary experiments, when added to rinsed root segments causes a concentration dependent leakage in Tms, but not N, corn at the first time period examined (Table 2). In other experiments direct determination of Na + and Ca + + have shown 2- to lo-fold increases of these ions when compared to controls after 15 h. The results appear to be at variance with the non-specific induction of leakage reported by Keck & Hodges [16] but purity of the toxin preparations may be a factor. The data of Table 2 were obtained with segments cut from the second cm behind the root tip. Similar, but less obvious effects, were obtained from the first cm (root tip) segment, which is distinguished physiologically from the second segment by little or no vacuolation and hence fewer or no tonoplast membranes. In our experi- ence, considerably higher rates of endogenous leakage are found for the root tip and, in addition, endogenous leakage continues for at least 24 hours. In contrast, 2nd cm root segments begin to re-absorb ions starting 7 or 8 h after excision. These effects are illustrated by comparing the conductivity at 23 h for 2nd cm segments and 1 cm root tips (in parenthesis, Table 2).

TABLE 3

Effect of aeration for 4 h in distilled water on changes in conductiuity (pmhos) of solutions bathing root segments in the absence or presence of race I toxin (500 ng ml- ‘)

Tissue Tips (1 cm) 2nd segment (1 cm) Aeration - - + + - - + + Time intoxin (h) 3 7 3 7 3 7 3 7

Control 224 305 59 85 168 197 65 77 Toxin 239 390 113 170 106 163 133 158 Toxin/control 1.07 1.28 1.91 2.00 1.23 1.33 2.05 2.05


% m- f ._> t 2 60- s 0

40 -

20 -

L I I I I I I I I 2 3 4 5 6 7

Time (h)

FIG. 1. 2nd cm root segments of Tms corn aerated in either water or 10 -* M CaCl, for 4 h, then transferred to 3 ml of either water or CaCl,. Each curve is identified by the aeration solution followed by an arrow indicating the experimental solution used for conductivity measurements. The fourth treatment (CaCl,+CaCl,), is omitted to avoid overlaps with upper curve (HsO-CaCI,). Toxin at 50 ng ml-r. A, CaC1,-+CaCl,; A, H,O-+CaCl,; o, HsO+HsO; 0, CaCl,+HsO.

In several experiments, root tips appeared to be less affected by toxin but aeration for 4 h in distilled water in the absence of toxin reduced endogenous leakage in both types of segments, so that for a given toxin concentration the quantitative responses are nearly the same (Table 3). By reducing endogenous leakage through aeration, more rapid responses to low concentration of toxin could be established.

The most rapid induction of ion leakage was obtained if toxin addition was delayed until the high initial endogenous leakage fell to rates of 1-O uS h-1 or less (Fig. 1). Second segments were excised and aerated either in distilled water or 1 OV4 M CaCl,. After 4 h, segments from each treatment were assayed for the effect of toxin at 50 ng ml-r either in water or 10e4 M CaCl,. Assaying in the presence of CaCl, resulted in higher background conductivity and perhaps a slightly initial rate of leakage but, regardless of treatment, a sharp increase in rates of leakage was evident at the next conductivity reading. If rates were 3.4 1.6 h- l or more at time of addition, the attainment of new rates was more difficult to establish. Delayed addition of toxin permitted detection of toxin effects at low toxin concentrations (5 ng ml-l) at 10 min from addition. This was the earliest time the sensitivity of the instrument would allow with the rates obtained.

Table 4 shows that either on a weight, or especially on a molar, basis race T toxin (7 x 10-s M) is 10 times more effective in inducing rapid ion leakage than is the synthetic respiratory uncoupler, 2, 4-dinitrophenol (DNP) ( low6 M). Further, even

Host-specific pathotoxin from Bipolaris maydis

TABLE 4

233

Comparison of ion leakage (,umhos) f ram Tms corn root segments caused by toxin and DNP added at zero time

Cont. Time (h) ---h---_7 Y-------- * 1

wt ml-’ M 1.0 1.5 2.0 2.5 3.0 19.5

Disk H,O - - 49 54 60 61 64 33 Toxin 50 ng 7x 10-84 52 60 74 85 92 119 DNP 184 ng 10-n 52 59 63 67 70 28 DNP 18.4 pg 10-a 77 90 100 108 118 36

a For a mol. wt of 768 daltons (17).

6 6 4

2 0

-2

IQ” 10-s 10-7

Molar

Hours ofter fresh solution (10T4 Y CaC12 t Toxin)

FIG. 2. The effect of long exposures (20 h) of Tms root segments to various concentrations oftoxin(seetextfordetails). 0, 5Ongrnl-l; n ,5ngml-l;O, 500 pgml-I; A, 5Opgml-l; A, control.

at 10s times the molar concentration of toxin, ions are reabsorbed from DNP solutions. In these experiments, there were indications that corn roots may metabolize or accumulate DNP from solution at later stages of exposure.

In addition to producing rapid effects on ion leakage at 5 to 50 ng ml-l, Fig. 2 shows that even lower concentrations are effective acting over time intervals compar- able to those required for symptom development. Segments of 1.5 cm were excised starting at 1.0 cm behind the root tip, aerated for 2 h in 10m4 M CaCl, and placed directly in 10W6 M Call, containing 50 pg, 500 pg, 5 ng and 50 ng per ml of toxin.


80 -

c .o

5 60- .E u _-e-m B g 40-

i!

20 - (2

1.0 2.0

Log ng ml-’

FIG. 3. Log dosage plot of inhibition of dark CO, fixation of leaf discs of Tms corn. Concentrations in ng ml-l shown in parentheses. (50% inhibition at 24 ng ml-‘.)

After 4 h, the leakage from control and these toxin concentrations were 62, 72, 78 104 and 127 pmhos, respectively. At 20 h, fresh solutions of the same composition replaced the original solution. The slopes of Fig. 2, calculated by linear regression, are plotted in the insert of Fig. 2. The correlation coefficients were O-95 or greater, except for 500 pg ml- 1 which was only 0.4. Poor correlation coefficients are expected when slopes approach zero (C. R. Daly, pers. comm.). The slopes for 0, 50 and 500 pg of toxin correspond to re-absorption of ions at rates of 2.12, l-17, O-1 7. Slopes for 5 to 50 ng ml-1 corresponded to continued leakage at rates of 1.2 and 7-3 l.tmhos h-l The molarities shown in the insert are based on a molecular weight of 768 daltons.

Dark CO, Jixation Using a 6 h pre-incubation in the light, followed by l-5 hours for dark r4C0, fixation [6], the results of Fig. 3 were obtained. Other tests resulted in 50% inhibition of this process by concentrations of toxin ranging from 10 to 30 ng ml-l. Similar effective concentrations were noted in earlier work using toxin prepared by different methods [61* Mitochondrial oxidation Studies of the effect of toxin on mitochondrial oxidation of several intermediates are described elsewhere [ZZ]. Generally, 5 to 10 ng toxin per ml was required to cause 50% stimulation of state 4 oxidation [8] of NADH, first described by Miller & Koeppe [,?I] and extended by others [I, 9, 10, 12, 18, 231. In the present studies, mitochondria were put through two cycles of state 3 (50 mM ADP) and stage 4 (ADP limiting) to establish respiratory control and ADP/O ratios, requiring a total of 8 to 10 min. At the end of the second state 4 period, toxin was added. Increased oxygen uptake was apparent al,most immediately, but the rate shown were measured only at 2 min after toxin addition. After 3 to 4 min, another cycle of state 3 and state 4 was measured.

Host-specific pathotoxin from Bipolaris maydis 235

TABLE 5

The effect of 10 ng ml -l of toxin or its individual compottents on the rate’ of oxidation of exogenous JVADH by mitochondria from etiolated shoots of Tms corn seedlings

Run no. ha

Toxin y. added stim. S3 %

Controls 1 270 100 100 - 270 105 13 258 123 123 -

Toxin 2 270 94 146 55 316 223

1: 240 287 100 94 159 152 62 59 281 310 199 182

Band 1 8 258 100 176 76 305 199 12 234 100 164 64 246 188

Band 2 4 250 94 141 50 305 176 9 252 100 152 52 270 164

Band 3 6 299 94 199 112 - 11 246 105 176 68 281 188

Bands 5, 6, 7 7 270 100 176 76 328 223

LI Rate in nmol 0, min-r mg protein-‘. b S,=state 3; S,=state 4 [S].

TABLE 6

The effect of 30 ng toxin per ml and its ixdividual components on the rate’ of odation of malate by mitochondria from etiolated shoots of Tms corn seedlings.

Cycle 1 S,

Cycle 2 S*

Cycle 3 S,b

Cycle 4 o/0 Inhibition SS hC

Control 147 125 107 89 0 l/2/78 Toxin 147 129 107 71 20 Band 1 147 116 103 63 19 Band 2 143 134 116 80 10 Band 3 143 125 112 71 20 Fraction 5 138 125 107 71 20

E nmoles 0s min-r mg protein-r. b Toxin added during this state 3. ’ Inhibition of the state 3 rate following toxin treatment, relative to the non-toxin treated control.

For the experiment of Table 5, approximately 100 mg of toxin was separated into individual components by thin layer chromatography as described [17]. Second or 3rd stage chromatography was employed until single components were ensured, and each was identified as before 117-J. Solutions were prepared by weighing 100 to 600 pg samples on a Cahn electrobalance. Sufficient amounts of the major components (bands 1 C,,H,sO,, and 3, Cs,H,,O,,) were recovered for separate testing. There was not enough of the next largest component (band 4) to permit accurate weighing and it was necessary to combine bands 5, 6 and 7.


During the course of the experiment of Table 5, nontreated controls were run at the beginning and at the end, and original toxin (l/2/78 toxin) was tested three times as indicated by the heading ‘run number’. Except for the first test of band 3 (run number 6) all observed stimulations of NADH oxidation by original toxin and its individual components were similar, and the variation is within the biological error (note first cycle S, values). A repetition of the experiment several days later with only one test for each component, but two for band 3, also showed no significant differences. Run 6 of band 3 (Table 5) most likely involved a pipetting error.

During this second test the toxin components were also examined for their activity on malate oxidation (Table 6). Mitochondria were treated as above except toxin was added during the third state 3 cycle. The rate of subsequent state 3 was compared to that of mitochondria in the absence of toxin. Once again the differences in the activities of the components were small and within biological error.

DISCUSSION

Our data show that the toxin described by Kono & Daly [17] is extremely active and host-specific for a number of physiological processes in T cytoplasm corn. Two other toxin structures have been proposed. Aranda et al. [3] have described race T toxin as an acetate ester of mannitol covalently linked with two .%formy-L-valine residues. Karr, Karr & Strobe1 [15], on the other hand, found no evidence for amino acids and have suggested the toxin consists of a sterol or related glycosides. The high biological activity and specificity of the toxin produced by the procedure of Kono & Daly [17] support the physical and chemical evidence for the purity of the preparation upon which the evidence for the structure was obtained.

Berville [5], for example, reported that 1 to 2 pg ml-l of the Aranda et al. preparation was required to observe minimal effects on mitochondria; maximal effects required 10 l,tg ml- 1. Table 5 showing significant effects at 10 ng ml-1 with our preparation suggests the preparation used by the French group was less than 1 o/o pure [3, 151. In addition, no evidence for carbohydrates or amino acids as components has been obtained in either our laboratory [17] or that of Dr C. Tipton of Iowa State University.

It is not possible to compare our data directly with that of Karr et al. [15] because the only biological activity examined by them was leaf necrosis. These workers state [15] that approximately 10-s mol of toxin caused a typical lesion when applied in a 5 ~1 drop to a puncture made in the leaf. Since they believed the molecular weight of the sterol was 388 daltons [15], this number of moles represents 3.9 pg in 5 ~1 or a solution of 780 l,tg ml- 1. However, several laboratories have found the leaf puncture assay to be relatively insensitive [S, 251. It should be noted that uptake from solution of 4 to 8 ng of our preparation is sufficient to kill a first leaf of corn in 3 days.

Similarly, it is difficult to relate our data to that of workers who have used the same isolation procedures or toxin fractions reported by Karr et al. [15]. Mertz & Arntzen [19], for example, used preparations that personal communication from Karr indicated were largely ‘Toxin II’, but the results were expressed as the percentage dilution of a lo-fold concentrate of crude culture filtrate. The glucose content of a 1% dilution of the concentrate was 0.69 mM [17], from which it can be calculated

Host-specific pathotoxin from Bipolaris maydis 237

that biologically effective dilutions [19] must have contained a minimum of 1200 ltg of dry matter per ml.

Another possible criterion for purity is the differential toxicity of preparations toward N and T corn. All of the processes we have studied are unaffected in N corn at toxin concentrations at least 1000 times greater than those required to give a substantial response in T corn. The maximal differential is difficult to establish because of potential problems with toxin solubility at concentrations above 50 pg ml-l. In contrast to our observations, Mertz and Arntzen [19, ,201 found 50% inhibition of root growth of N corn at only 8 times the concentration required for 50% inhibition in T corn seedlings. Inhibition of K+ accumulation and membrane depolarization also [18] were relatively non-selectively affected by their toxin preparations.

The existence of multiple toxin species has been reported for other host-specific toxins [24], but their biological significance for H. maydis race T toxin is unknown. At least seven components can be separated and each appears to have the same toxicity in mitochondrial oxidation assays (Tables 5, 6). Detailed examination of chromatographic behavior on plates or columns by one of us (H.W.K.) suggests the possibility that some may not be natural fungal products. Interaction with the chromatographic support might lead to inter-conversions by dehydration and cycliza- tion of at least some of the components observed on thin layer plates.

At present, it is not possible to relate any of the toxin fractions of Table 5 to the four toxin fractions described by Karr et al. [15] and used by other workers [19, 261. There is little resemblance in the NMR spectra obtained with our preparations [17] and that shown for Toxin II [15]. Using the same chromatographic systems described in their report (solvents A, F, G and H, support-Absorbosil-3) [15], our preparation gave a single spot near the solvent front. In addition, the ultraviolet extinction coefficients and solubilities in various solvents are quite different. For example, the original toxin and individual components used for the experiment of Table 5 were obtained by precipitation from acetone and the components are not readily soluble (ug ml-l) in water or acetone at room temperature. Fractions designated Cl.51 as Toxins I and II were soluble in acetone, Toxins II and IV were soluble in water [15], but the apparent ease of solution and the amount dissolved in 5 l.d [1.5] may be only a reflection of the solubilities of contaminants.

Although the original race T toxin mixture appears to have the same specific activity (wt ml-l) as the individual components (Table 5, 6), difficulty in obtaining the latter has precluded tests on processes other than mitochondrial oxidation. The activities of individual components on growth, ion leakage or uptake, etc. may be useful in distinguishing primary from secondary effects.

It was unexpected to find that inhibition of each of the processes studied in uivo occurred at nearly the same concentration of toxin, despite potentially different barriers to toxin penetration (floating leaf discs versus mitochondria, for example). In the cases of dark CO, fixation and coleoptile growth, reliable assays have required extended time of pre-incubation with toxin. In both cases, however, more rapid responses can be observed. Inhibition of coleoptile growth, measured with an auxino- meter, required 2 to 5 minutes for 500 ng ml-1 (D. Parrish &J. M. Daly, unpublished observations). In an earlier report [16] inhibition of CO, fixation occurred within


16 min, the earliest feasible time for measurement. In that study [16], the concentration of toxin was unknown. More recently, Barna & Daly (unpublished data) have found appreciable inhibition of dark CO, fixation in thin leaf slices supplied NaH1* CO, at 2 and 20 ng ml-l with only 30 min pre-incubation in toxin.

It is tempting to speculate that a primary effect on mitochondria leads to subsequent growth inhibition, ion leakage, etc. but it would be unusual if all the tissues came to passive equilibrium, either slowly (growth inhibition) or rapidly (ion leakage), with the approximate toxin concentrations (5 to 20 ng ml-l) required for interference with respiration of isolated mitochondria. It should also be noted that Bednarski et al. [4] did not find stimulation of respiration in toxin-treated root tips, although other root segments did respond. It is possible that respiration control is different in root tips [a], but toxin-induced ion leakage from root tips was consistently observed in the present study. Obviously more research is required before the primary site of toxin action can be established in susceptible corn.

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7. DALY, J. M. (1976). The carbon balance of diseased plants: Changes in respiration, photosynthesis and translocation. In Encyclopedia of Plant Physiology, Vol. 4, Physiological Plant Pathology, Ed. by R. Heitefuss & P. H. Williams, pp. 450-479. Springer-Verlag, New York.

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10. GENGENBACH, B. G. MILLER, R. J., KOEPPE, D. E. & ARNTZEN, C. J. (1973). The effect of toxin from Helminthosporium maydis (race T) on isolated corn mitochondria: swelling. Canadian Jountal of Botany 51, 2119-2125.

11. GRAOEN, V. E., GROGAN, C. 0. & FORSTER, M. J. (1972). Permeability changes induced by Helminthospwium maydis, race T, toxin. Canadian 3ournal of Botany 50,2167-2170.

12. GREGORY, P., MATTHEWS, D. E., YORK, D. W., EARLE, E. D. & GRAOBN, V. E. (1978). Southern corn leaf blight disease: studies on rnitochondrial biochemistry and ultrastructure. M&O- @zologica 66, 105-l 12.

13. HALLOIN, J. M., COMSTOCK, J. C., MARTINSON, C. A. & TIPTON, C. L. (1973). Leakage from corn tissues induced by Helminthosporium maydis race T toxin. Phytopathology 63,640-642.

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