Phenylalanine ammonia-lyase activity in soybean hypocotyls and leaves following
infection with Phytophthora megasperma f.sp. glycinea
M. K. BHATTACHARYYA
Department of Plant Sciences, University of Western Ontario, London, Ont., Canada N6A 5B7
AND
E. W. B. WARD'
Research Centre, Agriculture Canada, University Sub Post Ofice, London, Ont., Canada N6A 5B7
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Received January 6, 1986
M. K., and WARD,E. W. B. 1988. Phenylalanine ammonia-lyase activity in soybean hypocotyls and leaves
BHATTACHARYYA,
following infection with Phytophthora megasperma f.sp. glycinea. Can. J. Bot. 66: 18-23.
Phenylalanine ammonia-lyase activity increased rapidly beginning 2 h after inoculation with Phytophthora megasperma
(Drechs.) f.sp. glycinea (Hildeb.) Kuan & Erwin race 1 in unwounded hypocotyls of soybean cv. Harosoy 63 (resistant) but
did not change significantly in cv. Harosoy (susceptible). Small increases in phenylalanine ammonia-lyase activity also were
caused by wounding. Activity increased more slowly in hypocotyls (cv. Harosoy 63) wounded just before inoculation than in
intact inoculated hypocotyls, but most activity developed in hypocotyls wounded 12 h before inoculation. There were comparable effects of wounding on symptom development. Trifoliolate leaves of 14-day-old cv. Harosoy 63 plants are resistant,
but trifoliolate leaves of 12-day-old cv. Harosoy 63 plants and of 14-day-old cv. Harosoy plants are susceptible to race 1.
Increases in phenylalanine ammonia-lyase activity following inoculation were demonstrated only in 14-day-old Harosoy 63
plants but not until 24-36 h after the inoculation. Significant accumulation of glyceollin occurred by 24 h. Susceptible
trifoliolate leaves of 12-day-old cv. Harosoy 63 plants produced only low levels of glyceollin following either infection or
treatment with the abiotic elicitor AgNO,, whereas trifoliolate leaves of 14-day-old cv. Harosoy plants produced high levels of
glyceollin in response to AgNO,. It is concluded that trifoliolate leaves of 12-day-old, as opposed to 14-day-old, cv. Harosoy
63 plants have not developed mechanisms that trigger responses to either infection or the abiotic elicitor or they are deficient in
metabolic processes that support glyceollin biosynthesis or other defense-related responses.
M. K., et WARD,E. W. B. 1988. Phenylalanine ammonia-lyase activity in soybean hypocotyls and leaves
BHATTACHARYYA,
following infection with Phytophthora megasperma f.sp. glycinea. Can. J. Bot. 66 : 18-23.
L'activitC de l'ammonium phCnylalanine lyase augmente rapidement B partir de la 2' h aprks l'inoculation avec le Phytophthora megasperma (Dreschs.) f.sp. glycinea (Hildeb.) Kuan & Erwin race 1 sur hypocotyle de soja non bless6 du cv. Harosoy
63 (resistant); cependant, cette activitC ne change pas significativement sur le cv. Harosoy (susceptible). La blessure
occasionne Cgalement une faible augmentation de 1'activitC de l'ammonium phknylalanine lyase. L'activitC augmente plus
lentement chez les hypocotyles qui sont blesses juste avant l'inoculation (cv. Harosoy 63) que chez les hypocotyles intacts au
moment de l'inoculation, mais 1'activitC la plus ClevCe apparait chez les hypocotyles blessks 12 h avant l'inoculation. On
retrouve des effets comparables de la blessure sur le dCveloppement des sympt6mes. Les feuilles du cv. Harosoy 63 agCes de
14jours sont resistantes mais celles de 12 jours de ce m&mecultivar ainsi que celles du cv. Harosoy agkes de 14jours sont susceptible~B la race 1. L'augmentation de l'activitk de l'ammonium phknylalanine lyase suite B l'inoculation n'a CtC dCmontrCe
que chez les plants du cv. Harosoy 63 ages de 14 jours et pas avant 24-36 h. On note une augmentation significative de la
glycColline autour de 24 h. Les feuilles susceptibles agCes de 12 jours du cv. Harosoy 63 ne produisent que de faibles quantitCs
de glyckolline B la suite soit de l'infection ou soit d'un traitement avec l'kliciteur abiotique AgNO,, alors que les feuilles de
plants ages de 14 jours du cv. Harosoy produisent de grandes quantitks de glyckolline lorsqu'elles sont traitCes avec AgN0,.
Les auteurs concluent que les feuilles de plants du cv. Harosoy 63 ages de 12 jours, contrairement B celles de plants agCs de 14
jours, ne posskdent pas les mkcanismes qui dCclenchent les rkactions soit B l'infection soit B l'kliciteur abiotique, ou encore que
ces plants ne posskdent pas les processus mktaboliques qui supportent la biosynthkse de la glycColline ou d'autres rCaction
relikes B la defense.
[Traduit par la revue]
Introduction
The association of phenylalanine ammonia-lyase (PAL)
activity with the accumulation of phytoalexins was reported by
Hadwiger (1967) for peas and by Rahe et al. (1969) for beans.
The enzyme catalyzes the first step of the phenylpropanoid
pathway and hence it would be expected that activity should be
correlated with production of isoflavonoid phytoalexins in
incompatible host -pathogen interactions. This has been found
to the the case for elicitor-treated cell suspensions of beans and
soybeans (e.g., Ebel et al. 1976; Dixon and Bendall 1978;
Dixon and Lamb 1979; Lawton et al. 1983; Ebel et al. 1984;
Cramer et al. 1985; Robbins et al. 1985). Results with whole
plants have been less consistent. Recently PAL activity was
'Author to whom correspondence should be addressed.
Prinlcd in Canada / Imprim6 au Canada
correlated with glyceollin production in soybean (Glycine max
(L.) Merr.) roots (Bonhoff et al. 1986); however Partridge and
Keen (1977) concluded earlier that PAL activity was not
related to glyceollin synthesis in soybean hypocotyls wounded
and inoculated with P. megasperma (Drechs.) f.sp. glycinea
(Hildeb.) Kuan & Erwin. They found little difference between
PAL activity in wounded controls, in which glyceollin did not
accumulate, and that in inoculated hypocotyls in which it did
accumulate. Borner and Grisebach (1982), in a study of the
same interaction, detected little PAL activity in wounds but
reported a gradual increase in activity following inoculation.
They also found that activity was similar in both compatible
and incompatible interactions for the first 14 h. This was consistent with their data for glyceollin accumulation but not with
the data of others (Yoshikawa et al. 1978, 1979), or with
development of early differences in the extent of tissue
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BHATTACHARYYA AND WARD
colonization in compatible and incompatible interactions in
soybean hypocotyls o r roots in which glyceollin is presumed to
play a causal role (Yoshikawa et al. 1978; Hahn et al. 1985).
Furthermore, a recent report (Esnault et al. 1987) has provided
evidence for gene transcription of P A L m R N A in etiolated
soybean hypocotyls within 3 h of inoculation in the incompatible but not in the c o m ~ a t i b l einteraction.
W e reported recentl; that the Rps, gene for resistance to
Phytophthora megasperma f.sp. glycinea race 1 canied by cv.
Harosoy 63 is expressed in leaves in addition t o hypocotyls
(Bhattacharyya and Ward 1986). However, this was influenced
greatly by the age of the plant and the maturity of the leaves.
Thus, while leaves from 14-day-old cv. Harosoy 63 plants
were resistant and accumulated glyceollin, leaves from
12-day-old plants of the same cultivar were susceptible. In this
paper, therefore, w e have examined P A L activity in leaves as
well as in wounded and intact hypocotyls following inoculation with Phytophthora megasperma f.sp. glycinea.
Materials and methods
Host
Seeds of the near-isogenic soybean cultivars 'Harosoy' and
'Harosoy 63' were provided by R. I. Buzzell, Research Station, Agriculture Canada, Harrow, Ont. Etiolated seedlings were grown in trays
of vermiculite for 6 days in the dark as described previously (Ward
et al. 1979). Trifoliolate leaves were obtained from 14-day-old cv.
Harosoy and 12- and 14-day-old cv. Harosoy 63 plants grown in soil
(black muck - peat moss - sand, 5:2:1, by volume) that had been
pasteurized for 30 min at 100°C. The plants were grown in a growth
room at 95% RH with a daily 15-h light period (approximately
150 pE. m+. s" of photosynthetically active radiation) and daily temperatures of 23: 16°C (1ight:dark).
Pathogen
Phytophthora megasperma f.sp. glycinea race 1 (Ward and Buzzell
1983) was mainatined on V8-juice agar at 25°C. This race is virulent
on cv. Harosoy (rps,) and avirulent on cv. Harosoy 63 (Rps,). Zoospores were produced as described previously (Ward et al. 1979). A
10-pL drop of zoospore suspension (105/mL) was used as inoculum.
Elicitor
An abiotic elicitor of glyceollin, AgNO, (lo-, M) (Stossel 1982),
was applied in 10-pL drops to the upper surface of leaves.
Inoculation and incubation
Etiolated hypocotyls from 6-day-old seedlings were arranged horizontally in glass trays as described previously (Ward et al. 1979) and
two drops of zoospore suspension were placed on each hypocotyl
about 2 cm below the cotyledons and about 0.7 cm apart. Drops of
sterile distilled water were added to control hypocotyls. In hypocotyls
that were wounded, two surface wounds (about 3 mm long and
0.5 mm deep) were made about 2 cm below the cotyledons and about
0.7 cm apart immediately prior to inoculation, except in one experiment, in which hypocotyls were wounded 12 h prior to inoculation. In
that experiment sterile distilled water (10 pL) was placed in each
wound to prevent desiccation during the 12-h period. Wounds were
inoculated with 10 pL of zoospore suspension or sterile distilled water
in controls. Ten hypocotyls (20 inoculated or control sites) were used
for each treatment. After inoculation or wounding, hypocotyls were
incubated in the dark at 25OC and 100% RH.
Trifoliolate leaves were arranged on wet Cellucotton in glass trays
as described previously (Bhattacharyya and Ward 1986). Six or eight
drops of zoospore suspension, AgNO, solution, or sterile distilled
water were placed on adaxial surfaces of leaflets of 12-day-old plants;
20 drops (to give comparable surface distribution) were used on the
larger leaflets of 14-day-old plants. Eighty inoculated sites were used
for each treatment (10 leaflets from 12-day-old plants, 4 leaflets from
14-day-old plants). Trays were closed immediately with plastic film
19
and left undisturbed on a laboratory bench for 3-4 h. Thereafter,
they were incubated in a growth cabinet at 100% RH with a daily 16-h
light period (fluorescent lamps; approximately 33 pE . m+. S-I), at
23: 16°C (1ight:dark).
Determination of phenylalanine ammonia-lyase activity
For determinations of activity in hypocotyls, sections approximately 1.75 cm long containing the wounded and inoculated sites
were used. For leaves, the tissues of 80 lesions for each treatment
were excised carefully with a scalpel eliminating noninfected tissue;
in controls and AgN0,-treated leaves the tissues covered by the drops
of water or AgNO, solution were excised. The tissues were weighed,
immediately frozen in liquid nitrogen, and stored at -70°C. The
tissues were ground with a mortar and pestle with 0.1 M sodium
borate buffer, pH 8.8, containing 2 mM mercaptoethanol (Lamb
et al. 1979). The slurry was centrifuged in a microcentrifuge at
15000 rpm for 4 min. The supernatant was collected and, after its
volume was recorded, immediately frozen in liquid nitrogen and
stored at -70°C until required. PAL activity in the supernatant was
determined by measuring spectophotomet~callythe production of
cinnamic acid from L-phenvlalanine (Lamb et al. 1979). The reaction
mixture contained 300 p ~ s o d i u mborate, pH 8.8, 30 pM L-phenylalanine, and 0.5 (hypocotyl) or 1 mL (leaf) of supernatant in a total
volume of 3 mL. After incubation for 1 h at 40°C the absorbance at
290 nm was read against an identical mixture in which D-phenylalanine was substituted for L-phenylalanine. The enzyme activity was
expressed as nanomole cinnamic acid produced per minute per gram
fresh weight of tissue.
Determination of glyceollin
Excised tissues from lesions in leaves inoculated with zoospores or
treated with AgN0, solution (lo', M) were extracted with 5 mL 95%
ethanol by boiling for 2 min. The ethanol was decanted and combined
with two ethanol rinses (2 mL) of the tissues. The tissues were dried
and weighed. The ethanol was reduced nearly to dryness and the
residue extracted three times with 2 mL of ethyl acetate. The ethyl
acetate soluble fraction was dried and redissolved in 100 pL ethyl
acetate and together with two 200-pL rinses applied to a thin-layer
chromatography plate (silica, Whatman LK6DF, 250 pm thick). The
plates were developed in benzene-methanol (95:8, vlv) and glyceollin was detected by fluorescence quenching under ultraviolet light.
Silica bands containing glyceollin were eluted with ethyl acetate.
After evaporating the ethyl acetate and redissolving the residue in
ethanol, glyceollin (a mixture of three isomers) was determined from
its absorption at 285 nm and the extinction coefficient (Ayers et al.
1976). Glyceollin concentrations are expressed as micrograms per
gram fresh weight of tissue. Efficiency of the procedure was assessed
by analyzing leaf tissue to which purified glyceollin I had been added.
Recovery was from 83 to 87 % of glyceollin I added in the concentration range of 250 to 3000 pg. g fresh weight-'. Data are presented
without correction.
Determination of reducing sugars
Reducing sugars in the supernatant used in the assay for PAL activity were determined using the arseno-molybdate method of Nelson
( 1944).
Statistical analysis
Data were analyzed in a completely randomized block design
(Little and Hills 1978).
Results
~
~ ~ypocoty~s
~
~
l
~
~
~
In intact etiolated ~ypocotyls, symptoms following inoculation were consistent with those reported previously (Ward
et al. 1979). In the incompatible interaction (CV.~ a r o s 63)
o~
brown spots were visible after 5-6 h , while in the compatible
interaction (CV. Harosoy), surfaces of inoculated sites developed a transparency that preceded the general water soaking
d
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TABLE 1. Phenylalanine ammonia-lyase activity (nmol cinnamic acid. min-'. g
fresh wt.?) in trifoliolate leaves of soybean inoculated with Phytophthora megaspenna f.sp. glycinea
Cultivar
Plant age
(days)
Harosoy
Harosoy 63
Harosoy 63
14
12
14
Inoculated*
24 h t
36 h
20.6k9.73 9.4k3.7
10.5k6.3 16.3k12.2
9.9k3.5 84.2k13.3
Control
24 h
36 h
26.1k10.9
8.7k1.5
9.9k3.6
8.2k2.2
10.2k2.0
9.2k3.0
*Trifoliolate leaves were inoculated by placing 10-pL drops of zoospore suspension (IOs/mL) of Ptlyropt~.
rhora tnegaspentro f.sp. glycinea race 1 on the adaxial surface. Drops of water (10 pL) were applied to
controls.
tIncubation period following inoculation until analysis.
$Data are the means and standard errors from two experiments. Data were analyzed in a completely randomized block design treating each experiment as a replicate; the LSD value ( p = 0.05) was calculated to be 18.7.
with browning that was prevalent after 11 - 12 h. By comparison, the development of symptoms was much delayed in
hypocotyls wounded just before inoculation. Browning could
not be detected in the resistant response in wounds in cv.
Harosoy 63 until 8-9 h after inoculation. There also was a
reduction in the amount of browning in the compatible interaction (cv. Harosoy). Hypocotyls wounded 12 h prior to inoculation, however, responded very rapidly to infection. In
resistant responses in such hypocotyls of cv. Harosoy 63,
tissues became brown 4 h after inoculation.
In intact etiolated hypocotyls PAL activity increased rapidly
from 2 h after inoculation in the resistant response (cv.
Harosoy 63) to P. megasperma f.sp. glycinea race 1 (Fig. la)
but not in the susceptible response (cv. Harosoy). At 8 h after
inoculation PAL activity in the resistant response was more
than six times greater than that in the susceptible response or
control hypocotyls. Wounding caused a small increase in PAL
activity over that in intact hypocotyls (Figs. l a , lb). Significant differences in PAL activity between resistant and susceptible responses in wounded hypocotyls did not develop until
after 6 h and stimulation in the resistant response (cv. Harosoy
63) was much smaller than in unwounded hypocotyls. In hypocotyls wounded 12 h before inoculation the trends to increased
PAL activity in the controls continued (Fig. lc). There was
also a rapid increase in activity in the resistant response, and
this was significantly greater than that in intact hypocotyls. In
hypocotyls wounded 12 h before inoculation there was also a
significant stimulation of activity in the susceptible response at
8 h after inoculation above that in the control (Fig. lc).
Leaves
The selection of leaves used for determination of PAL activity was based upon observations of symptoms reported previously (Bhattacharyya and Ward 1986). Trifoliolate leaves
from 14-day-old cv. Harosoy plants were susceptible and
developed pale brown lesions by 24 h, which started to spread
by 36 h, with the development of soft rotten tissues. Trifoliolate leaves from 12-day-old cv. Harosoy 63 were susceptible
also and lesions were similar to those in cv. Harosoy. However, by 14 days trifoliolate leaves of cv. Harosoy 63 were
resistant. Brown spots appeared beneath inoculum drops 12 h
after inoculation and conspicuous dark brown to reddish brown
lesions, more or less restricted to the inoculated area, developed by 24 h.
There was no significant change in PAL activity in any of
the infected leaves until 24 h after inoculation (Table 1). At 36
h after inoculation there was a major increase in activity in
TABLE2. Accumulation of glyceollin (pg .g fresh wt.-') in leaves of
soybeans following inoculation with Phytophthora megasperma f.sp.
glycinea race 1 or AgN03*
Zoospores
Cultivar
Age (day)
24 h t
36 h
AgN02,
62 h
Harosoy
Harosoy 63
Harosoy 63
14
12
14
593 k453
205 k 4
928+ 128
400k4
336+151
1097+94
2876k243
556k2
2755k301
*Trifoliolate leaves were inoculated by placing 10-pL drops of a zoospore suspension
(105/mL or a AgNO, solution (10.' M) on the adaxial surface of detached leaves.
tIncubation period following inoculation or AgNO, treatment until analysis.
$Data are the means and standard errors from two replicate determinations. Data were
analyzed in a completely randomized block design; the LSD value ( p = 0.05) was
calculated to be 240. No glyceollin was detected in leaves that received water drops only.
leaves of 14-day-old cv. Harosoy 63 plants (resistant) but not
in leaves of other plants.
Following zoospore inoculation much more glyceollin
accumulated in trifoliolate leaves from 14-day-old cv. Harosoy
63 plants than in those from either 12-day-old cv. Harosoy 63
plants or 14-day-old cv. Harosoy plants (Table 2). At 24 h, but
not 36 h, after inoculation, glyceollin accumulation was
significantly less in 12-day-old cv. Harosoy 63 trifoliolate
leaves than in cv. Harosoy trifoliolate leaves. With AgN03
treatment (Table 2) amounts of glyceollin produced in trifoliolate leaves of 14-day-old plants of both cultivars were similar
but only about one-fifth as much was produced in trifoliolate
leaves of 12-day-old plants of cv. Harosoy 63.
No differences in reducing sugar levels were found among
the three groups of trifoliolate leaves (Table 3).
Discussion
The rapid increase in PAL activity in the resistant response
but not in the susceptible response of intact etiolated soybean
hypocotyls demonstrated here (Fig. la) is comparable with
that in intact roots of soybean seedlings reported by Bonhoff
et al. (1986). It is consistent also with evidence for early
increases in gene transcription for mRNA for PAL in resistant
but not in susceptible reactions reported by Esnault et al.
(1987) and with our unpublished evidence that rates of biosynthesis and accumulation of glyceollin I are higher in resistant
than in susceptible inoculated hypocotyls.
In hypocotyls wounded just prior to inoculation, symptom
development was delayed compared with that in intact hypocotyls. Similarly, PAL activity in the resistant response in
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BHATTACHARYYA AND WARD
Incubation period
(h)
FIG. 1. Phenylalanine ammonia-lyase activity in etiolated soybean
hypocotyls unwounded and wounded and inoculated with Phytophthora megaspenna f.sp. glycinea race 1. (a) Unwounded inoculated
hypocotyls of cv. Harosoy ( 0 )and cv. Harosoy 63 (0)and watertreated controls (cv. Harosoy, +; cv. Harosoy 63, 0). (b) Hypocotyls
of cv. Harosoy ( W ) and cv. Harosoy 63 (0)wounded and inoculated
immediately (0 h) and water-treated wounded controls (cv. Harosoy,
A;cv. Harosoy 63, A). (c) Hypocotyls of soybean cv. Harosoy ( W )
and cv. Harosoy 63 (0)wounded 12 h prior to inoculation at 0 h and
water-treated controls (cv. Harosoy, A ;cv. Harosoy 63, A). Points
on the graphs are the means of determinations from two experiments.
21
wounded hypocotyls increased at a lower rate than in intact
hypocotyls. These differences were due evidently to the
removal of the epidermal layer and a few cortical cell layers by
wounding. This suggests that the epidermal cell layer is important for early and rapid stimulation of PAL activity in response
to infection.
PAL activity in wounded tissues treated with water only was
slightly higher than that of intact hypocotyls (Figs. la, lb).
Bomer and Grisebach (1982) also observed only a low level of
PAL activity, but Partridge and Keen (1977) reported a high
level of PAL activity in wounded green hypocotyls of soybeans treated with water. Neither of the two studies, however,
observed any difference in PAL activity between resistant and
susceptible responses during early stages following inoculation. This contrasts with the present study, in which PAL
activity in wounded inoculated hypocotyls was significantly
lower in the susceptible response than in the resistant response.
Both studies used fragmented mycelium for inoculum, which
may have contained nonspecific elicitors that caused similar
responses in both resistant and susceptible tissues. They also
used green hypocotyls and it is possible that, as we observed
for leaves, green tissues have a sufficient basal level of PAL to
support biosynthesis of glyceollin during the first few hours of
the host -pathogen interaction.
Wounding of hypocotyls 12 h prior to inoculation (Fig. lc)
enhanced the development of symptoms and PAL activity significantly compared with wounding immediately before inoculation. This appears to be similar to the effects of wounding on
the development of hypersensitivity in potatoes reported by
Tomiyama (1960). There was a gradual increase in PAL activity due to wounding alone and by the time of inoculation this
was appreciably higher in hypocotyls wounded 12 h previously
than in unwounded hypocotyls. Following inoculation, PAL
activity reached a higher level in the hypocotyls wounded 12 h
before inoculation than in the unwounded hypocotyls. In these
hypocotyls also we have observed accelerated production of
glyceollin (M. K. Bhattacharyya and E. W. B. Ward, unpublished data). Possibly, the wound response involves the priming of cellular mechanisms for synthesis of PAL and other
enzymes that are rapidly mobilized on infection. Similar conclusions were reached by Inoue et al. (1984) for enhanced
activity of enzymes for furanoterpenoid biosynthesis in
wounded sweet potato roots. As they suggested, the enhanced
response may increase resistance to wound-invading microorganisms.
In leaves, PAL activity increased after 24 h only in the resistant response of 14-day-old trifoliolate leaves of cv. Harosoy
63. Therefore, accumulation of glyceollin that was demonstrated at 24 h occurred without stimulation of PAL activity
over background levels. This indicates that background levels
of PAL are sufficient for initial accumulations of glyceollin
and suggests that the control of pathways after PAL, leading to
other phenylpropanoids, may differ in leaves from that in
etiolated hypocotyls, e.g., there may be more demands for precursors for lignification in hypocotyls than in leaves. It suggests also that the Rps gene for resistance in cv. Harosoy 63
does not directly control glyceollin biosynthesis at the PAL
step, but that changes in PAL activity occur secondarily in
Data from each incubation period were analyzed in a completely randomized block design considering each experiment as a replication.
LSD values ( p = 0.05) calculated were 1.4, 3.8, 4.1, 9.6, and 6.7
for 0-, 2-, 4-, 6-, and 8-h incubation periods, respectively.
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TABLE3. Total reducing sugar concentrations (mg.g fresh wt.?) in soybean leaves
following inoculation with Phytophthora megasperma f.sp. glycinea race l *
Inoculated
Control
Cultivar
Plant age
(days)
24 hf
36 h
24 h
36 h
Harosoy
Harosoy 63
Harosoy 63
14
12
14
4.35+0.20$
4.45k0.23
4.87k0.91
3.96kO.04
3.85k0.31
4.47k0.11
3.61 k0.54
4.33k0.54
4.08k0.20
4.19k0.47
4.48k0.52
4.06k0.22
'Trifoliolate leaves were inoculated by placing 10-pL drops of a zoospore suspension (10' mL-') of Pttyrophrhora tnegaspemtu f.sp. glycittea race 1 on the adaxial surface. Drops of water (10 pL) were applied to
controls.
tlncubation period following inoculation until analysis.
$Data are the means and standard errors from two replicate determinations; Data were analyzed in a completely randomized block design; no significant variation (at p = 0.05) was observed among the treatments.
response to demand and may b e under allosteric control.
In contrast to the resistant response of trifoliolate leaves of
14-day-old plants of cv. Harosoy 63 to race 1, trifoliolate
leaves of 12-day-old plants of this cultivar were susceptible
and developed P A L activity comparable with that in 14-dayold trifoliolate leaves of the susceptible cultivar, Harosoy.
T h e relatively small differential in glyceollin concentrations
between resistant and susceptible responses in leaves has been
discussed elsewhere (Bhattacharyya and Ward 1986). Glyceollin concentrations in trifoliolate leaves of 12-day-old cv.
Harosoy 63 plants were reduced also at both 24 and 36 h after
inoculation and were less than in susceptible trifoliolate leaves
of cv. Harosoy. Moreover, when treated with AgNO, solution,
an abiotic elicitor of glyceollin (Stossel 1982), the level of
glyceollin accumulation in these leaves was only one-fifth that
of that in AgN0,-treated trifoliolate leaves of 14-day-old
plants of both cultivars. T h e leaves did not differ in levels of
reducing sugars that may b e regarded as general precursors for
phenylpropanoid biosynthesis as well as for many other processes. It appears that 12-day-old leaves have not developed
mechanisms for response to either infection o r the abiotic
elicitor o r they are deficient in metabolic processes to support
glyceollin synthesis o r other defense-related processes.
Acknowledgments
W e are grateful to R. I. Buzzell for supplying soybean seeds
and A. Gaidauskas for technical assistance. M . K. Bhattacharyya was the recipient of a Commonwealth Scholarship for
postgraduate studies.
AYERS,A. R., EBEL,J., FINELLI,F., BERGER,N., and ALBERSHEIM,P. 1976. Host-pathogen interactions. IX. Quantitative
assays of elicitor activity and characterization of the elicitor present
in the extracellular medium of cultures of Phytophthora megas p e r m var. sojae. Plant Physiol. 57: 75 1 -759.
BHATTACHARYYA,
M. K., and WARD,E. W. B. 1986. Expression of
gene-specific and age-related resistance and the accumulation of
glyceollin in soybean leaves infected with Phytophthora megas p e r m f. sp. glycinea. Physiol. Mol. Plant Pathol. 29: 105 - 113.
BONHOFF,A., LOYAL,R., EBEL, J., and GRISEBACH,
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