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Biochemical comparisons of resistance to wheat stem rust disease controlled by the Sr6 or Sr11 alleles
Phytial.
PI. Path.
(1971).
1, 397-407
Biochemical comparisons of resistance to wheat stem rust disease controlled by the Sr6 or Srll alleles J. M. DALY, P. LUDDEN and P.
SEEVJZRS
Department of Biochemistry and Xtrition, University of .Nebraska, Lincoln, Neb. 68503,
U.S.A.
(Accepted for publication
March
1971)
Near-isogenic lines of wheat carrying the Srll and sell alleles for resistance and susceptibility to race 56 of Puccinia graminis tritici were examined for total phenolic compounds and total peroxidase activity during infection. As in the case of the Sr6 allele, no significant changes in phenolic components were detected. Increases in total peroxidase with resistant reactions controlled by the Srll allele were similar to those found previously for the Sr6 allele and the same isozyme was responsible for the increase. Previous evidence obtained with the Sr6 allele indicated that this isozyme is not a causal factor in resistance. Because the genetic and physiological basis for resistance controlled by the Sr6 and Srll alleles is distinct, it is concluded that increased activity of the same isozyme in both instances is a result of a nonspecific event analogous to wounding. Infected plants carrying the sr6 allele, with low peroxidase activity, produced much more ethylene than resistant infected plants. The relationships among ethylene production, disease reaction and peroxidase activity are not easily resolved.
INTRODUCTION
studies [S, 15, 16, 17l, comparisons have been made of chemical and during infection of near-isogenic lines of wheat carrying the dominant Sr6 allele for resistance and the recessive sr6 allele for susceptibility to race 56 of Puccinia graminis Pers. Esp. tritici Eriks. & E. Henn. When grown at 20 “C, there were no significant increases in phenolic compounds with infection of either line [15], although the infection types [18] of each are quite dissimilar (resistant line immune to 0; susceptible line 3 to 4). Peroxidase activity in extracts of the resistant infected line showed marked increases over the susceptible infected line. This increase occurred at the time when mechanisms determining resistance or susceptibility should be apparent (days 3 to 4 after inoculation) and when indoleacetic acid decarboxylation shows even greater increase [2]. Evidence has been presented to show that much of the increase in peroxidase activity in extracts from resistant tissue is caused by the activation of a single peroxidase isozyme [17]. The mechanisms controlled at the Sr6 locus for resistance are influenced by temperature [2, II] and ethylene [5]. Higher temperatures (25 to 26” C) or ethylene treatment initiated at 3 or 4 days after inoculation cause resistant reactions to revert to susceptibility, a phenomenon analogous to “challenge” infection [S]. Such treatments do not cause total peroxidase activity [16] or the activity of individual isozymes induced by resistance [17] to change significantly despite reversal of disease In
previous
enzymic
changes
398
J.
M. Daly et al.
reaction. Consequently, it appears that the increase in peroxidase levels with resistant reactions is a response to, rather than a cause of, resistance. The relatively unusual effects of temperature and ethylene, in addition to the contrast with other data on biochemical changes in resistance [lo], raise a question as to whether the reactions controlled by the Sr6 allele are unique [15]. If so, the data obtained might not be applicable to other varietal-race combinations in the wheat-stem rust disease complex. In the present study, we have examined a genetically different locus (Srll alleles) for reaction to the same pathogen (race 56 of Puccinia gruminis Pers. f.sp. tritici Erick. & E. Henn) in order to obtain a partial answer to the question. MATERIALS
AND
METHODS
The near isogenic lines of wheat used were: susceptible srll (C.I. 14172), resistant Srll (C.I. 14171), susceptible sr6 (C.I. 14164), resistant S76 (C.I. 14163). Their histories are given by Loegering & Harmon [II]. The general procedures for growth and inoculation of plants and extraction of leaves for phenols are given in a previous paper [15]. The assays for phenolic compounds were restricted to the Folin-Denis method with absorbance measured at 660 nm and results expressed as chlorogenic acid equivalents. In a previous paper [17], a number of parameters in peroxidase extraction, calorimetric assay and electrophoretic assays on gels were examined and the procedures employed in this study are a consequence of that examination. Peroxidase extraction, employing two media, was accomplished with the same tissue disintegrator used previously [ 161. M e d ium A was that of Staples & Stahmann [19] ; O-1 M-Tris, O-5 M-sucrose, 0.1 o/o ascorbic acid and O-1 o/o cysteine HCl, final pH adjusted to 8.0. Medium B was 0.25 M-sucrose in 0.1 M-KHsPO, (pH 4.6). All data presented for calorimetric determination of total peroxidase activity employed the p-phenylenediamine assay [16, 171. The use of extraction medium A results in higher total peroxidase activity than medium B and is generally more suitable for gel electrophoresis. However, the kinetics of p-phenylenediamine oxidation in extracts prepared with medium A are slightly curvilinear, while medium B gives a linear reaction [17]. The departure from linearity through use of medium A was so slight that differences in activity among tissues could be established by measuring the overall change in absorbance for 5 min and expressing the rates as AA/min. Extracts prepared with medium A were applied directly to the top of the 7% running gel and electrophoresis was carried out in the modified Davis system [17]. Standard diameter tubes 10 cm in length were used for typical runs of 2.5 h at 3 mA/tube. The longer electrophoresis time permitted adequate separation of those enzymes specifically increased in resistant disease reactions, although it did result in loss of four rapidly moving isozymes migrating from the end of the gel. A shorter electrophoresis time permitted an analysis of the latter components but they increased non-specifically with infection of both resistant and susceptible plants. After removal of the gels from tubes, they were reacted for four hours in 10 ml of O-2 M-sodium acetate buffer (pH 5.0) containing O-250/O benzidine to which 0.02 ml of 1.5% H,O, was added just prior to gel immersion. The gels then were placed in 0.2 M-sodium acetate buffer (pH 5.0) until scanned at 340 nm at a speed
Wheat
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rust
399
resistance
of 1 cm/min in a Gilford scanning spectrophotometer with a range of 0 to 2.5 absorbancy units. The area of each isozyme peak was determined by tracing it on uniform bond paper and weighing the trace to the nearest mg. For reasons given previously [16], total peroxidase activity in extracts is expressed in units of change in absorbancy (AA) per min per leaf. In electrophoresis of peroxidase from different tissues, volumes of extract containing equivalent amounts of the original leaf material were used. Normally, the amount of extract applied (50 to 100 ~1) was equivalent to 5 x 10e4 g fresh weight or 5 x 10V3 parts of an intact primary leaf. Measurement of ethylene production was carried out as in an earlier paper [5] with minor modifications. Ethylene from excised leaves was collected for a period of 22 h each day and measured by gas chromatography. The column used was 1.83 m x 6.35 mm and contained 120 to 150 mesh Porapak Q-S. The quantity of ethylene produced was determined by comparing peak areas of samples with those of known amounts of ethylene standards. RESULTS Neither growth at 25 “C nor treatment with 80 parts ethylene/million at 20 “C caused significant change in infection type when resistance to race 56 is controlled by the Srll allele, although lines carrying the Sr6 alIele for resistance reverted to susceptibility under these conditions. A direct comparison was necessary (Table l), TABLE
1
Comparison under various conditions of infection Qpes produced on near-kogenic lines of wheat possessing either Sr6 or Srl 1 alleles for rust reaction to race 56 qf Puccinia graminis tritici
Growth conditions 25 “C 20 “C parts 20 X+80 ethylene/million
Sr6
4 0 4
Alleles sr6 Srll Infection type 4 4 4
otozotozotoz-
ST11
4 4 4
as Loegering & Harmon [11] have noted a range of infection types from 0; to 2 in reaction with the Srll allele, apparently owing to environmental effects. Under our conditions, lines carrying the Srll allele for resistance typically showed infection type 0; through the seventh or eighth day after inoculation. Subsequently, a small percentage of the infected sites sporulated and these were classified as pustules of 2-type although they were not entirely typical infection type 2 in that they did not possess the characteristic ring of secondary sporulation. At 20 “C, during the period (days 5 to 6) when the resistant infection type on lines carrying the Sr6 allele for resistance become visible, infected sites on the wheat line carrying the Sell allele for resistance were nearly identical visually, except that they were somewhat larger. Table 2 summarizes changes in phenolic compounds for lines possessing the $11 alleles (for two separate experiments) with a slight difference in the infection density
400
J. M. Daly
ef al.
between the two sets of plants. The concentration of phenolic compounds, expressed as chlorogenic acid equivalents, does not vary significantly among any of the tissues examined, especially from the third to sixth day after infection, when mechanisms of resistance or susceptibility are being developed [Z]. The absolute quantities are comparable to those determined with the Folin-Denis procedure for near-isogenic lines of wheat carrying the Sr6 alleles [15], although they are slightly lower. In Table 3, data on peroxidase activity in leaves from the same infected plants of Table 2, experiment 2, are combined with data from a third group of plants and TABLE
Concentrations
2
of phenolic compoundr during infection of near-isogenic lines of wheat the Srl 1 alleles for rust reaction to race 56 of Puccinia graminis tritici
pmol Tissue6 1
3
chlorogenic acid equivalents per g of tissue on day 4 5 6
Exfieriment I. I.47 1.26 1.52 1.42
R RI s SI R RI S SI
2.12 2.10 2.58 2.17
Q R and S refer to non-inoculated b Determined by the Folin-Denis
Experiment 2.22 2.18 2.29 2.56 leaves; reagent.
a
77 pustules/leaf I.22 1.26 l-36 1.49
1.27 1.15 1.16 1.31
1.37 1.25 1.13 1.40
2. 93 pustules/leaf 1.70 1.73 2.17 2-01
1.45 1.63 1.88 2.08
la35 l-70 1.57 1.93
RI
and
SI to inoculated
canying
leaves.
expressed as percentage of the activity in extracts from non-infected control leaves. In both sets of plants, the non-infected leaves of plants carrying either the Srll or srll allele had nearly identical activity, ranging from 10 to 15 peroxidase units over the period of time given in the Table. It is to be noted that these values are higher than those reported for comparable non-infected leaves with the Sr6 alleles, values which ranged from 5 to 10 peroxidase units per leaf [16]. TABLE
3
Percentage change in peroxidase activity, relative to non-infected controls, in extracts Prepared from infected near-isogenic lines of wheat with the Sr 11 alleles for rust reaction to Puccinia graminis tritici (race 56).
Tissuea
1
2
SI RI
114 128
112
108
a SI and RI refer to susceptible b Flecks observed on both lines occurred on day 5 or 6.
Days 3 124 154
after
infectiona 4
5
6
7
a
a2 156
95 188
118 260
100 210
105 138
or resistant infected by the fourth day,
tissue. sporulation
on the susceptible
lines
Wheat
stem
rust
resistance
401
The relationships shown in Table 3 are similar in several respects to those reported previously for Sr6 alleles [If?]. Slight increases in peroxidase activity are found in both susceptible and resistant reaction in the early stages of infection (days 1 to 2). Subsequently, infected resistant leaves increase in peroxidase, while no significant differences were apparent during the onset of sporulation in susceptible tissue.
IO lb)
IO
6 6
9
5
5
9 0.00 L
0
I
234
5 Fisk
FIG. 1. gels. Wheat tritici. SrlI day 10; (b) on ordinate
gel length
lIJ!Lk 2
3
4
5
6
(cm)
Typical isoperoxidase patterns obtained by electrophoretic separation on acrylamide leaves extracted 10 days after inoculation with race 56 of Puccinia p-aminis plants showed resistant reactions; srlI plants were susceptible. (a) ,911, inoculated, $711, inoculated, day 10; (c) Srll, control, day 10; (d) srll, control, day 10. 0 cm indicates top of gel.
There are some minor differences to be noted when peroxidase data for Srll reactions are compared to previous data. The increases in the early stages of infection (days 1 to 3) are not as large in the Sell reactions (Table 3). With resistance controlled at the Srll allele, the increase in peroxidase in the later stages of infection is not as great as with the Sr6 allele when expressed as a percentage of control activity (TabIe 3) or in terms of absolute values. As an example, after the fourth day of infection of plants with the Sr6 allele, only two values out of eight values recorded [16] were lower than 30 peroxidase units/leaf while with the Srll allele for resistance only two values out of the seven used for the calculation of the data of Table 3
402
J. M. Daly
et al.
exceeded 30 peroxidase units/leaf, despite the higher basal activity observed in noninoculated plants. The most conspicuous difference, however, was the fact that peroxidase activities in infected leaves of Srll plants reached a maximum on day 5 or 6 (Table 3) and then declined, while infected leaves of Sr6 plants continued to increase until at least the 15th day after inoculation [16]. These differences suggested the possibility that infections of plants carrying either the Sr6 or Srll alleles might differ in the number [I] or type [5] of peroxidase isozymes affected by disease in resistant plants. Figure 1 shows isozyme patterns for healthy and infected resistant or susceptible leaves of Srll plants 10 days after inoculation. As in previous studies [17], healthy leaves contained at least 14 isozymes
Time FIG.
resistant in peak
after
inoculation
(days)
2. Relative activity, measured by peak area, of four isozymes during and susceptible near-isogenic lines of wheat. Data as percentage increase area of infected tissue relative to healthy tissue. q , Sr 11; q , srl I.
infection of or decrease
and there did not appear to be any significant qualitative or quantitative differences between Sr6 and Srl 1 plants in the bands of activity observed on gels. Of the 14 isozymes detected, only those labelled bands 9 and 10 were consistently increased by infection. Isozyme bands 5 and 6 usually showed an increase but since the activity of these isozymes tends to decrease in normal tissues with age, the increase was variable [ 171. The increase in peroxidase in extracts from resistant infected leaves was due largely to bands 9 and IO. Figure 2 illustrates data from a different set of healthy and inoculated plants carrying the Srll alleles. The percentage change in enzyme activity measured by areas of isozyme bands 5, 6, 9 and 10 is shown as a function of
Wheat
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rust
403
resistance
disease development in resistant (black bars) or susceptible (white bars) lines. The activity represented by the area of peak 9 in resistant infected leaves rose to 250% of the corresponding non-infected line, while susceptible reactions caused a smaller increase but only during the first days after infection. The same general trends were observed in a second experiment and in that instance susceptible infected tissue showed somewhat higher activity and for a longer period of time.
(b)
2.50
6 1 a. .t z! -z < .'i 5
0.50 0.00 2.50 (dl
2.00
Disk
gel length
FIG. 3. Direct comparison of isoperoxidase patterns obtained healthy lines of wheat carrying the Sr6 or Srll alleles for resistance (a) Srll, inoculated, 0; day 8; (b)‘Sr6, inoculated, 0; day 8; (c) control, day 8. 0 cm on ordinate indicates top of gel.
from resistant infected or to wheat stem rust disease. Srll, control, day 8; (d) Sr6,
Figure 3 shows gel patterns obtained in a direct comparison of resistant healthy and infected plants carrying either the Sr6 or Sr1l alleles. The plants were inoculated at the same time and thus were infected to the same degree. Fric & Fuchs [7] have shown the importance of infection intensity in measurements of peroxidase activity during infection. Figure 3 shows that the same isozymes are activated during resistant disease reaction and also illustrates the generally higher peroxidase values with the Sr6 allele. Differential rates of production of ethylene by wheat carrying the Sr.Zl alleles and showing resistant (RI) or susceptible (SI) reactions are shown in Table 4. The
404
J. M. Daly
higher rate with susceptible reactions and the very large increase in rates initiation of sporulation (125 h and later) on susceptible lines was similar patterns reported for the Sr6 alleles [5]. Furthermore, there did not appear significant quantitative differences between resistant infected plants with SW or Sell alleles or between the lines carrying the recessive alleles (Table reference [5j .) TABLE
ef al.
at the to the to be either 4 and
4
Ethylene production at 20 “C by primary leaves of healthy or rust-affected resistant and susceptible lines of wheat possessing the Srl 1 alleles for rust reaction to race 56 of Puccinia gram&s tritici Ethylene
‘“R,
produced/leaf from
for 22 h at given intervals inoculation chamber
Tissue
77 to 99 h
101 to 123 h
125 to 147 h
Ra RI SI
1750 1280 1760
1720 2080 3100
1680 2180 7360
resistant
non-inoculated
leaves;
RI,
resistant leaves.
infected
after
removal
149 to 171 h 1440 3060 11500
leaves;
SI,
resistant
infected
DISCUSSION The present results with the Srll allele for disease reaction indicate that the Sr6 allele for resistance is not unique with respect to phenolic biosynthesis [15]. The data for both Sr6 and SrlI alleles are in contrast to other reports showing marked increases in total phenolics in rust-affected wheat varieties with either resistant or susceptible disease reactions [S, 131. It is possible that the use of near-isogenic lines, rather than varieties with heterogeneous genetic backgrounds, reduces spurious biochemical responses arising from infection. The earlier reports by Canadian workers [12] of greater synthesis in some fractions of aromatic compounds (measured by incorporation of radioactivity, not total phenolic compounds) are not in conflict with our results. Turnover of radioactive components does not mean necessarily that there is net synthesis. More recently, the same workers have reported net synthesis of two putrescine derivatives of ferulic and coumaric acids [.24]. These compounds, as pointed out by the investigators, do not appear to be responsible for resistance since they are produced in infected susceptible plants at 25 “C and nonspecifically by other pathogens and by stress conditions. [13] The analytical methods employed in our studies may have precluded detection of putrescine derivatives especially if they were present in small amounts relative to total aromatic components. It should be noted, however, that environmental conditions influence aromatic biosynthesis in normal plants [S, 201. Environmental factors may condition tissues so that in some instances [3, 8, 211 phenolics are synthesized in greater amounts as a consequence of disease. The Canadian group made several observations that suggested that stress and especially light are important for biosynthesis of the putrescine derivatives [14]. In our studies, light intensity was much lower than they report. Further, leaves were collected within 1 h of the start
Wheat
stem
rust
resistance
405
of the photoperiod, while at least 4 h of light apparently is optimal for detection of that these compounds were not putrescine compounds [ 141. It is conceivable synthesized in measurable amounts, if at all, in the biological material we assayed. Increases in peroxidase activity of resistant over susceptible inoculated wheat lines carrying the Sr6 alleles has also been demonstrated by Fric & Fuchs [7]. The fact that they used race 21 of Puccinia graminis tritici is further evidence that such increases are not unique to the specific host-parasite combination previously used [IS]. Although the magnitude of the increases in peroxidase activity was not identical in resistant reactions controlled by the Sr6 and Srll alleles, the same isozyme (band 9) is involved. In a previous detailed study, the problems in attempting to correlate isozyme patterns with activity in crude extracts were discussed [ 171. Evidence obtained by partial purification of several isozymes led to the conclusion that isozyme band 9 is responsible for most, if not all, of the increased peroxidase activity found in extracts from resistant plants. The fact that the same isozyme is activated in both instances is of some importance in understanding the possible roles of peroxidase in resistance. With the Sr6 allele for resistance, reversion to susceptibility by temperature or by ethylene treatments does not result in a loss of activity in isozyme 9, as would be expected if it were a cause of resistance. On the other hand, there is no ready way to break the correlation between peroxidase activity and disease resistance with the Srll allele. The two alleles are not only genetically distinct, they appear biochemically divergent in that the Srll gene is not sensitive to temperature or ethylene. The results with both alleles imply strongly that increases in peroxidase are a non-specific end-result of biochemical resistance, not a cause. It should be emphasized that such a conclusion cannot be supported unless all of the evidence, especially the temperature and ethylene sensitivity of the Sr6 alleles, from both sets of near-isogenic lines is considered. Attempts to identify chemical compounds causing disease resistance are summarized in recent reviews [4, 9, 10, 131. A major obstacle to full acceptance of the role of specific inhibitory compounds in resistance is the possibility that they arise from cell necrosis after invasion of the pathogen has been restricted by other mechanisms. The use of genetically defined sources of resistance and susceptibility may help to resolve this problem, especially if biological systems are available for which disease reaction can be altered after infection, as in the case of the Sr6 alleles [Z, 5, II]. Although disease resistance sometimes has been altered dramatically by temperature [ 101, there are no reported cases where this has been done after resistance has been expressed during infection. Usually, temperature treatments are applied prior to inoculation. Consequently, the tissue is susceptible during all stages of the infection process and it is impossible to logically decide that failure to subsequently detect antifungal compounds [4] is responsible for lack of resistance. In the case of the resistant Sr6 allele, exposure to 25 “C from the day of inoculation results in infections which are indistinguishable in isozyme patterns, as well as in disease reaction, from infections on the normally susceptible sr6 lines of wheat [17]. It could be argued from these data that failure to induce isozyme band 9 is connected with change to susceptibility; yet transfers to 25 “C after induction of this isozyme clearly show it does not decrease with the onset of susceptibility [17l.
J. M. Daly et al.
406
“Challenge” inoculations [9] suffer from similar uncertainty. It is assumed, a priori, that antifungal compounds induced by a first inoculation with an avirulent pathogen are responsible for the subsequent inhibition of normally virulent organisms. In view of many other possible metabolic changes, such proof is hardly sufficient. Although the present studies were concerned only with enzymic changes, the observations of the Canadian workers [I41 indicate that manipulation of disease reaction during the development of the disease may be a useful tool to exploit in studies of chemically based resistance. The patterns of ethylene production in infected near-isogenic lines of wheat are of some interest. In susceptible reactions controlled either by the sr6 or srll alleles, ethylene production increases markedly just prior to sporulation. Because of the effect of ambient ethylene in causing reversion from resistance to susceptibility and because of the low rates of ethylene production in the case of the Sr6 allele, it was suggested that ethylene may be necessary for the development of susceptible reactions [S]. Resistance controlled by the S-11 allele also is characterized by low rates of ethylene production, but it is not affected by added ethylene. If ethylene is required for the ultimate expression of susceptibility, the mechanism controlled by Srll allele for resistance must parallel, or be prior to, the biochemical sequence in susceptibility which is affected by ethylene. In view of the obvious discrepancies arising from (i) peroxidase activation by ambient ethylene [5], (ii) the high production rates for ethylene in susceptible tissue and (iii) the low peroxidase in such tissue, there is as yet no ready physiological explanation of the role of ethylene in rust diseases. This work has been approved by the Director of the Nebraska Agricultural Experiment Station as Paper No. 3084, Journal Series. The research reported was conducted near Project No. 15-17 and was supported in part by the Nebraska Wheat Commission. REFERENCES 1. ANDREEV, L. & SHAW, M. (1965). A note on the effect of rust infection on peroxidase isozymes in Aax. Can. 3. Bot. 43, 1479-1483. 2. ANTONELLI, E. & DALY, J. M. (1966). Decarboxylation of indoleacetic acid by near-isogenic lines of wheat resistant or susceptible to Puccinia gaminis f.sp. tritici. Phytajathology 56, 610-618. 3. BIEHN, W. L., KoE, J. & WILLIAMS, E. B. (1968). Accumulation of phenols in resistant plant-fungi interactions. Phytopatholopy 58, 1255-1260. 4. CRUICKSHANK, I. A. M. (1963). Disease resistance in plants-a review of some recent developments. J..Aust. Inst. a&c. &. 29, 23-30. 5. DALY. T. M.. SEEVERS, P. M. & LUDDEN, P. (1970). Studies on wheat stem rust resistance controlled at the Sr6 locus. III. Ethylene and disease reaction. Phytopatholop 60, 1648-1652. 6. EL BASYOUNI, S. & TOWERS, G. N. H. (1964). The phenolic acids in wheat. I. Changes during growth and development. Can. 3. Biochem. 42, 203-2 10. 7. FRIC, F. & FUCHS, W. H. (1970). Veranderugen der Aktivitat einiger Enzyme im Weizenblatt in Abhangigkeit van der temperaturlabilen VertrPglichkeit fiir Puccinia graminis tritici. Phytofiathol. <. 67, 161-174. 8. KIRALY, Z. & FARKAS, G. L. (1962). Relation between phenol metabolism and stem rust resistance in wheat. Phytopatholo,g 52, 657-664. 9. Ku6, J. (1966). Resistance of plants to infectious agents. A. Rev. Microbial. 20, 337-370. 10. KU& J. (1968). Biochemical control of disease resistance in plants. World Review of Pest Control 7, 42-55. Il. LOEGERING, W. Q. & HARMON, D. L. (1969). Wheat lines near-isogenic for reaction to Puccinia graminis tritici. Phytopathology 59, 456-459. *-
I
I
.
,
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rust
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12. ROHRINGER, R., FUCHS, A., LUNDERSTAD, J. & SAMBORSKI, D.J. (1967). Metabolism of aromatic compounds in healthy and rust-infected primary leaves. I. Studies with l*COs, quinate-ur*C and shikimate-U-14C as precursors. Can. J. Bot. 45, 863-869. 13. ROHRINGER, R. & SAMBORSKI, D. J. (1967). Aromatic compounds in the host-parasite interaction. Annual Review of PhytopatholoQ 5, 77-86. 14. SAMBORSKI, D. J. & ROHRINGER, R. (1970). Abnormal metabolites of wheat: Occurrence, isolation and biogenesis of 2-hydroxyputrescene amides Phytochemistry 9, 1939-1945. 15. SEEVERS, P. M. & DALY, J. M. (1970). Studies on wheat stem rust resistance controlled at the Sr6 locus. I. The role of phenolic compounds. Phytopathology 60, 1322-1328. 16. SEEVERS, P. M. & DALY, J. M. (1970). Studies on wheat stem rust resistance controlled at the Sr6 locus. II. Peroxidase activities. Phytopatholopy 60, 1642-1647. 17. SEEVERS, P. M., DALY, J. M. & CATEDRAL, F. F. (1971). The role of peroxidase isozymes in resistance to wheat stem rust disease. PI. Physiol., Lancaster (in the press). 18. STAKMAN, E. C., STEWART, D. M. & LOEGERING, W. Q. (Revised 1962). Identification of physiologic races of Puccinia graninis var. tritici. Bulletin. United States Department of Agriculture, Agriculture Research Service E6I7. 19. STAPLES, R. C. & STAHMANN, M. A. (1964). Changes in protein and several enzymes in susceptible bean leaves after infection by the bean rust fungus. Phytopatholopy 54, 760-764. 20. TAYLOR, A. 0. (1965). Some effects of photoperiod on the biosynthesis of phenylpropane derivatives in Xanthium. PI. Physiol., Lancaster 40, 273-280. 21. TOMIYAMA, K., SAKUMA, T., ISHIGAKA, N., SATO, N., KATSUI, N., TAKASUGI, M. & MASAMUNE, T. (1968). A new antifungal substance isolated from resistant potato tuber tissue infected by pathogens. Phytopathologv 58, 115-l 16.
PI. Path.
(1971).
1, 397-407
Biochemical comparisons of resistance to wheat stem rust disease controlled by the Sr6 or Srll alleles J. M. DALY, P. LUDDEN and P.
SEEVJZRS
Department of Biochemistry and Xtrition, University of .Nebraska, Lincoln, Neb. 68503,
U.S.A.
(Accepted for publication
March
1971)
Near-isogenic lines of wheat carrying the Srll and sell alleles for resistance and susceptibility to race 56 of Puccinia graminis tritici were examined for total phenolic compounds and total peroxidase activity during infection. As in the case of the Sr6 allele, no significant changes in phenolic components were detected. Increases in total peroxidase with resistant reactions controlled by the Srll allele were similar to those found previously for the Sr6 allele and the same isozyme was responsible for the increase. Previous evidence obtained with the Sr6 allele indicated that this isozyme is not a causal factor in resistance. Because the genetic and physiological basis for resistance controlled by the Sr6 and Srll alleles is distinct, it is concluded that increased activity of the same isozyme in both instances is a result of a nonspecific event analogous to wounding. Infected plants carrying the sr6 allele, with low peroxidase activity, produced much more ethylene than resistant infected plants. The relationships among ethylene production, disease reaction and peroxidase activity are not easily resolved.
INTRODUCTION
studies [S, 15, 16, 17l, comparisons have been made of chemical and during infection of near-isogenic lines of wheat carrying the dominant Sr6 allele for resistance and the recessive sr6 allele for susceptibility to race 56 of Puccinia graminis Pers. Esp. tritici Eriks. & E. Henn. When grown at 20 “C, there were no significant increases in phenolic compounds with infection of either line [15], although the infection types [18] of each are quite dissimilar (resistant line immune to 0; susceptible line 3 to 4). Peroxidase activity in extracts of the resistant infected line showed marked increases over the susceptible infected line. This increase occurred at the time when mechanisms determining resistance or susceptibility should be apparent (days 3 to 4 after inoculation) and when indoleacetic acid decarboxylation shows even greater increase [2]. Evidence has been presented to show that much of the increase in peroxidase activity in extracts from resistant tissue is caused by the activation of a single peroxidase isozyme [17]. The mechanisms controlled at the Sr6 locus for resistance are influenced by temperature [2, II] and ethylene [5]. Higher temperatures (25 to 26” C) or ethylene treatment initiated at 3 or 4 days after inoculation cause resistant reactions to revert to susceptibility, a phenomenon analogous to “challenge” infection [S]. Such treatments do not cause total peroxidase activity [16] or the activity of individual isozymes induced by resistance [17] to change significantly despite reversal of disease In
previous
enzymic
changes
398
J.
M. Daly et al.
reaction. Consequently, it appears that the increase in peroxidase levels with resistant reactions is a response to, rather than a cause of, resistance. The relatively unusual effects of temperature and ethylene, in addition to the contrast with other data on biochemical changes in resistance [lo], raise a question as to whether the reactions controlled by the Sr6 allele are unique [15]. If so, the data obtained might not be applicable to other varietal-race combinations in the wheat-stem rust disease complex. In the present study, we have examined a genetically different locus (Srll alleles) for reaction to the same pathogen (race 56 of Puccinia gruminis Pers. f.sp. tritici Erick. & E. Henn) in order to obtain a partial answer to the question. MATERIALS
AND
METHODS
The near isogenic lines of wheat used were: susceptible srll (C.I. 14172), resistant Srll (C.I. 14171), susceptible sr6 (C.I. 14164), resistant S76 (C.I. 14163). Their histories are given by Loegering & Harmon [II]. The general procedures for growth and inoculation of plants and extraction of leaves for phenols are given in a previous paper [15]. The assays for phenolic compounds were restricted to the Folin-Denis method with absorbance measured at 660 nm and results expressed as chlorogenic acid equivalents. In a previous paper [17], a number of parameters in peroxidase extraction, calorimetric assay and electrophoretic assays on gels were examined and the procedures employed in this study are a consequence of that examination. Peroxidase extraction, employing two media, was accomplished with the same tissue disintegrator used previously [ 161. M e d ium A was that of Staples & Stahmann [19] ; O-1 M-Tris, O-5 M-sucrose, 0.1 o/o ascorbic acid and O-1 o/o cysteine HCl, final pH adjusted to 8.0. Medium B was 0.25 M-sucrose in 0.1 M-KHsPO, (pH 4.6). All data presented for calorimetric determination of total peroxidase activity employed the p-phenylenediamine assay [16, 171. The use of extraction medium A results in higher total peroxidase activity than medium B and is generally more suitable for gel electrophoresis. However, the kinetics of p-phenylenediamine oxidation in extracts prepared with medium A are slightly curvilinear, while medium B gives a linear reaction [17]. The departure from linearity through use of medium A was so slight that differences in activity among tissues could be established by measuring the overall change in absorbance for 5 min and expressing the rates as AA/min. Extracts prepared with medium A were applied directly to the top of the 7% running gel and electrophoresis was carried out in the modified Davis system [17]. Standard diameter tubes 10 cm in length were used for typical runs of 2.5 h at 3 mA/tube. The longer electrophoresis time permitted adequate separation of those enzymes specifically increased in resistant disease reactions, although it did result in loss of four rapidly moving isozymes migrating from the end of the gel. A shorter electrophoresis time permitted an analysis of the latter components but they increased non-specifically with infection of both resistant and susceptible plants. After removal of the gels from tubes, they were reacted for four hours in 10 ml of O-2 M-sodium acetate buffer (pH 5.0) containing O-250/O benzidine to which 0.02 ml of 1.5% H,O, was added just prior to gel immersion. The gels then were placed in 0.2 M-sodium acetate buffer (pH 5.0) until scanned at 340 nm at a speed
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of 1 cm/min in a Gilford scanning spectrophotometer with a range of 0 to 2.5 absorbancy units. The area of each isozyme peak was determined by tracing it on uniform bond paper and weighing the trace to the nearest mg. For reasons given previously [16], total peroxidase activity in extracts is expressed in units of change in absorbancy (AA) per min per leaf. In electrophoresis of peroxidase from different tissues, volumes of extract containing equivalent amounts of the original leaf material were used. Normally, the amount of extract applied (50 to 100 ~1) was equivalent to 5 x 10e4 g fresh weight or 5 x 10V3 parts of an intact primary leaf. Measurement of ethylene production was carried out as in an earlier paper [5] with minor modifications. Ethylene from excised leaves was collected for a period of 22 h each day and measured by gas chromatography. The column used was 1.83 m x 6.35 mm and contained 120 to 150 mesh Porapak Q-S. The quantity of ethylene produced was determined by comparing peak areas of samples with those of known amounts of ethylene standards. RESULTS Neither growth at 25 “C nor treatment with 80 parts ethylene/million at 20 “C caused significant change in infection type when resistance to race 56 is controlled by the Srll allele, although lines carrying the Sr6 alIele for resistance reverted to susceptibility under these conditions. A direct comparison was necessary (Table l), TABLE
1
Comparison under various conditions of infection Qpes produced on near-kogenic lines of wheat possessing either Sr6 or Srl 1 alleles for rust reaction to race 56 qf Puccinia graminis tritici
Growth conditions 25 “C 20 “C parts 20 X+80 ethylene/million
Sr6
4 0 4
Alleles sr6 Srll Infection type 4 4 4
otozotozotoz-
ST11
4 4 4
as Loegering & Harmon [11] have noted a range of infection types from 0; to 2 in reaction with the Srll allele, apparently owing to environmental effects. Under our conditions, lines carrying the Srll allele for resistance typically showed infection type 0; through the seventh or eighth day after inoculation. Subsequently, a small percentage of the infected sites sporulated and these were classified as pustules of 2-type although they were not entirely typical infection type 2 in that they did not possess the characteristic ring of secondary sporulation. At 20 “C, during the period (days 5 to 6) when the resistant infection type on lines carrying the Sr6 allele for resistance become visible, infected sites on the wheat line carrying the Sell allele for resistance were nearly identical visually, except that they were somewhat larger. Table 2 summarizes changes in phenolic compounds for lines possessing the $11 alleles (for two separate experiments) with a slight difference in the infection density
400
J. M. Daly
ef al.
between the two sets of plants. The concentration of phenolic compounds, expressed as chlorogenic acid equivalents, does not vary significantly among any of the tissues examined, especially from the third to sixth day after infection, when mechanisms of resistance or susceptibility are being developed [Z]. The absolute quantities are comparable to those determined with the Folin-Denis procedure for near-isogenic lines of wheat carrying the Sr6 alleles [15], although they are slightly lower. In Table 3, data on peroxidase activity in leaves from the same infected plants of Table 2, experiment 2, are combined with data from a third group of plants and TABLE
Concentrations
2
of phenolic compoundr during infection of near-isogenic lines of wheat the Srl 1 alleles for rust reaction to race 56 of Puccinia graminis tritici
pmol Tissue6 1
3
chlorogenic acid equivalents per g of tissue on day 4 5 6
Exfieriment I. I.47 1.26 1.52 1.42
R RI s SI R RI S SI
2.12 2.10 2.58 2.17
Q R and S refer to non-inoculated b Determined by the Folin-Denis
Experiment 2.22 2.18 2.29 2.56 leaves; reagent.
a
77 pustules/leaf I.22 1.26 l-36 1.49
1.27 1.15 1.16 1.31
1.37 1.25 1.13 1.40
2. 93 pustules/leaf 1.70 1.73 2.17 2-01
1.45 1.63 1.88 2.08
la35 l-70 1.57 1.93
RI
and
SI to inoculated
canying
leaves.
expressed as percentage of the activity in extracts from non-infected control leaves. In both sets of plants, the non-infected leaves of plants carrying either the Srll or srll allele had nearly identical activity, ranging from 10 to 15 peroxidase units over the period of time given in the Table. It is to be noted that these values are higher than those reported for comparable non-infected leaves with the Sr6 alleles, values which ranged from 5 to 10 peroxidase units per leaf [16]. TABLE
3
Percentage change in peroxidase activity, relative to non-infected controls, in extracts Prepared from infected near-isogenic lines of wheat with the Sr 11 alleles for rust reaction to Puccinia graminis tritici (race 56).
Tissuea
1
2
SI RI
114 128
112
108
a SI and RI refer to susceptible b Flecks observed on both lines occurred on day 5 or 6.
Days 3 124 154
after
infectiona 4
5
6
7
a
a2 156
95 188
118 260
100 210
105 138
or resistant infected by the fourth day,
tissue. sporulation
on the susceptible
lines
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The relationships shown in Table 3 are similar in several respects to those reported previously for Sr6 alleles [If?]. Slight increases in peroxidase activity are found in both susceptible and resistant reaction in the early stages of infection (days 1 to 2). Subsequently, infected resistant leaves increase in peroxidase, while no significant differences were apparent during the onset of sporulation in susceptible tissue.
IO lb)
IO
6 6
9
5
5
9 0.00 L
0
I
234
5 Fisk
FIG. 1. gels. Wheat tritici. SrlI day 10; (b) on ordinate
gel length
lIJ!Lk 2
3
4
5
6
(cm)
Typical isoperoxidase patterns obtained by electrophoretic separation on acrylamide leaves extracted 10 days after inoculation with race 56 of Puccinia p-aminis plants showed resistant reactions; srlI plants were susceptible. (a) ,911, inoculated, $711, inoculated, day 10; (c) Srll, control, day 10; (d) srll, control, day 10. 0 cm indicates top of gel.
There are some minor differences to be noted when peroxidase data for Srll reactions are compared to previous data. The increases in the early stages of infection (days 1 to 3) are not as large in the Sell reactions (Table 3). With resistance controlled at the Srll allele, the increase in peroxidase in the later stages of infection is not as great as with the Sr6 allele when expressed as a percentage of control activity (TabIe 3) or in terms of absolute values. As an example, after the fourth day of infection of plants with the Sr6 allele, only two values out of eight values recorded [16] were lower than 30 peroxidase units/leaf while with the Srll allele for resistance only two values out of the seven used for the calculation of the data of Table 3
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J. M. Daly
et al.
exceeded 30 peroxidase units/leaf, despite the higher basal activity observed in noninoculated plants. The most conspicuous difference, however, was the fact that peroxidase activities in infected leaves of Srll plants reached a maximum on day 5 or 6 (Table 3) and then declined, while infected leaves of Sr6 plants continued to increase until at least the 15th day after inoculation [16]. These differences suggested the possibility that infections of plants carrying either the Sr6 or Srll alleles might differ in the number [I] or type [5] of peroxidase isozymes affected by disease in resistant plants. Figure 1 shows isozyme patterns for healthy and infected resistant or susceptible leaves of Srll plants 10 days after inoculation. As in previous studies [17], healthy leaves contained at least 14 isozymes
Time FIG.
resistant in peak
after
inoculation
(days)
2. Relative activity, measured by peak area, of four isozymes during and susceptible near-isogenic lines of wheat. Data as percentage increase area of infected tissue relative to healthy tissue. q , Sr 11; q , srl I.
infection of or decrease
and there did not appear to be any significant qualitative or quantitative differences between Sr6 and Srl 1 plants in the bands of activity observed on gels. Of the 14 isozymes detected, only those labelled bands 9 and 10 were consistently increased by infection. Isozyme bands 5 and 6 usually showed an increase but since the activity of these isozymes tends to decrease in normal tissues with age, the increase was variable [ 171. The increase in peroxidase in extracts from resistant infected leaves was due largely to bands 9 and IO. Figure 2 illustrates data from a different set of healthy and inoculated plants carrying the Srll alleles. The percentage change in enzyme activity measured by areas of isozyme bands 5, 6, 9 and 10 is shown as a function of
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disease development in resistant (black bars) or susceptible (white bars) lines. The activity represented by the area of peak 9 in resistant infected leaves rose to 250% of the corresponding non-infected line, while susceptible reactions caused a smaller increase but only during the first days after infection. The same general trends were observed in a second experiment and in that instance susceptible infected tissue showed somewhat higher activity and for a longer period of time.
(b)
2.50
6 1 a. .t z! -z < .'i 5
0.50 0.00 2.50 (dl
2.00
Disk
gel length
FIG. 3. Direct comparison of isoperoxidase patterns obtained healthy lines of wheat carrying the Sr6 or Srll alleles for resistance (a) Srll, inoculated, 0; day 8; (b)‘Sr6, inoculated, 0; day 8; (c) control, day 8. 0 cm on ordinate indicates top of gel.
from resistant infected or to wheat stem rust disease. Srll, control, day 8; (d) Sr6,
Figure 3 shows gel patterns obtained in a direct comparison of resistant healthy and infected plants carrying either the Sr6 or Sr1l alleles. The plants were inoculated at the same time and thus were infected to the same degree. Fric & Fuchs [7] have shown the importance of infection intensity in measurements of peroxidase activity during infection. Figure 3 shows that the same isozymes are activated during resistant disease reaction and also illustrates the generally higher peroxidase values with the Sr6 allele. Differential rates of production of ethylene by wheat carrying the Sr.Zl alleles and showing resistant (RI) or susceptible (SI) reactions are shown in Table 4. The
404
J. M. Daly
higher rate with susceptible reactions and the very large increase in rates initiation of sporulation (125 h and later) on susceptible lines was similar patterns reported for the Sr6 alleles [5]. Furthermore, there did not appear significant quantitative differences between resistant infected plants with SW or Sell alleles or between the lines carrying the recessive alleles (Table reference [5j .) TABLE
ef al.
at the to the to be either 4 and
4
Ethylene production at 20 “C by primary leaves of healthy or rust-affected resistant and susceptible lines of wheat possessing the Srl 1 alleles for rust reaction to race 56 of Puccinia gram&s tritici Ethylene
‘“R,
produced/leaf from
for 22 h at given intervals inoculation chamber
Tissue
77 to 99 h
101 to 123 h
125 to 147 h
Ra RI SI
1750 1280 1760
1720 2080 3100
1680 2180 7360
resistant
non-inoculated
leaves;
RI,
resistant leaves.
infected
after
removal
149 to 171 h 1440 3060 11500
leaves;
SI,
resistant
infected
DISCUSSION The present results with the Srll allele for disease reaction indicate that the Sr6 allele for resistance is not unique with respect to phenolic biosynthesis [15]. The data for both Sr6 and SrlI alleles are in contrast to other reports showing marked increases in total phenolics in rust-affected wheat varieties with either resistant or susceptible disease reactions [S, 131. It is possible that the use of near-isogenic lines, rather than varieties with heterogeneous genetic backgrounds, reduces spurious biochemical responses arising from infection. The earlier reports by Canadian workers [12] of greater synthesis in some fractions of aromatic compounds (measured by incorporation of radioactivity, not total phenolic compounds) are not in conflict with our results. Turnover of radioactive components does not mean necessarily that there is net synthesis. More recently, the same workers have reported net synthesis of two putrescine derivatives of ferulic and coumaric acids [.24]. These compounds, as pointed out by the investigators, do not appear to be responsible for resistance since they are produced in infected susceptible plants at 25 “C and nonspecifically by other pathogens and by stress conditions. [13] The analytical methods employed in our studies may have precluded detection of putrescine derivatives especially if they were present in small amounts relative to total aromatic components. It should be noted, however, that environmental conditions influence aromatic biosynthesis in normal plants [S, 201. Environmental factors may condition tissues so that in some instances [3, 8, 211 phenolics are synthesized in greater amounts as a consequence of disease. The Canadian group made several observations that suggested that stress and especially light are important for biosynthesis of the putrescine derivatives [14]. In our studies, light intensity was much lower than they report. Further, leaves were collected within 1 h of the start
Wheat
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of the photoperiod, while at least 4 h of light apparently is optimal for detection of that these compounds were not putrescine compounds [ 141. It is conceivable synthesized in measurable amounts, if at all, in the biological material we assayed. Increases in peroxidase activity of resistant over susceptible inoculated wheat lines carrying the Sr6 alleles has also been demonstrated by Fric & Fuchs [7]. The fact that they used race 21 of Puccinia graminis tritici is further evidence that such increases are not unique to the specific host-parasite combination previously used [IS]. Although the magnitude of the increases in peroxidase activity was not identical in resistant reactions controlled by the Sr6 and Srll alleles, the same isozyme (band 9) is involved. In a previous detailed study, the problems in attempting to correlate isozyme patterns with activity in crude extracts were discussed [ 171. Evidence obtained by partial purification of several isozymes led to the conclusion that isozyme band 9 is responsible for most, if not all, of the increased peroxidase activity found in extracts from resistant plants. The fact that the same isozyme is activated in both instances is of some importance in understanding the possible roles of peroxidase in resistance. With the Sr6 allele for resistance, reversion to susceptibility by temperature or by ethylene treatments does not result in a loss of activity in isozyme 9, as would be expected if it were a cause of resistance. On the other hand, there is no ready way to break the correlation between peroxidase activity and disease resistance with the Srll allele. The two alleles are not only genetically distinct, they appear biochemically divergent in that the Srll gene is not sensitive to temperature or ethylene. The results with both alleles imply strongly that increases in peroxidase are a non-specific end-result of biochemical resistance, not a cause. It should be emphasized that such a conclusion cannot be supported unless all of the evidence, especially the temperature and ethylene sensitivity of the Sr6 alleles, from both sets of near-isogenic lines is considered. Attempts to identify chemical compounds causing disease resistance are summarized in recent reviews [4, 9, 10, 131. A major obstacle to full acceptance of the role of specific inhibitory compounds in resistance is the possibility that they arise from cell necrosis after invasion of the pathogen has been restricted by other mechanisms. The use of genetically defined sources of resistance and susceptibility may help to resolve this problem, especially if biological systems are available for which disease reaction can be altered after infection, as in the case of the Sr6 alleles [Z, 5, II]. Although disease resistance sometimes has been altered dramatically by temperature [ 101, there are no reported cases where this has been done after resistance has been expressed during infection. Usually, temperature treatments are applied prior to inoculation. Consequently, the tissue is susceptible during all stages of the infection process and it is impossible to logically decide that failure to subsequently detect antifungal compounds [4] is responsible for lack of resistance. In the case of the resistant Sr6 allele, exposure to 25 “C from the day of inoculation results in infections which are indistinguishable in isozyme patterns, as well as in disease reaction, from infections on the normally susceptible sr6 lines of wheat [17]. It could be argued from these data that failure to induce isozyme band 9 is connected with change to susceptibility; yet transfers to 25 “C after induction of this isozyme clearly show it does not decrease with the onset of susceptibility [17l.
J. M. Daly et al.
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“Challenge” inoculations [9] suffer from similar uncertainty. It is assumed, a priori, that antifungal compounds induced by a first inoculation with an avirulent pathogen are responsible for the subsequent inhibition of normally virulent organisms. In view of many other possible metabolic changes, such proof is hardly sufficient. Although the present studies were concerned only with enzymic changes, the observations of the Canadian workers [I41 indicate that manipulation of disease reaction during the development of the disease may be a useful tool to exploit in studies of chemically based resistance. The patterns of ethylene production in infected near-isogenic lines of wheat are of some interest. In susceptible reactions controlled either by the sr6 or srll alleles, ethylene production increases markedly just prior to sporulation. Because of the effect of ambient ethylene in causing reversion from resistance to susceptibility and because of the low rates of ethylene production in the case of the Sr6 allele, it was suggested that ethylene may be necessary for the development of susceptible reactions [S]. Resistance controlled by the S-11 allele also is characterized by low rates of ethylene production, but it is not affected by added ethylene. If ethylene is required for the ultimate expression of susceptibility, the mechanism controlled by Srll allele for resistance must parallel, or be prior to, the biochemical sequence in susceptibility which is affected by ethylene. In view of the obvious discrepancies arising from (i) peroxidase activation by ambient ethylene [5], (ii) the high production rates for ethylene in susceptible tissue and (iii) the low peroxidase in such tissue, there is as yet no ready physiological explanation of the role of ethylene in rust diseases. This work has been approved by the Director of the Nebraska Agricultural Experiment Station as Paper No. 3084, Journal Series. The research reported was conducted near Project No. 15-17 and was supported in part by the Nebraska Wheat Commission. REFERENCES 1. ANDREEV, L. & SHAW, M. (1965). A note on the effect of rust infection on peroxidase isozymes in Aax. Can. 3. Bot. 43, 1479-1483. 2. ANTONELLI, E. & DALY, J. M. (1966). Decarboxylation of indoleacetic acid by near-isogenic lines of wheat resistant or susceptible to Puccinia gaminis f.sp. tritici. Phytajathology 56, 610-618. 3. BIEHN, W. L., KoE, J. & WILLIAMS, E. B. (1968). Accumulation of phenols in resistant plant-fungi interactions. Phytopatholopy 58, 1255-1260. 4. CRUICKSHANK, I. A. M. (1963). Disease resistance in plants-a review of some recent developments. J..Aust. Inst. a&c. &. 29, 23-30. 5. DALY. T. M.. SEEVERS, P. M. & LUDDEN, P. (1970). Studies on wheat stem rust resistance controlled at the Sr6 locus. III. Ethylene and disease reaction. Phytopatholop 60, 1648-1652. 6. EL BASYOUNI, S. & TOWERS, G. N. H. (1964). The phenolic acids in wheat. I. Changes during growth and development. Can. 3. Biochem. 42, 203-2 10. 7. FRIC, F. & FUCHS, W. H. (1970). Veranderugen der Aktivitat einiger Enzyme im Weizenblatt in Abhangigkeit van der temperaturlabilen VertrPglichkeit fiir Puccinia graminis tritici. Phytofiathol. <. 67, 161-174. 8. KIRALY, Z. & FARKAS, G. L. (1962). Relation between phenol metabolism and stem rust resistance in wheat. Phytopatholo,g 52, 657-664. 9. Ku6, J. (1966). Resistance of plants to infectious agents. A. Rev. Microbial. 20, 337-370. 10. KU& J. (1968). Biochemical control of disease resistance in plants. World Review of Pest Control 7, 42-55. Il. LOEGERING, W. Q. & HARMON, D. L. (1969). Wheat lines near-isogenic for reaction to Puccinia graminis tritici. Phytopathology 59, 456-459. *-
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12. ROHRINGER, R., FUCHS, A., LUNDERSTAD, J. & SAMBORSKI, D.J. (1967). Metabolism of aromatic compounds in healthy and rust-infected primary leaves. I. Studies with l*COs, quinate-ur*C and shikimate-U-14C as precursors. Can. J. Bot. 45, 863-869. 13. ROHRINGER, R. & SAMBORSKI, D. J. (1967). Aromatic compounds in the host-parasite interaction. Annual Review of PhytopatholoQ 5, 77-86. 14. SAMBORSKI, D. J. & ROHRINGER, R. (1970). Abnormal metabolites of wheat: Occurrence, isolation and biogenesis of 2-hydroxyputrescene amides Phytochemistry 9, 1939-1945. 15. SEEVERS, P. M. & DALY, J. M. (1970). Studies on wheat stem rust resistance controlled at the Sr6 locus. I. The role of phenolic compounds. Phytopathology 60, 1322-1328. 16. SEEVERS, P. M. & DALY, J. M. (1970). Studies on wheat stem rust resistance controlled at the Sr6 locus. II. Peroxidase activities. Phytopatholopy 60, 1642-1647. 17. SEEVERS, P. M., DALY, J. M. & CATEDRAL, F. F. (1971). The role of peroxidase isozymes in resistance to wheat stem rust disease. PI. Physiol., Lancaster (in the press). 18. STAKMAN, E. C., STEWART, D. M. & LOEGERING, W. Q. (Revised 1962). Identification of physiologic races of Puccinia graninis var. tritici. Bulletin. United States Department of Agriculture, Agriculture Research Service E6I7. 19. STAPLES, R. C. & STAHMANN, M. A. (1964). Changes in protein and several enzymes in susceptible bean leaves after infection by the bean rust fungus. Phytopatholopy 54, 760-764. 20. TAYLOR, A. 0. (1965). Some effects of photoperiod on the biosynthesis of phenylpropane derivatives in Xanthium. PI. Physiol., Lancaster 40, 273-280. 21. TOMIYAMA, K., SAKUMA, T., ISHIGAKA, N., SATO, N., KATSUI, N., TAKASUGI, M. & MASAMUNE, T. (1968). A new antifungal substance isolated from resistant potato tuber tissue infected by pathogens. Phytopathologv 58, 115-l 16.