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Hydrogen cyanide detoxification by Gloeocercospora sorghi
P&-iological Plant Pat/w&~ (1975)
7,23-33
Hydrogen cyanide detoxification W. E.
FRY
and D. C.
MUNCH
D+artment of Plant Pathlogy, Corn& University, (Acceptid fw publhtion
by Gloeocercospora sorghi
Ithaca, New rork 14853, U.S.A.
February 1975)
Glocvc~cvsrpora sorgki D. Bain & Edg., the causal agent of aonate leaf spot of Svrgham spp., is adaptively tolerant of hydrogen cyanide. Associited with adaptation to hydrogen cyanide is the appearance of formamide hydra-lyase. The enzyme is induced by hydrogen cyanide and converts hydrogen cyanide to formamide, a compound which is relatively non-toxic. Formamide hydro-lyase from G. swghi has characteristics of a high molecular weight enzyme, is stable to storage at 4 “C, has optimal activity in the range of pH 7 to 9, and has a K,,, of approximately 30 msr-hydrogen cyanide. Sorghum tissue infected with C. svrghi has formamide hydro-lyase activity whereas healthy sorghum does not. ,Formamide hydra-lyase .from infected tissue had properties similar to that produced by G. sorghi in v&o.
INTRODUCTION The capability of some plants to produce cyanogenic glycosides has intrigued plant scientists for nearly 150 years. Injury to plant cells by such factors as water stress, frost and infection results in degradation of the glycosides and release of hydrogen cyanide (HCN) [3, 1.2, 201. A possible function of the cyanogenic glycosides is that they protect plants from bacteria, fungi, vertebrate and invertebrate animals [7, Kj. However, data which critically support this hypothesis are lacking. Several investigators have attempted to determine whether cyanogenic glycosides are responsible for resistance to plant pathogens. The experimental approach has been to correlate the quantity of cyanogenic glycoside with resistance to a specific pathogen. In most of these studies resistance and cyanogenic glycoside content were not positively correlated [II, 17, 191, but in other studies a positive correlation was reported [17, 181. However, none of these investigators considered the possibility that pathogens of cyanogenic plants might be tolerant of HCN. Millar and coworkers have shown that Stemfihylium loti, a leaf-spotting pathogen of birdsfoot trefoil (Lotus cornicdatus L.), is less sensitive to HCN than are S. botryosum and S. sarcinaGforme which are not pathogenic to trefoil [I, 121. Tolerance to HCN in S. Zoti is due apparently to HCN-induced formamide hydro-lyase which converts HCN to HCONH, (formamide) [KJ. These studies suggested that the lack of correlation between cyanogenic glycoside content and resistance might be caused by HCN tolerance in the pathogen and that for such pathogens, cyanogenic glycoside level in the host might be irrelevant. Consequently, we are investigating the possibi that pathogen tolerance of HCN (perhaps by detoxification of the released HCN) is necessary for a compatible interaction [sem Kiraly, 81. This paper reports on the HCN tolerance and mechanism of HCN detoxification in Gloeocercosfiorasorghi D. Bain & Edg. G. sorghi is a pathogen of sorghum, a cyanogenic plant.
24
W. E. Fry and D. C. Munch
MATERIALS
AND
METHODS
D. Bain & Edg. was isolated from diseased sorghum leaves provided by R. A. Frederiksen, Texas A & M University, College Station, Texas 77840, U.S.A. The fungus was maintained on V-8 juice agar [13] under continuous fluorescent light at 22 to 25 “C. Mycelium was harvested after 7 to 10 days from cultures grown in V-8 juice broth on a rotary shaker (c. 100 rotations/ min) at 21 to 26 “C under natural light augmented with fluorescent light. Virulence of the pathogen was maintained by re-isolating it from inoculated plants every 3 to 4 months. The host plant was a sorghum-Sudan hybrid (S. vulgare x S. vulgare var. sudanense “Grazer”) maintained in the greenhouse at c. 30 “C with supplemental fluorescent light. To confirm that our isolate of G. sorghi was pathogenic to this hybrid, 3-week-old “Grazer” seedlings were inoculated by spraying with G. sorghi conidia (10 000 to 50 OOO/ml) and then maintained at c. 24 “C and at 100% r.h. for 48 h. Lesions were first visible within 20 h as water-soaked areas 2 to 3 mm in diameter and within 48 h these water-soaked areas had expanded to 3 to 5 mm diameter and had become rust red. Oxygen uptake determinations were made on mycelium from shake cultures which had been fragmented in a Waring blendor (10 s), washed with deionized water and then resuspended in 50 mM-phosphate buffer pH 7.5. Measurements were made either with a Gilson differential respirometer (model GRP 20) or polarographically with a Gilson oxygraph (model KM), equipped with a Clark Electrode [5]. Gloeocercospora
Analytical
sorghi
procedure
Hydrogen cyanide concentration was determined calorimetrically using alkaline sodium picrate (picric acid, 21.8 mmol; NasCO,, 235.8 mmol; H,O, 2 1) as described previously [4]. Formamide was detected by g.1.c. utilizing a Perkin Elmer Gas chromatograph (model 900) with a flame ionization detector. The 1830 x 2 mm glass column was packed with Tenax GC, 60 to 80 mesh (Applied Science Laboratories Inc., State College, Pennsylvania 16802, U.S.A.). The instrument was temperature programmed from 140 to 170 “C at 3 “C/min. Under these conditions the retention time of formamide was 4.8 min. Formamide concentration was also measured calorimetrically by producing the hydroxamic acid derivative as described previously [S]. Protein concentration was estimated according to the method of Lowry et al. [IO] using bovine serum albumin as a standard. Formamide hydro-lyase (FHL) activity was measured by determining the amount of formamide produced per min at 25 “C. Unless stated otherwise, reaction mixtures contained FHL, 70 to 100 pmol HCN, 0.05 mmol Tris (hydroxymethyl) amino methane (Fisher) at pH 8-O in a total volume of 1-Oml. FHL activity is expressed in units such that 1 unit of activity is equal to 1 pmol of formamide produced per min at pH 7 to 8 in the presence of 70 to 100 mr+HCN. Preparation
and putijcation
of formamide
hydra-lyase
All procedures except tissue fragmentation in the Waring c. 4 “C, and the buffer utilized was 50 mr+Tris, at pH 8.0.
blendor were done at
Hydrogen cyanide detoxification
25
Fungal mycelium, diseased sorghum or healthy sorghum were fragmented for 30 s in a Waring blendor and then aliquots (15 to 20 ml) were homogenized with glass beads ( 10 to 12 ml 0.25 or 0*45-0*50 mm diameter) for 60 s at 4000 cycles/min in a Bronwill MSK cell homogenizer. After removal of the liquid the beads were washed twice (5.0 ml/wash) with buffer, and the washings were combined with the previous supernatant. This crude cell homogenate was then centrifuged for 20 min at 13 000 rev/min (20 000 g) in a Sorvall Super-speed RCB-B centrifuge with a SS-34 rotor. All preparations were concentrated c. lo-fold with a Diaflo ultrafiltration membrane UM 20 E (Amicon Corporation, Lexington, Mass. 02173, U.S.A.). The concentrated FHL preparations were then passed through a column (2.6 x 34 cm) of Sephadex G-100, and the filtrate was collected in 5-ml fractions. Fractions containing most of the FHL activity were combined and applied to a column of DEAE-Sephadex A-25 (1.5 x 30 cm), which was eluted first with Tris buffer (50 ml) and then with a NaCl gradient (0.00 to 0.75 M) in Tris (300 ml). The NaCl gradient eluant was collected in 5-ml fractions and those with FHL activity were dialyzed against 25 to 50 volumes of buffer. The dialyzed fractions were then concentrated c. IO-fold as described above. For molecular weight estimation, crude or purified FHL preparations were applied to a column ( 1.5 x 43.5 cm) of Bio-Gel A-l 5 M (Bio Rad) . The column was eluted with 50 mM-Tris buffer (pH 8) in O-1 N-Nacl. Blue Dextran 2000 (Pharmacia) was used to determine the void volume of the columns, and the concentration of Blue Dextran was determined by measuring the absorbance of each fraction at 630 nm. All experiments were performed at least twice with or without modifications. Figures and tables present results of typical experiments. RESULTS
Sensitivity of G. sorghi to HCN Some preliminary experiments measuring growth of G. sorghi on media containing HCN indicated that G. sorghi was tolerant of HCN. The sensitivity of G. sorghi to HCN was also measured by determining the effect of HCN on oxygen uptake. Fragmented G. sorghi mycelium was exposed to 0, 0.1, 0.5, 1.0 and 2.2 rm+HCN in the respirometer. HCN concentration was maintained by an appropriate mixture of Ca(OH),.Ca(CN), in the center well [15]. HCN at all concentrations initially inhibited oxygen uptake, but within 5 h oxygen uptake of mycelium exposed to 0.1 mr+HCN was stimulated (P< 0.05). The initial respiratory response of G. sorghi to HCN was measured polarographically, and rates of 0, uptake were obtained within 5 min of the addition of HCN to the electrode chamber. Two types of G. sorghi hyphae were utilized; one type was obtained from shake cultures which had not been exposed to HCN (nonadapted), and a second type was obtained from shake cultures (25 ml) to which HCN (25 ~01 in 2.5 ml H,O) had been added 16 h previously (adapted). The mycelium in each case was washed thoroughly and fragmented before being tested in the oxygraph. The results of a typical experiment are given in Table 1. Oxygen uptake of non-adapted hyphae was inhibited by I.0 and 2-O mM-HCN, whereas
W. E. Fry and D. C. Munch
26
oxygen uptake of adapted hyphae was stimulated by 1-OmM-HCN. However, 2-O mr+HCN decreased the oxygen uptake of adapted hyphae. These results corroborate the data obtained with the respirometer. TABLET Initial
rate
of 0, u/duke by G. sorghi Control
Qo.
Treatment Cells not previously exposed to HCN Cells exposed to HCN (1.0 mu) 16 h prior to test
% Of Control
cells ar affectcd by HCN” Rate of 0, uptake 1.0 mu-HCN % of (Lo, Control
2.0 nnr-HCN % of Qo, Control
0.109
100
o-033
30
o-030
27
0.109
100
0.130
119
0493
85
Q 0s uptake was measured polarographically with a Gilson oxygraph model KM. The sample consisted of c. 1.6 ml of fragmented 0. sorghi mycelium (2.6 mg dry wt) in 0.05 M-phosphate buffer pH 7.5 to which 16 or 32 pl 100 mu-HCN was added to achieve 1.0 or 2-O mar-HCN in the sample. Rates of 0s uptake were determined within 5 min of addition of HCN. &, is here defined as pl 0s utihaed/min per mg dry wt.
Detection of formamide
hydra-lyase
Because the reaction of G. sorghi to HCN was very similar to that of S. loti [4] adapted and non-adapted G. sorghi mycelium was assayed for formamide hydro-lyase (FHL) activity. Mycelium of adapted G. sorghi was capable of metabolizing HCN to HCONH, (formamide) very rapidly. Cell-free homogenates from adapted G. sorghi also exhibited FHL activity. Purification of FHL was attempted by means of gel filtration through Sephadex G-100 and chromatography with DEAE-Sephadex (Figs 1 and 2). FHL activity was recovered in a single peak from each column but the degree of purification was only 3 to 6-fold for Sephadex G-100 and 1.5 to 5-fold for DEAE-Sephadex. In non-adapted G. sorghi mycelium FHL activity was absent or at a very low level ( < 1y. of that detected in adapted G. sorghi mycelium). FHL was produced by G. sorghi in the absence of other demonstrable organisms. HCN solution was prepared by adding KCN (Mallinckrodt; purified granular) aseptically to sterile water. This HCN solution (25 pm01 in 2.5 ml HsO) was added asceptically to shake cultures (25 ml) of G. sorghi. Sixteen h later the mycelium was washed and fragmented aseptically, and then incubated for 30 min in a medium containing O-5 mmol phosphate buffer pH 7 and 200 pmol HCN in a total volume of 20 ml. FHL activity was detected, but aliquots (1.0 ml) of the culture filtrates or reaction mixtures yielded no organisms other than G. sorghi when plated onto nutrient agar and incubated for 24 h at c. 25 “C. The product of FHL activity on HCN was positively identified as formamide by g.1.c. with authentic formamide as standard. FHL purified by gel filtration and DEAE chromatography (specific activity 395 lJ.mol/min per mg protein) was incubated at 25 “C and at pH 8 in the presence of 100 mM-HCN. HCN was periodically added to the reaction mixture to maintain the HCN concentration at 100 mM.
Hydrogen cyanide detoxification
27
.U.‘..U ci’
Fmction
Xu
no.
FIG. 1. Elution pattern through a column of Sephadex G-100 of FHL activity in a crude homogenate of adapted C. sorghi. Column size was 2-6 x 34 cm and the filtrate was collected in 5-ml fractions. Elution pattern of Blue Dextran 2000 was the same as FHL activity. Protein was estimated accordii to the method of Lowry ct al [IO]. Visible brown pigments (presumably phenols) eluting in fractions 20 to 25 probably caused the estimations of protein to be erroneously high. This curve is typical of each of the several times this experiment was performed.
300
-“o
E
l i
- 500 Protein 4.. 100 -
O-
1
_e - -
_ M -*.+ f
. . .n.. . . . ..+-***n.
p”“’ *..* *.-* &-“---.:. c
5
I IO
_--1 15
20 Fmctii
FIG. 2. Elution pattern FHL was eluted from the collected in 5 ml fractions. column of Sephadex G-100. experiment was performecl.
.... .
25
30
35
40
no.
of FHL from a column (1.5 x 30 cm) of DEAE-Sephadex A-25. column by a salt gradient in 0.05 sa-Tris, pH 8.0. Eluant was Source of FHL was the pooled FHL-containing fractions from a Results are typical of those obtained each of several times this
W.
28
E. Fry and
D. C. Munch
After 3.5 h the reaction medium contained 60 mr+formamide as determined by calorimetric measurement. The remaining HCN was removed by bubbling filtered air through the reaction mixture. Aliquots of this reaction medium were then analyzed by g.1.c. Samples of water solutions (1 to 4 ~1) containing standard formamide were also analyzed. The retention times of the compounds in each of these solutions was 4.8 min, and when the two solutions were co-chromatographed, only one peak resulted. Conversion of HCN to formamide by purified FHL or by adapted mycelium was stoichiometric (Fig. 3) ; for each pmol of HCN metabolized, Formamide was the sole detectable product of a pmol of formamide was detected. the reaction.
10
min
FIG. 3. Stoichiometry of the reaction catalyzed by formamide hydro-lyase. Each reaction mixture (pH 8) contained 0.49 units FHL, 11 umol HCN, 55 pmol Tris buffer in a total volume of 1.1 ml and was maintained at 25 “Cl. Individual reaction mixtures were assayed for HCN and HCONH, at the times indicated. The line marked by HCNf HCONH, indicates the sum of the concentrations of HCN and HCONH, at the times indicated. Control reaction mixtures contained autoclaved FHL. FHL utilized in this experiment had been purified by means of Sephadex G-100 gel filtration and by chromatography on DEAE-Sephadex. Formamide since culture whereas FHL Character-i&s
hydro-lyase is apparently intracellular or bound to cells of G. sorghi, medium separated from G. sorghi mycelium lacked FHL activity, activity was associated with the mycelium. of formamide
hydro-&se
produced by G. sorghi
Activity of FHL from G. sorghi was optimal from pH 7 to about pH 9 (Fig. 4). The enzyme was saturated with substrate at 50 to 60 mr.+HCN. The K, was estimated to be 27 mu-HCN (Fig. 5). FHL produced by G. sorghi was excluded from Sephadex G-100.
Hydrogen
cyanide detoxification
c-
29
0’ I
40-
.c .E Al 309
l
8 l
2 20a . IO 0
L 1 ‘45676
1. .. 0
I
I
I
FIG. 4. Activity of formamide hydro-lyase at different pH levels. Buffers used were sodium citrate-citric acid (O-O), phosphate (O-O), Tris-HCl (O-O), and glycine-NaOH (e---e). Reaction mixtures contained buffer (100 pool) and HCN (80 pool) in a total volume of 1.0 ml. FHL activity was measured by determining the amount of HCONH, produced after 30 min at 25 “C. The results illustrated were obtained with FHL in unpurified cell-free homogenate. The same curve resulted when the FHL had been purified by means of Sephadex G-100 filtration and DEAE-Sephadex chromatography.
To estimate the molecular weight of FHL, enzyme preparations were passed through Bio-Gel A-15 M in the presence of O-1 M-NaCl. FHL was eluted slightly behind the void volume (Fig. 6). The elution pattern of Blue Dextran 2000 in Fig. 6 is typical for this material which is composed of a population of dextrans of different molecular weights. Purified and crude homogenates from adapted G. sorghi containing FHL activity were centrifuged at 100 000 g for 6 h (Beckman model L-4 Ultracentrifuge with SW65 rotor at 37 500 rev/min). The proportion of FHL sedimented varied from 50 to 95%. The data available are insufficient to estimate accurately the molecular weight of FHL, but these initial findings suggest it might be as great as 2-10 x lo6 daltons. Stability of FHL was assessed by measuring the effects of a variety of factors on its activity. No FHL activity was recovered from either the supernatant or the resuspended, dialyzed precipitates resulting from treatment of crude homogenates with (NH,),SO1. At a final concentration of 1-OmM- (pH 8-O) NaF, NaN,, EDTA, NaSCN, HOCHsCHsSH did not inhibit FHL activity. However, 10 mM-NaF decreased FHL activity by 50%. The other compounds had little or no effect at IOIllM.
W. E. Fry and D. C. Munch
(b)
-0.02
I 20
0
I 40
0.02
0.04 I/S
I 80
I 80
0.06
0.06
0.1
I loo
m HCN Saturation kinetics of formamide hydro-lyase. Reaction mixtures contained FHL (purified by means of Sephadex G-100 and DEAE-Sephadex chromatography), phosphate buffer (50 pmol) and 0 to 100 ~01 HCN in a total volume of 1.0 ml at pH 7.0. Formamide was measured after 30 min at 25 “C. (a) Velocity vs substrate concentration. (b) LineweaverBurk plot of data in (a). FIG.
5.
Fraction
no.
Fro. 6. Elution pattern of formamide hydro-lyase Column size was 1.5 x 43.5 cm, and the eluant was was prepared in and eluted with 0.05 r+c-Tris pH 8 crude homogenate containing 28.8 units FHL and Blue Dextran 2000 (average molecular weight of 2 column. The amount of protein detectable in the estimation.
through a column of Bio-Gel A-15 M. collected in 3.3~ml fractions. The column in 0.1 N-Nacl. The sample was 2.0 ml of 0*24mg protein. The first appearance of 000 000) indicates the void volume of the fractions was below the limits of protein
Hydrogen cyanide detoxif’ication
31
Activity of FHL in crude homogenates generally decreased c. 50% within the first 24 h after cell breakage. The cause of this loss of activity is unknown. After the initial loss of activity, the enzyme was stable if maintained at 4 or -20 “C. A preparation of FHL after Sephadex G-100 gel filtration and DEAE-Sephadex chromatography retained about 60, 30 and 10% of its activity after 2 weeks at 4, - 20 and 25 “C, respectively. And after 6 weeks 60,5 and 0% of the activity remained in preparations stored at 4, -20 and 25 “C, respectively. Formamide
hydro-&se
in diseased sorghum tissue
To determine if FHL could be functioning in pathogenesis, FHL activity was measured in sorghum leaves infected with G. sorghi. Leaves inoculated with 2 to 5 x 10s conidia/ml (in O.l”h Tween 20) were homogenized when the tissue was about 50% water-soaked (24 to 36 h after inoculation). In addition, FHL activity was determined for healthy leaves, leaves sprayed with autoclaved G. sorghi conidia, leaves inoculated with S. sarcinutforme (50 000 conidia/ml) known to be incapable of producing FHL (unpublished results) or for leaf sections incubated in 1.0 mu-HCN for 16 h. Formamide hydro-lyase activity was detected only in infected tissue (I.1 7 ~ol/min per mg protein). Formamide hydro-lyase and most of the darkcolored compounds from diseased tissue were adsorbed onto DEAE-Sephadex. FHL was selectively removed by eluting with 0.5 M-NaCI or by batch-wise treatment with 0.5 M-NaCl and subsequent filtration of the resin. FHL preparations decolorized in this way were utilized to characterize the enzyme from diseased tissue. FHL from diseased tissue had the following characteristics: optimum activity at c. pH 7 to 9; a K, of about 40 mr+HCN, exclusion from Sephadex G-1 00 and elution from DEAESephadex A-25 with c. 0.25 r+NaCl. Consequently, FHL found in diseased tissue was probably produced by G. sorghi. The effect of formamide on radial growth of G. sorghi was measured to determine whether this compound was toxic. Formamide was incorporated into V-8 juice agar at final concentrations of 0, 10, 25, 50, 100 and 200 mM. Five days after transfer, growth of G. sorghi was slightly stimulated at 25 and 50 mu formamide, and there was little inhibitory effect at the higher concentrations. After 11 days, however, 100 and 200 mM formamide had reduced growth to 64 and 54% of control, respectively.
DISCUSSION
To our knowledge G. sorghi D. Bain & Edg. is only the second organism demonstrated capable of producing formamide hydro-lyase (FHL). This enzyme was first discovered in S. loti, and similarities for the two pathogens are striking. Each fungus is a pathogen of a cyanogenic plant, and produces FHL in reponse to HCN. Each fungus is apparently exposed to HCN upon pathogenesis: HCN was detected from trefoil leaves inoculated with S. loti [12], and FHL (presumably induced by HCN) was detected in sorghum leaves infected by G. sorghi. Characteristics of FHL from each pathogen are similar in terms of preliminary estimates of molecular weight, affinity for DEAE-Sephadex or DEAE-cellulose, pH optimum, K, and stability.
32
W. E. Fry and D. C. Munch
Experiments currently in progress indicate that the capability to produce FHL may be common among fungi. Pathogens of sorghum and birdsfoot trefoil and some fungi which are not reported pathogens of cyanogenic plants can be induced to produce FHL. Certain pathogens of non-cyanogenic plants are unable to produce FHL. Because FHL activity may be widespread among fungi and is not associated solely with pathogens of cyanogenic plants, other types of activity of this enzyme are being investigated. For example, other nitriles such as acetonitrile, propionitrile or p-hydroxymandelonitrile (the aglycone of dhurrin-the cyanogenic glycoside in sorghum) might serve as inducers and/or as substrates for FHL. A question yet unanswered concerns the mechanism of HCN-insensitive oxygen uptake in adapted G. sorghi and adapted S. loti. The induction of a cyanideinsensitive respiration was apparently separable from FHL production in S. loti [5]. Therefore, cyanide-insensitive respiration is probably not due to HCN detoxification by FHL. It is possible that an alternate pathway of electron transport to 0, may develop when cytochrome oxidase is inhibited by cyanide. Such cyanide-insensitive oxidases have been demonstrated in mung bean, in skunk cabbage mitochondria [ 11 and in ~eurosporu crussa mycelium [Z] . The role of HCN in diseases of cyanogenic plants is yet unclear. However, the finding that some pathogens of cyanogenic plants can detoxify and hence tolerate HCN calls for a re-examination of the role of HCN in diseases of such plants. For example, Snyder [17] f ound no correlation between dhurrin (the cyanogenic glycoside in sorghum) content and resistance to Helminthosporium turcicum. However, dhurrin content was correlated with resistance to G. sorghi. Our results indicate that at least our isolate of G. sorghi is tolerant of HCN; consequently, the correlation between dhurrin content and resistance to G. sorghi may have been meaningless. We are currently testing the hypothesis that HCN may contribute to the inhibitory environment of a plant and in order for a pathogen to grow in such an environment it must be capable of tolerating the environment (i.e. detoxify HCN, in this case). Cyanogenesis might contribute to resistance of the plant since it necessitates that pathogens (which stimulate HCN release) tolerate HCN. A second hypothesis is that HCN may aid the pathogen by injuring the host relatively more severely than the pathogen. Such a result would be somewhat analogous to the role suggested for HCN in injury to peach roots caused by nematode feeding [I41 or to the role of HCN in a snow mold disease caused by an unidentified psychrophilic basidiomycete [9]. In the case of the snow mold, however, HCN was produced by the pathogen under a snow cover. HCN accumulated in plant tissues (alfalfa) and was more injurious to the plant than to the pathogen. In the case of sorghum and G. sorghi, the relative sensitivity to HCN of host and pathogen is not yet known, and should be determined to clarify the role of HCN in pathogenesis. REFERENCES 1. BENDALL, D. S. & BONNER, W. D. JR (1971). Cyanide-insensitive respiration in plant mitochondria. Plant Physiology 47, 236245. 2. EDWAFLDS, D. L., ROSENBERG, E. & ~~ARONEY, P. A. (1974). Induction of cyanide-insensitive respiration in Jveurospora ~a.s~a. 3ouwd of Biological Chemistry 249, 3551-3556. 3. FRANZICE, C. J., PUHR, L. F. & HUME, A. N. (1939). A study of sorghum with reference to the content of HCN. South Dakota Agridtural Expmiment Shtion Technical Bulletin. 1, 51 pp.
Hydrogen 4. FRY,
cyanide detoxification W.
E. & MILLAR,
R.
33 L.
(1971).
Cyanide
tolerance
in Stemphylium
l&i.
Phytopathology
494-500. 5. FRY, W. E. & MILLAR, R. L. (1971). Development of cyanide tolerance in Stemphylium Phytopathology 61, 501-506. 6. FRY, W. E. & M~LI..+R, R. L. (1972). Cyanide degradation by an enzyme from Stemphylium Archives of Biochemisby and Biophysics 151,468-474.
61, loti. loti.
7. Jonas, D. A. (1973). Co-evolution and cy-anogenesis. In Taxonomy and Ecology, Ed. by V. H. Heywood, Special Vol. No. 5, pp. 213-242. Academic Press, New York. 8. KIRALY, Z. (1973). Developing concepts of plant resistance to infections. Acta phytopathologica Academiae scientianan hungaticae 8,381-390. 9. L+EBEAU, J. B. & DICILWN, J. G. (1955). Physiology and nature of disease development in winter crown rot of alfalfa. Phytopathology 45, 667-673. 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. Jownal of Biological Chmdtty 193,265-275. 11. LiiDTTLE, M. & HAHN, H. (1953). Uber den Linamaringehalt gesunder turd von Colletotrichum lini befallener junger Leinpflanaen. Biochemische ,+tsc~Ql324,433442. 12. ~IILLAR, R. L. & HKXXNS, V. J. (1970). Association of cyanide with infection of birdsfoot trefoil by Stemphylium lofi. Phytopathology 60, 104-l 10. 13. MILLER, P. M. (1955). V-8 juice agar as a general purpose medium for fungi and bacteria. PhytopatholoBy 45,461-462. 14. MOUNTAIN, W. B. & PATRICK, Z. A. (1959). The peach replant problem in Ontario. VII. The pathogcnicity of Pratylenchus penetrans (Cobb, 1917) Filip & Stek 1941. Canadian 3ownal of Botany 37,45!%470. 15. ROBBIE, W. A. (1948). Use of cyanide in tissue respiration studies. In Methods in Medical Research, Ed. by V. R. Potter, Vol. I, pp. 307-316. Yearbook Publications, Chicago. 16. ROBINSON, M. E. (1930). Cyanogensis in plants. Biological Review 5, 126-141. 17. SNYDER, E. B. ( 1950). Inheritance and associations of hydrocyanic acid potential, disease reactions, and other characters in Sudan grass., Sorghum vulgare var. sudanensis. Ph.D. Thesis, University of Wisconsin, Madison. 18. TIMOMN, M. I. (1941). The interaction of higher plants and soil microorganisms. III. Effect of by-products of plant growth on activity of fungi and actinomycetcs. Soil Science 52, 395-414. 19. TRIONE, E. J. (1960). The HCN content of flax in relation to flax wilt resistance. Phytopathology 50,462-466. 20. WAI-I-ENBARGER, D. W., GRAY, E., RICE, J. S. & REYNOLDS, J. H. (1968). Effects of frost and freezing on hydrocyanic acid potential of sorghum plants. Crop Scieme 8, 526-528.
3
7,23-33
Hydrogen cyanide detoxification W. E.
FRY
and D. C.
MUNCH
D+artment of Plant Pathlogy, Corn& University, (Acceptid fw publhtion
by Gloeocercospora sorghi
Ithaca, New rork 14853, U.S.A.
February 1975)
Glocvc~cvsrpora sorgki D. Bain & Edg., the causal agent of aonate leaf spot of Svrgham spp., is adaptively tolerant of hydrogen cyanide. Associited with adaptation to hydrogen cyanide is the appearance of formamide hydra-lyase. The enzyme is induced by hydrogen cyanide and converts hydrogen cyanide to formamide, a compound which is relatively non-toxic. Formamide hydro-lyase from G. swghi has characteristics of a high molecular weight enzyme, is stable to storage at 4 “C, has optimal activity in the range of pH 7 to 9, and has a K,,, of approximately 30 msr-hydrogen cyanide. Sorghum tissue infected with C. svrghi has formamide hydro-lyase activity whereas healthy sorghum does not. ,Formamide hydra-lyase .from infected tissue had properties similar to that produced by G. sorghi in v&o.
INTRODUCTION The capability of some plants to produce cyanogenic glycosides has intrigued plant scientists for nearly 150 years. Injury to plant cells by such factors as water stress, frost and infection results in degradation of the glycosides and release of hydrogen cyanide (HCN) [3, 1.2, 201. A possible function of the cyanogenic glycosides is that they protect plants from bacteria, fungi, vertebrate and invertebrate animals [7, Kj. However, data which critically support this hypothesis are lacking. Several investigators have attempted to determine whether cyanogenic glycosides are responsible for resistance to plant pathogens. The experimental approach has been to correlate the quantity of cyanogenic glycoside with resistance to a specific pathogen. In most of these studies resistance and cyanogenic glycoside content were not positively correlated [II, 17, 191, but in other studies a positive correlation was reported [17, 181. However, none of these investigators considered the possibility that pathogens of cyanogenic plants might be tolerant of HCN. Millar and coworkers have shown that Stemfihylium loti, a leaf-spotting pathogen of birdsfoot trefoil (Lotus cornicdatus L.), is less sensitive to HCN than are S. botryosum and S. sarcinaGforme which are not pathogenic to trefoil [I, 121. Tolerance to HCN in S. Zoti is due apparently to HCN-induced formamide hydro-lyase which converts HCN to HCONH, (formamide) [KJ. These studies suggested that the lack of correlation between cyanogenic glycoside content and resistance might be caused by HCN tolerance in the pathogen and that for such pathogens, cyanogenic glycoside level in the host might be irrelevant. Consequently, we are investigating the possibi that pathogen tolerance of HCN (perhaps by detoxification of the released HCN) is necessary for a compatible interaction [sem Kiraly, 81. This paper reports on the HCN tolerance and mechanism of HCN detoxification in Gloeocercosfiorasorghi D. Bain & Edg. G. sorghi is a pathogen of sorghum, a cyanogenic plant.
24
W. E. Fry and D. C. Munch
MATERIALS
AND
METHODS
D. Bain & Edg. was isolated from diseased sorghum leaves provided by R. A. Frederiksen, Texas A & M University, College Station, Texas 77840, U.S.A. The fungus was maintained on V-8 juice agar [13] under continuous fluorescent light at 22 to 25 “C. Mycelium was harvested after 7 to 10 days from cultures grown in V-8 juice broth on a rotary shaker (c. 100 rotations/ min) at 21 to 26 “C under natural light augmented with fluorescent light. Virulence of the pathogen was maintained by re-isolating it from inoculated plants every 3 to 4 months. The host plant was a sorghum-Sudan hybrid (S. vulgare x S. vulgare var. sudanense “Grazer”) maintained in the greenhouse at c. 30 “C with supplemental fluorescent light. To confirm that our isolate of G. sorghi was pathogenic to this hybrid, 3-week-old “Grazer” seedlings were inoculated by spraying with G. sorghi conidia (10 000 to 50 OOO/ml) and then maintained at c. 24 “C and at 100% r.h. for 48 h. Lesions were first visible within 20 h as water-soaked areas 2 to 3 mm in diameter and within 48 h these water-soaked areas had expanded to 3 to 5 mm diameter and had become rust red. Oxygen uptake determinations were made on mycelium from shake cultures which had been fragmented in a Waring blendor (10 s), washed with deionized water and then resuspended in 50 mM-phosphate buffer pH 7.5. Measurements were made either with a Gilson differential respirometer (model GRP 20) or polarographically with a Gilson oxygraph (model KM), equipped with a Clark Electrode [5]. Gloeocercospora
Analytical
sorghi
procedure
Hydrogen cyanide concentration was determined calorimetrically using alkaline sodium picrate (picric acid, 21.8 mmol; NasCO,, 235.8 mmol; H,O, 2 1) as described previously [4]. Formamide was detected by g.1.c. utilizing a Perkin Elmer Gas chromatograph (model 900) with a flame ionization detector. The 1830 x 2 mm glass column was packed with Tenax GC, 60 to 80 mesh (Applied Science Laboratories Inc., State College, Pennsylvania 16802, U.S.A.). The instrument was temperature programmed from 140 to 170 “C at 3 “C/min. Under these conditions the retention time of formamide was 4.8 min. Formamide concentration was also measured calorimetrically by producing the hydroxamic acid derivative as described previously [S]. Protein concentration was estimated according to the method of Lowry et al. [IO] using bovine serum albumin as a standard. Formamide hydro-lyase (FHL) activity was measured by determining the amount of formamide produced per min at 25 “C. Unless stated otherwise, reaction mixtures contained FHL, 70 to 100 pmol HCN, 0.05 mmol Tris (hydroxymethyl) amino methane (Fisher) at pH 8-O in a total volume of 1-Oml. FHL activity is expressed in units such that 1 unit of activity is equal to 1 pmol of formamide produced per min at pH 7 to 8 in the presence of 70 to 100 mr+HCN. Preparation
and putijcation
of formamide
hydra-lyase
All procedures except tissue fragmentation in the Waring c. 4 “C, and the buffer utilized was 50 mr+Tris, at pH 8.0.
blendor were done at
Hydrogen cyanide detoxification
25
Fungal mycelium, diseased sorghum or healthy sorghum were fragmented for 30 s in a Waring blendor and then aliquots (15 to 20 ml) were homogenized with glass beads ( 10 to 12 ml 0.25 or 0*45-0*50 mm diameter) for 60 s at 4000 cycles/min in a Bronwill MSK cell homogenizer. After removal of the liquid the beads were washed twice (5.0 ml/wash) with buffer, and the washings were combined with the previous supernatant. This crude cell homogenate was then centrifuged for 20 min at 13 000 rev/min (20 000 g) in a Sorvall Super-speed RCB-B centrifuge with a SS-34 rotor. All preparations were concentrated c. lo-fold with a Diaflo ultrafiltration membrane UM 20 E (Amicon Corporation, Lexington, Mass. 02173, U.S.A.). The concentrated FHL preparations were then passed through a column (2.6 x 34 cm) of Sephadex G-100, and the filtrate was collected in 5-ml fractions. Fractions containing most of the FHL activity were combined and applied to a column of DEAE-Sephadex A-25 (1.5 x 30 cm), which was eluted first with Tris buffer (50 ml) and then with a NaCl gradient (0.00 to 0.75 M) in Tris (300 ml). The NaCl gradient eluant was collected in 5-ml fractions and those with FHL activity were dialyzed against 25 to 50 volumes of buffer. The dialyzed fractions were then concentrated c. IO-fold as described above. For molecular weight estimation, crude or purified FHL preparations were applied to a column ( 1.5 x 43.5 cm) of Bio-Gel A-l 5 M (Bio Rad) . The column was eluted with 50 mM-Tris buffer (pH 8) in O-1 N-Nacl. Blue Dextran 2000 (Pharmacia) was used to determine the void volume of the columns, and the concentration of Blue Dextran was determined by measuring the absorbance of each fraction at 630 nm. All experiments were performed at least twice with or without modifications. Figures and tables present results of typical experiments. RESULTS
Sensitivity of G. sorghi to HCN Some preliminary experiments measuring growth of G. sorghi on media containing HCN indicated that G. sorghi was tolerant of HCN. The sensitivity of G. sorghi to HCN was also measured by determining the effect of HCN on oxygen uptake. Fragmented G. sorghi mycelium was exposed to 0, 0.1, 0.5, 1.0 and 2.2 rm+HCN in the respirometer. HCN concentration was maintained by an appropriate mixture of Ca(OH),.Ca(CN), in the center well [15]. HCN at all concentrations initially inhibited oxygen uptake, but within 5 h oxygen uptake of mycelium exposed to 0.1 mr+HCN was stimulated (P< 0.05). The initial respiratory response of G. sorghi to HCN was measured polarographically, and rates of 0, uptake were obtained within 5 min of the addition of HCN to the electrode chamber. Two types of G. sorghi hyphae were utilized; one type was obtained from shake cultures which had not been exposed to HCN (nonadapted), and a second type was obtained from shake cultures (25 ml) to which HCN (25 ~01 in 2.5 ml H,O) had been added 16 h previously (adapted). The mycelium in each case was washed thoroughly and fragmented before being tested in the oxygraph. The results of a typical experiment are given in Table 1. Oxygen uptake of non-adapted hyphae was inhibited by I.0 and 2-O mM-HCN, whereas
W. E. Fry and D. C. Munch
26
oxygen uptake of adapted hyphae was stimulated by 1-OmM-HCN. However, 2-O mr+HCN decreased the oxygen uptake of adapted hyphae. These results corroborate the data obtained with the respirometer. TABLET Initial
rate
of 0, u/duke by G. sorghi Control
Qo.
Treatment Cells not previously exposed to HCN Cells exposed to HCN (1.0 mu) 16 h prior to test
% Of Control
cells ar affectcd by HCN” Rate of 0, uptake 1.0 mu-HCN % of (Lo, Control
2.0 nnr-HCN % of Qo, Control
0.109
100
o-033
30
o-030
27
0.109
100
0.130
119
0493
85
Q 0s uptake was measured polarographically with a Gilson oxygraph model KM. The sample consisted of c. 1.6 ml of fragmented 0. sorghi mycelium (2.6 mg dry wt) in 0.05 M-phosphate buffer pH 7.5 to which 16 or 32 pl 100 mu-HCN was added to achieve 1.0 or 2-O mar-HCN in the sample. Rates of 0s uptake were determined within 5 min of addition of HCN. &, is here defined as pl 0s utihaed/min per mg dry wt.
Detection of formamide
hydra-lyase
Because the reaction of G. sorghi to HCN was very similar to that of S. loti [4] adapted and non-adapted G. sorghi mycelium was assayed for formamide hydro-lyase (FHL) activity. Mycelium of adapted G. sorghi was capable of metabolizing HCN to HCONH, (formamide) very rapidly. Cell-free homogenates from adapted G. sorghi also exhibited FHL activity. Purification of FHL was attempted by means of gel filtration through Sephadex G-100 and chromatography with DEAE-Sephadex (Figs 1 and 2). FHL activity was recovered in a single peak from each column but the degree of purification was only 3 to 6-fold for Sephadex G-100 and 1.5 to 5-fold for DEAE-Sephadex. In non-adapted G. sorghi mycelium FHL activity was absent or at a very low level ( < 1y. of that detected in adapted G. sorghi mycelium). FHL was produced by G. sorghi in the absence of other demonstrable organisms. HCN solution was prepared by adding KCN (Mallinckrodt; purified granular) aseptically to sterile water. This HCN solution (25 pm01 in 2.5 ml HsO) was added asceptically to shake cultures (25 ml) of G. sorghi. Sixteen h later the mycelium was washed and fragmented aseptically, and then incubated for 30 min in a medium containing O-5 mmol phosphate buffer pH 7 and 200 pmol HCN in a total volume of 20 ml. FHL activity was detected, but aliquots (1.0 ml) of the culture filtrates or reaction mixtures yielded no organisms other than G. sorghi when plated onto nutrient agar and incubated for 24 h at c. 25 “C. The product of FHL activity on HCN was positively identified as formamide by g.1.c. with authentic formamide as standard. FHL purified by gel filtration and DEAE chromatography (specific activity 395 lJ.mol/min per mg protein) was incubated at 25 “C and at pH 8 in the presence of 100 mM-HCN. HCN was periodically added to the reaction mixture to maintain the HCN concentration at 100 mM.
Hydrogen cyanide detoxification
27
.U.‘..U ci’
Fmction
Xu
no.
FIG. 1. Elution pattern through a column of Sephadex G-100 of FHL activity in a crude homogenate of adapted C. sorghi. Column size was 2-6 x 34 cm and the filtrate was collected in 5-ml fractions. Elution pattern of Blue Dextran 2000 was the same as FHL activity. Protein was estimated accordii to the method of Lowry ct al [IO]. Visible brown pigments (presumably phenols) eluting in fractions 20 to 25 probably caused the estimations of protein to be erroneously high. This curve is typical of each of the several times this experiment was performed.
300
-“o
E
l i
- 500 Protein 4.. 100 -
O-
1
_e - -
_ M -*.+ f
. . .n.. . . . ..+-***n.
p”“’ *..* *.-* &-“---.:. c
5
I IO
_--1 15
20 Fmctii
FIG. 2. Elution pattern FHL was eluted from the collected in 5 ml fractions. column of Sephadex G-100. experiment was performecl.
.... .
25
30
35
40
no.
of FHL from a column (1.5 x 30 cm) of DEAE-Sephadex A-25. column by a salt gradient in 0.05 sa-Tris, pH 8.0. Eluant was Source of FHL was the pooled FHL-containing fractions from a Results are typical of those obtained each of several times this
W.
28
E. Fry and
D. C. Munch
After 3.5 h the reaction medium contained 60 mr+formamide as determined by calorimetric measurement. The remaining HCN was removed by bubbling filtered air through the reaction mixture. Aliquots of this reaction medium were then analyzed by g.1.c. Samples of water solutions (1 to 4 ~1) containing standard formamide were also analyzed. The retention times of the compounds in each of these solutions was 4.8 min, and when the two solutions were co-chromatographed, only one peak resulted. Conversion of HCN to formamide by purified FHL or by adapted mycelium was stoichiometric (Fig. 3) ; for each pmol of HCN metabolized, Formamide was the sole detectable product of a pmol of formamide was detected. the reaction.
10
min
FIG. 3. Stoichiometry of the reaction catalyzed by formamide hydro-lyase. Each reaction mixture (pH 8) contained 0.49 units FHL, 11 umol HCN, 55 pmol Tris buffer in a total volume of 1.1 ml and was maintained at 25 “Cl. Individual reaction mixtures were assayed for HCN and HCONH, at the times indicated. The line marked by HCNf HCONH, indicates the sum of the concentrations of HCN and HCONH, at the times indicated. Control reaction mixtures contained autoclaved FHL. FHL utilized in this experiment had been purified by means of Sephadex G-100 gel filtration and by chromatography on DEAE-Sephadex. Formamide since culture whereas FHL Character-i&s
hydro-lyase is apparently intracellular or bound to cells of G. sorghi, medium separated from G. sorghi mycelium lacked FHL activity, activity was associated with the mycelium. of formamide
hydro-&se
produced by G. sorghi
Activity of FHL from G. sorghi was optimal from pH 7 to about pH 9 (Fig. 4). The enzyme was saturated with substrate at 50 to 60 mr.+HCN. The K, was estimated to be 27 mu-HCN (Fig. 5). FHL produced by G. sorghi was excluded from Sephadex G-100.
Hydrogen
cyanide detoxification
c-
29
0’ I
40-
.c .E Al 309
l
8 l
2 20a . IO 0
L 1 ‘45676
1. .. 0
I
I
I
FIG. 4. Activity of formamide hydro-lyase at different pH levels. Buffers used were sodium citrate-citric acid (O-O), phosphate (O-O), Tris-HCl (O-O), and glycine-NaOH (e---e). Reaction mixtures contained buffer (100 pool) and HCN (80 pool) in a total volume of 1.0 ml. FHL activity was measured by determining the amount of HCONH, produced after 30 min at 25 “C. The results illustrated were obtained with FHL in unpurified cell-free homogenate. The same curve resulted when the FHL had been purified by means of Sephadex G-100 filtration and DEAE-Sephadex chromatography.
To estimate the molecular weight of FHL, enzyme preparations were passed through Bio-Gel A-15 M in the presence of O-1 M-NaCl. FHL was eluted slightly behind the void volume (Fig. 6). The elution pattern of Blue Dextran 2000 in Fig. 6 is typical for this material which is composed of a population of dextrans of different molecular weights. Purified and crude homogenates from adapted G. sorghi containing FHL activity were centrifuged at 100 000 g for 6 h (Beckman model L-4 Ultracentrifuge with SW65 rotor at 37 500 rev/min). The proportion of FHL sedimented varied from 50 to 95%. The data available are insufficient to estimate accurately the molecular weight of FHL, but these initial findings suggest it might be as great as 2-10 x lo6 daltons. Stability of FHL was assessed by measuring the effects of a variety of factors on its activity. No FHL activity was recovered from either the supernatant or the resuspended, dialyzed precipitates resulting from treatment of crude homogenates with (NH,),SO1. At a final concentration of 1-OmM- (pH 8-O) NaF, NaN,, EDTA, NaSCN, HOCHsCHsSH did not inhibit FHL activity. However, 10 mM-NaF decreased FHL activity by 50%. The other compounds had little or no effect at IOIllM.
W. E. Fry and D. C. Munch
(b)
-0.02
I 20
0
I 40
0.02
0.04 I/S
I 80
I 80
0.06
0.06
0.1
I loo
m HCN Saturation kinetics of formamide hydro-lyase. Reaction mixtures contained FHL (purified by means of Sephadex G-100 and DEAE-Sephadex chromatography), phosphate buffer (50 pmol) and 0 to 100 ~01 HCN in a total volume of 1.0 ml at pH 7.0. Formamide was measured after 30 min at 25 “C. (a) Velocity vs substrate concentration. (b) LineweaverBurk plot of data in (a). FIG.
5.
Fraction
no.
Fro. 6. Elution pattern of formamide hydro-lyase Column size was 1.5 x 43.5 cm, and the eluant was was prepared in and eluted with 0.05 r+c-Tris pH 8 crude homogenate containing 28.8 units FHL and Blue Dextran 2000 (average molecular weight of 2 column. The amount of protein detectable in the estimation.
through a column of Bio-Gel A-15 M. collected in 3.3~ml fractions. The column in 0.1 N-Nacl. The sample was 2.0 ml of 0*24mg protein. The first appearance of 000 000) indicates the void volume of the fractions was below the limits of protein
Hydrogen cyanide detoxif’ication
31
Activity of FHL in crude homogenates generally decreased c. 50% within the first 24 h after cell breakage. The cause of this loss of activity is unknown. After the initial loss of activity, the enzyme was stable if maintained at 4 or -20 “C. A preparation of FHL after Sephadex G-100 gel filtration and DEAE-Sephadex chromatography retained about 60, 30 and 10% of its activity after 2 weeks at 4, - 20 and 25 “C, respectively. And after 6 weeks 60,5 and 0% of the activity remained in preparations stored at 4, -20 and 25 “C, respectively. Formamide
hydro-&se
in diseased sorghum tissue
To determine if FHL could be functioning in pathogenesis, FHL activity was measured in sorghum leaves infected with G. sorghi. Leaves inoculated with 2 to 5 x 10s conidia/ml (in O.l”h Tween 20) were homogenized when the tissue was about 50% water-soaked (24 to 36 h after inoculation). In addition, FHL activity was determined for healthy leaves, leaves sprayed with autoclaved G. sorghi conidia, leaves inoculated with S. sarcinutforme (50 000 conidia/ml) known to be incapable of producing FHL (unpublished results) or for leaf sections incubated in 1.0 mu-HCN for 16 h. Formamide hydro-lyase activity was detected only in infected tissue (I.1 7 ~ol/min per mg protein). Formamide hydro-lyase and most of the darkcolored compounds from diseased tissue were adsorbed onto DEAE-Sephadex. FHL was selectively removed by eluting with 0.5 M-NaCI or by batch-wise treatment with 0.5 M-NaCl and subsequent filtration of the resin. FHL preparations decolorized in this way were utilized to characterize the enzyme from diseased tissue. FHL from diseased tissue had the following characteristics: optimum activity at c. pH 7 to 9; a K, of about 40 mr+HCN, exclusion from Sephadex G-1 00 and elution from DEAESephadex A-25 with c. 0.25 r+NaCl. Consequently, FHL found in diseased tissue was probably produced by G. sorghi. The effect of formamide on radial growth of G. sorghi was measured to determine whether this compound was toxic. Formamide was incorporated into V-8 juice agar at final concentrations of 0, 10, 25, 50, 100 and 200 mM. Five days after transfer, growth of G. sorghi was slightly stimulated at 25 and 50 mu formamide, and there was little inhibitory effect at the higher concentrations. After 11 days, however, 100 and 200 mM formamide had reduced growth to 64 and 54% of control, respectively.
DISCUSSION
To our knowledge G. sorghi D. Bain & Edg. is only the second organism demonstrated capable of producing formamide hydro-lyase (FHL). This enzyme was first discovered in S. loti, and similarities for the two pathogens are striking. Each fungus is a pathogen of a cyanogenic plant, and produces FHL in reponse to HCN. Each fungus is apparently exposed to HCN upon pathogenesis: HCN was detected from trefoil leaves inoculated with S. loti [12], and FHL (presumably induced by HCN) was detected in sorghum leaves infected by G. sorghi. Characteristics of FHL from each pathogen are similar in terms of preliminary estimates of molecular weight, affinity for DEAE-Sephadex or DEAE-cellulose, pH optimum, K, and stability.
32
W. E. Fry and D. C. Munch
Experiments currently in progress indicate that the capability to produce FHL may be common among fungi. Pathogens of sorghum and birdsfoot trefoil and some fungi which are not reported pathogens of cyanogenic plants can be induced to produce FHL. Certain pathogens of non-cyanogenic plants are unable to produce FHL. Because FHL activity may be widespread among fungi and is not associated solely with pathogens of cyanogenic plants, other types of activity of this enzyme are being investigated. For example, other nitriles such as acetonitrile, propionitrile or p-hydroxymandelonitrile (the aglycone of dhurrin-the cyanogenic glycoside in sorghum) might serve as inducers and/or as substrates for FHL. A question yet unanswered concerns the mechanism of HCN-insensitive oxygen uptake in adapted G. sorghi and adapted S. loti. The induction of a cyanideinsensitive respiration was apparently separable from FHL production in S. loti [5]. Therefore, cyanide-insensitive respiration is probably not due to HCN detoxification by FHL. It is possible that an alternate pathway of electron transport to 0, may develop when cytochrome oxidase is inhibited by cyanide. Such cyanide-insensitive oxidases have been demonstrated in mung bean, in skunk cabbage mitochondria [ 11 and in ~eurosporu crussa mycelium [Z] . The role of HCN in diseases of cyanogenic plants is yet unclear. However, the finding that some pathogens of cyanogenic plants can detoxify and hence tolerate HCN calls for a re-examination of the role of HCN in diseases of such plants. For example, Snyder [17] f ound no correlation between dhurrin (the cyanogenic glycoside in sorghum) content and resistance to Helminthosporium turcicum. However, dhurrin content was correlated with resistance to G. sorghi. Our results indicate that at least our isolate of G. sorghi is tolerant of HCN; consequently, the correlation between dhurrin content and resistance to G. sorghi may have been meaningless. We are currently testing the hypothesis that HCN may contribute to the inhibitory environment of a plant and in order for a pathogen to grow in such an environment it must be capable of tolerating the environment (i.e. detoxify HCN, in this case). Cyanogenesis might contribute to resistance of the plant since it necessitates that pathogens (which stimulate HCN release) tolerate HCN. A second hypothesis is that HCN may aid the pathogen by injuring the host relatively more severely than the pathogen. Such a result would be somewhat analogous to the role suggested for HCN in injury to peach roots caused by nematode feeding [I41 or to the role of HCN in a snow mold disease caused by an unidentified psychrophilic basidiomycete [9]. In the case of the snow mold, however, HCN was produced by the pathogen under a snow cover. HCN accumulated in plant tissues (alfalfa) and was more injurious to the plant than to the pathogen. In the case of sorghum and G. sorghi, the relative sensitivity to HCN of host and pathogen is not yet known, and should be determined to clarify the role of HCN in pathogenesis. REFERENCES 1. BENDALL, D. S. & BONNER, W. D. JR (1971). Cyanide-insensitive respiration in plant mitochondria. Plant Physiology 47, 236245. 2. EDWAFLDS, D. L., ROSENBERG, E. & ~~ARONEY, P. A. (1974). Induction of cyanide-insensitive respiration in Jveurospora ~a.s~a. 3ouwd of Biological Chemistry 249, 3551-3556. 3. FRANZICE, C. J., PUHR, L. F. & HUME, A. N. (1939). A study of sorghum with reference to the content of HCN. South Dakota Agridtural Expmiment Shtion Technical Bulletin. 1, 51 pp.
Hydrogen 4. FRY,
cyanide detoxification W.
E. & MILLAR,
R.
33 L.
(1971).
Cyanide
tolerance
in Stemphylium
l&i.
Phytopathology
494-500. 5. FRY, W. E. & MILLAR, R. L. (1971). Development of cyanide tolerance in Stemphylium Phytopathology 61, 501-506. 6. FRY, W. E. & M~LI..+R, R. L. (1972). Cyanide degradation by an enzyme from Stemphylium Archives of Biochemisby and Biophysics 151,468-474.
61, loti. loti.
7. Jonas, D. A. (1973). Co-evolution and cy-anogenesis. In Taxonomy and Ecology, Ed. by V. H. Heywood, Special Vol. No. 5, pp. 213-242. Academic Press, New York. 8. KIRALY, Z. (1973). Developing concepts of plant resistance to infections. Acta phytopathologica Academiae scientianan hungaticae 8,381-390. 9. L+EBEAU, J. B. & DICILWN, J. G. (1955). Physiology and nature of disease development in winter crown rot of alfalfa. Phytopathology 45, 667-673. 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. Jownal of Biological Chmdtty 193,265-275. 11. LiiDTTLE, M. & HAHN, H. (1953). Uber den Linamaringehalt gesunder turd von Colletotrichum lini befallener junger Leinpflanaen. Biochemische ,+tsc~Ql324,433442. 12. ~IILLAR, R. L. & HKXXNS, V. J. (1970). Association of cyanide with infection of birdsfoot trefoil by Stemphylium lofi. Phytopathology 60, 104-l 10. 13. MILLER, P. M. (1955). V-8 juice agar as a general purpose medium for fungi and bacteria. PhytopatholoBy 45,461-462. 14. MOUNTAIN, W. B. & PATRICK, Z. A. (1959). The peach replant problem in Ontario. VII. The pathogcnicity of Pratylenchus penetrans (Cobb, 1917) Filip & Stek 1941. Canadian 3ownal of Botany 37,45!%470. 15. ROBBIE, W. A. (1948). Use of cyanide in tissue respiration studies. In Methods in Medical Research, Ed. by V. R. Potter, Vol. I, pp. 307-316. Yearbook Publications, Chicago. 16. ROBINSON, M. E. (1930). Cyanogensis in plants. Biological Review 5, 126-141. 17. SNYDER, E. B. ( 1950). Inheritance and associations of hydrocyanic acid potential, disease reactions, and other characters in Sudan grass., Sorghum vulgare var. sudanensis. Ph.D. Thesis, University of Wisconsin, Madison. 18. TIMOMN, M. I. (1941). The interaction of higher plants and soil microorganisms. III. Effect of by-products of plant growth on activity of fungi and actinomycetcs. Soil Science 52, 395-414. 19. TRIONE, E. J. (1960). The HCN content of flax in relation to flax wilt resistance. Phytopathology 50,462-466. 20. WAI-I-ENBARGER, D. W., GRAY, E., RICE, J. S. & REYNOLDS, J. H. (1968). Effects of frost and freezing on hydrocyanic acid potential of sorghum plants. Crop Scieme 8, 526-528.
3