Phenolic biosynthesis inhibitors suppress adult plant resistance to Erysiphe graminis in oat at 20...

Physiological and Molecular Plant Pathology (1996) 49, 121–141

Phenolic biosynthesis inhibitors suppress adult plant

resistance to Erysiphe graminis in oat at 20 °C and

10 °C

T. L. W. C*", L. Z", R. J. Z# and M. P. R"

" Institute of Grassland and En�ironmental Research, Aberystwyth, Dyfed SY23 3EB, U.K.and #Department of Plant Pathology, Uni�ersity of Minnesota, St Paul MN 55108, U.S.A.

(Accepted for publication May 1996)

Seedling and adult plant leaves of three oat genotypes, Selma, Maldwyn and OM1387 (a hybridderived from a Selma¬Maldwyn cross), were excised and treated with water or with α-amino-β-phenylpropionic acid (AOPP) to inhibit phenylalanine ammonia lyase activity, or with [[(2-hydroxyphenly)amino] sulfinyl]acid 1,1-dimethylethyl ester (OH-PAS) to inhibit cinnamylalcohol dehydrogenase activity. None of these genotypes possess any known major gene resistanceto the isolate of Erysiphe graminis f.sp. a�enae with which they were inoculated, although Maldwynis known to possess quantitative adult plant resistance that has proved durable since 1947. Allgenotypes showed some adult plant resistance which limited the proportion of fungal appressoriathat formed haustoria in water-treated leaves. However, this resistance was expressed morestrongly in Maldwyn and OM1387 than in Selma. Treatment with AOPP or OH-PAS increasedsubstantially the proportion of fungal appressoria that formed haustoria in adult as well asseedling leaves, indicating that phenolic compounds, probably products of the lignin biosyntheticpathway, contributed to resistance. However, AOPP-treated adult leaves remained more resistantto infection than AOPP-treated seedling leaves, suggesting that additional unknown factor(s)contributed to adult plant resistance. In all genotypes and in seedling and adult leaves, failure ofappressoria to infect was correlated to the localized accumulation of autofluorescent componentsin host cell papillae and cell wall regions subtending appressoria. This localized autofluorescencewas suppressed by AOPP treatment, and to a lesser extent by OH-PAS treatment, indicating thatthe fluorogens were phenolic compounds. In Maldwyn and OM1387 a proportion of host cellsshowed whole-cell autofluorescence, probably indicative of cell death, following attack byappressoria. Whole-cell autofluorescence was almost totally suppressed by AOPP or OH-PAStreatment, suggesting that this response was also dependent upon active synthesis of phenoliccompounds. The contribution of phenolic compound synthesis to disease resistance appeared tobe independent of temperature; AOPP treatment had similar effects in suppressing resistance inplants acclimated and incubated at 10 °C and at 20 °C. # 1996 Academic Press Limited

INTRODUCTION

It is well recognized that adult plant resistance to cereal powdery mildew and rusts is

commonly expressed in certain cultivars under field conditions. With powdery mildew,

caused by Erysiphe graminis DC (Blumeria graminis (DC) Speer), adult plant resistance

*To whom correspondence should be addressed.Abbreviations used in text : AOPP, α-amino-β-phenylpropionic acid; CAD, cinnamyl alcohol dehydro-

genase ; OH-PAS, [[(2-hydroxyphenyl) amino] sulfinyl]acid, 1,1-dimethylethyl ester ; PAL, phenyl-alanine ammonia lyase ; PGT, primary germ tube; p.f.d., photon flux density.

0885–5765}96}08012121 $25.00}0 # 1996 Academic Press Limited

122 T. L. W. Carver et al.

is characterized by delay in latent period of infection, and restriction of fungal colony

development and sporulation compared to susceptible cultivars. Unlike single gene

controlled resistance to powdery mildew, which is generally race-specific and ephemeral

in effect, adult plant resistance is often (but not exclusively) under the control of

multiple Mendelian factors and is referred to as polygene resistance. It is generally race

non-specific and durable. The physiological bases of adult plant resistance are poorly

understood.

The adult plant resistance of Maldwyn oat to E. graminis f.sp. a�enae Marchal has

been the subject of interest since this cultivar’s release in 1947 [21, 24, 31]. Maldwyn’s

resistance has remained field-effective [30] despite changes in the virulence of E.

graminis f.sp. a�enae which overcame many single gene resistances deployed in oat since

1947. At least six additive Mendelian factors contribute to Maldwyn’s resistance [22].

In Maldwyn and in a derivative hybrid, OM1387, sporulating colonies form on adult

leaves but the percentage of leaf area affected is lower than on seedling leaves or on the

adult leaves of more susceptible oat genotypes [21, 23, 24, 31]. The present studies

considered the possibility that phenolic compounds synthesized in response to attack by

E. graminis contribute to the adult plant resistance of Maldwyn oat.

Even in nominally susceptible seedling leaves of oat, barley or wheat attacked by

virulent isolates of Erysiphe graminis DC, only a proportion of fungal germlings

successfully penetrate host epidermal cells to form haustoria and establish infection.

The failure of some germlings to penetrate from appressoria to penetrate is in part due

to the intrinsic weakness of germlings (due, for example, to exhaustion of reserves).

However, other attacks fail because even nominally susceptible host cells express a

certain degree of resistance to infection. This can be recognized when experimental

treatments, e.g. the use of metabolic inhibitors, are shown to increase leaf susceptibility.

This phenomenon can be detected by histological analyses if sufficiently large numbers

of host parasite contact sites are characterized [8, 12–17, 35, 36].

We have shown previously that the proportion of appressoria successfully penetrating

nominally susceptible leaves of oat, barley and wheat inoculated with virulent,

appropriate formae speciales of E. graminis increased greatly when leaves were treated

with inhibitors of enzymes involved in phenolic compound biosynthesis [12, 14–17].

However, these studies were restricted to seedling leaves in all cases. In the present

study we examine the effects of inhibiting phenolic compound biosynthesis on the adult

plant resistance in Maldwyn and in a derivative hybrid genotype OM1387, and in the

susceptible oat cultivar Selma.

Our earlier work examined the effects on E. graminis}host interaction of α-amino-

β-phenylpropionic acid (AOPP), a potent and specific competitive inhibitor of

phenylalanine ammonia lyase (PAL) [3], effective against oat and barley PAL in �itro

[12, 35]. However, PAL catalyses the first committed step in phenylpropanoid

biosynthesis and so affects the synthesis of many different phenolic compounds. Thus,

the effects of PAL inhibition can be interpreted only broadly. Later, we also used

[[(2-hydroxyphenyl) amino] sulfinyl]acid, 1,1-dimethylethyl ester (OH-PAS), a known

inhibitor of cinnamyl alcohol dehydrogenase (CAD) [1, 19]. CAD is directly involved

with the synthesis of lignin precursors, and we showed that OH-PAS inhibited CAD

from oat and barley in �itro [12, 35]. When applied to nominally susceptible seedling

leaves of oat and barley, the effects of OH-PAS and AOPP were indistinguishable in

Phenolic biosynthesis inhibitors 123

increasing susceptibility to attempted penetration by E. graminis appressoria. This

suggested that products synthesized as part of the lignin biosynthetic pathway,

synthesized de no�o in response to pathogen attack, contribute to resistance to attempted

penetration from appressoria.

Mayama and Shisiyama [27] first reported the accumulation of autofluorogenic

compounds in host cell papillae and surrounding cell wall regions. From spectral

characteristics, they suggested that the autofluorogens were probably phenolic

compounds involved in resistance. In our previous experiments, increased susceptibility

to attempted penetration from appressoria following treatment with either AOPP or

OH-PAS, was associated with decreased frequency and intensity of localized

autofluorescent responses in host epidermal cells at sites of contact with fungal primary

germ tubes (PGTs) and appressoria. This provided supporting evidence that the

autofluorogens are indeed phenolic compounds with a role in resistance to penetration.

We also found that AOPP and OH-PAS suppressed host cell death associated with

hypersensitivity in barley possessing the Mla1 allele for resistance [35]. When Mla1

barley is challenged by an avirulent pathogen isolate, hypersensitive cell death is

characterized by bright whole-cell autofluorescence [26, 35]. It is also known [15, 26]

that even in genetically compatible interactions a small proportion of host epidermal

cells die in response to attack by E. graminis. In Maldwyn (used here) an early study,

that preceded our investigations of the involvement of plant phenolics in resistance,

reported that after staining around 10–20% of attacked cells may show signs of

cytoplasmic disorganization indicative of death when attacked by an oat mildew isolate

capable of forming a fully compatible infection [11]. However, in later studies of oat

which used AOPP and OH-PAS [12, 14, 15, 17], we did not examine their effects on

whole-cell autofluorescence, because with the equipment we used the autofluorescence

of dead oat cells was relatively faint and difficult to observe with certainty. In the

present study, we used an improved incident fluorescence microscope which allowed us

to study whole-cell autofluorescence responses (epidermal cell death) in oat.

Finally, in all previous studies examining the effects of AOPP and OH-PAS on E.

graminis development and host responses, we used a single temperature of 20 °C, the

optimal for powdery mildew development but in the current study we compared events

occurring in host leaves acclimated and incubated at 20 °C and at 10 °C. This allowed

us to judge the efficacy of resistance involving phenolic compound synthesis at different

temperatures likely to be encountered under field conditions.

MATERIALS AND METHODS

Host and pathogen material

Spring oat (A�ena sati�a L.) cvs Maldwyn and Selma and a transgressive segregant

(OM1387) derived from a Selma¬Maldwyn cross were used. None has any known

major gene resistance to race 5 of Erysiphe graminis DC f.sp. a�enae Marchal (Blumeria

graminis (DC) Speer f.sp. a�enae). Maldwyn has quantitative adult plant resistance [23]

under the control of at least six additive genetic factors [22]. This resistance has

remained effective in the field since 1947 [22]. In the field, OM1387 displays a higher

level of resistance to powdery mildew than either parent [30]. Selma has little field

resistance to E. graminis.


The E. graminis isolate was maintained on young Selma plants. One day before

inoculation, heavily sporulating leaves were shaken to displace older conidia, ensuring

that mainly young conidia were used for inoculation the following day.

Application of AOPP and OH-PAS to plant lea�es, and inoculation and incubation procedures

All experiments involved leaf uptake of enzyme inhibitors prior to and during E.

graminis attack. The phenylalanine ammonia lyase (PAL) inhibitor α-amino-β-

phenylpropionic acid (AOPP) [12] was purchased from Cambridge Research

Biochemicals Ltd. (Northwich, Cheshire, U.K.). The cinnamyl alcohol dehydrogenase

(CAD) inhibitor [[(2-hydroxyphenyl) amino] sulfinyl]acid, 1,1-dimethylethyl ester

(OH-PAS), was synthesized by D. A. Boyles [17]. Aqueous solutions of both inhibitors

were used at 1±0 m concentration [12, 17]. They were made by dissolution in distilled

water using continuous stirring for 60 min at room temperature. Distilled water was

the control treatment.

For application to first-formed leaves, the leaves were cut at soil level and their cut

ends trimmed beneath water to remove vascular tissue containing air embolisms to

prevent wilting [12, 14–17, 35]. The cut ends were then immersed in the required test

solution which was taken up in the transpiration stream. Where later-formed leaves

were needed, a 100 mm segment was cut from the centre of the leaf blade and the basal

end trimmed to remove vascular tissue containing air embolisms prior to immersing in

the required test solution. Leaves were allowed to take up test solutions for 24 h before

inoculation, at either 20 °C or 10 °C as appropriate (see experimental designs).

For inoculation, leaves were removed from test solutions, laid adaxial surface up in

a spore settling tower, and conidia from infected leaves were blown directly into the

tower [12, 14–17, 35]. A microscope slide placed among the leaves was used to monitor

inoculum density which was adjusted to 10–20 conidia mm−#. Inoculation took no

longer than 10 min, after which leaves were again trimmed to remove air embolisms,

replaced in the appropriate test solution, and returned to the appropriate controlled

environment for incubation.

Fixation, clearing and light and incident fluorescence microscopy

For microscopy, 30 mm leaf segments were cut from the centre of treated leaves, fixed

in acetic acid-ethanol and cleared in lactoglycerol by a previously described process

that avoids displacement of ungerminated conidia or loosely attached germlings [12].

For microscopy, cleared leaf segments were placed on a microscope slide without a

coverslip and observed by transmitted light microscopy using a ‘‘no coverslip ’’

40¬objective lens [12]. Germling structures including primary germ tubes (PGTs),

appressoria, haustoria and hyphae were clearly visible.

Our previous studies of autofluorescent responses of oat epidermal cells to attack by

E. graminis f.sp. a�enae [12, 14, 15, 17] used an Olympus BH-A microscope and blue light

excitation (Olympus Optical Co. Ltd., Tokyo, Japan). For the current studies we also

used an Olympus BH-2 microscope with an Olympus RFC reflected light fluorescence

attachment fitted with a blue-violet excitation filter (max. transmittance 440 nm) and

barrier filter with transmittance above 475 nm. The BH-2 system gave bright


fluorescent images for visualization of blue-white autofluorescence associated with

localized responses of host epidermal cells and whole-cell autofluorescence indicative of

cell death [26].

In most of our previous studies of oat [12, 14, 17] we had not considered whole-cell

autofluorescence in oat because it is often faint and very difficult to see. However, with

the BH-2 system used here, we could discern whole-cell autofluorescence with relative

ease. In barley, Koga et al. [26] showed that epidermal cells with whole-cell

autofluorescence also failed to take up the vital dye neutral red, and to respond to

plasmolysing treatment. They concluded that such cells were dead. In preliminary tests

we followed the procedures of Koga et al. [26] using unfixed leaves of oat cv. Maldwyn

inoculated 36 h prior to testing. Our results were similar to those from the barley

system [26]. We found that unattacked epidermal cells, or cells with localized

autofluorescence associated with appressorial contact, almost invariably took up

neutral red and responded to plasmolysing treatment, indicating that they were alive.

By contrast, cells showing whole-cell autofluorescence almost always failed to take up

the vital dye neutral red and to respond to plasmolysing treatment, indicating that

these cells were dead. There were a few cells that showed faint whole-cell

autofluorescence but the protoplast also showed faint neutral red staining and

appeared to be slightly plasmolysed with an irregular outline. We, like Koga et al. [26],

concluded that such cells were alive but dying. We therefore consider that whole-cell

autofluorescence is a useful indication of cell death in oat as well as in barley epidermal

cells.

Data collection and statistical analyses

In previous studies, treatments with AOPP and OH-PAS did not affect spore

germination [12, 14–17, 35]. To confirm that this was true in adult as well as seedling

leaves, 100 conidia were examined on each leaf segment in Experiment 1, to determine

whether they had germinated i.e. formed at least a primary germ tube.

In all experiments, we determined penetration success (haustorium formation) from

fungal appressoria by examining 100 germlings with mature appressoria on each

leaf. Only those germlings were assessed if their PGT and appressorium were on host

cells that had no contact with other germlings. In addition, PGT and appressorium

contact sites on epidermal cells were examined by incident fluorescence microscopy for

localized autofluorescent responses, and host cells subtending appressoria were

examined for whole-cell autofluorescence.

Mean replicate data for each category of observation were calculated as a

percentage. Percentages were transformed to arcsine square roots (transformed value

¯ 180}π¬arcsine [o(percent}100)]) to normalize data and stabilize the variance

throughout the data range prior to standard analysis of variance using appropriately

written Genstat programs [12, 14–17, 35].

For appressorium contact sites, the intensity of localized autofluorescent host cell

responses was ranked visually on a 0–3 scale where: 0¯no visible fluorescence; 1¯faint fluorescence; 2¯moderate fluorescence; and 3¯ intense fluorescence [12].

Fluorescence intensity data were not analyzed statistically because distributions were

often heavily skewed and data often had a high proportion of zero values.


The influence of AOPP on adult plant resistance in successi�ely-formed lea�es : Experiments 1

and 2

Experiments 1 and 2 were identical in design and were conducted in successive years.

We wished to produce plants reaching different growth stages, seedling to adult, at the

same time. This would allow comparison of fungal development and host responses in

leaves from different positions formed under the same environment. To produce such

plants, seeds were sown in succession over a 45 day period and plants were grown in

a spore-proof glasshouse under natural lighting conditionswith aminimum temperature

of 12 °C. For both experiments, pre-germinated seedlings of the susceptible genotype

Selma and the adult plant resistant genotype Maldwyn were planted separately, three

plants per 125 mm pot, in John Innes No. 3 potting compost. A pot of each oat

genotype was grown in each of three replicates of a randomized block. The first sowing

in each experiment was made in mid June, and freshly sown pots were incorporated

into the randomized block each week until the last week in July. Thus, successive

sowings produced plants at different growth stages with leaves at different ascending

positions on primary shoots reaching the same physiological age at the same time and

under the same environmental conditions.

In early August, the eighth-formed leaves on primary shoots were newly expanded

on plants from the earliest sowing, and seventh-formed leaves from these plants

provided segments (100 mm) for experimentation. At the same time, fifth-formed leaf

segments were cut from plants with newly expanded sixth leaves, third-formed leaf

segments were cut from plants with newly expanded fourth leaves, and first-formed

(seedling) leaves were cut from plants of the latest sowing where the second-formed leaf

was newly expanded.

In all cases, a leaf segment was taken from each of two phenotypically similar plants

grown in the same pot ; one leaf segment from each pot was used for AOPP treatment,

and the other as the water control. Thus, for each genotype and each leaf position,

three leaves (one from each replicate) were used for AOPP treatment, and three for

water controls. In total, therefore, there were six segments from leaves of the same

position (three replicate segments from each of the two oat genotypes) used for AOPP

treatment, and six for water control treatment. The cut ends of one set of six leaves from

each position were immersed in a vessel containing 20 ml of 1±0 m AOPP solution,

and their counterparts in a vessel containing 20 ml of distilled water.

All vessels were transferred to a growth cabinet at 20³0±5 °C, with light supplied by

warm white fluorescent tubes for 12 h day−" (9.00–21.00 h) at 150 µmol m−# s−" photon

flux density (p.f.d.) at leaf level. The first light period began immediately. After 24 h,

leaves were inoculated, returned to the appropriate solution and incubated in the

growth cabinet for a further 36 h before fixation.

Comparati�e effects of AOPP and OH-PAS on the resistance of fifth-formed lea�es : Experiment

3

In mid-April, pre-germinated seedlings of the susceptible genotype Selma and the

adult plant resistant genotype Maldwyn were planted separately, three plants per

125 mm pot, in John Innes No. 3 potting compost. Three pots of each genotype were


included in each of three replicates of a randomized block. Plants were grown in a

spore-proof glasshouse as for Experiments 1 and 2. By late May plants had formed

newly expanded sixth-formed leaves on the primary shoot. To ensure that leaves

destined for different experimental treatments were comparable, one plant was selected

from every pot on the basis of its phenotypic similarity to equivalent plants. A 100 mm

segment was cut from the fifth-formed leaf of each selected plant. The basal cut ends

of sets of three leaf segments of each genotype (one from each of the three replicates)

were immersed in 20 ml 1±0 m AOPP, 20 ml 1±0 m OH-PAS, or 20 ml distilled

water. Pre- and post-inoculation conditions were the same as for Experiments 1 and 2.

The influence of AOPP on first- and fifth-formed lea�es of plants acclimated at 10 °C or 20 °C:

Experiments 4 and 5

Experiment 4: first-formed lea�es. Two sets of plants with first-formed leaves of similar

physiological age were grown under the same lighting environment but acclimated to

10 °C or 20 °C temperature conditions. For the 10 °C requirement, six pre-germinated

seedlings of Selma, Maldwyn and OM1387 oat were planted in separate 125 mm pots

of John Innes No. 3 compost, and grown in a spore-proof glasshouse. When the first-

formed leaves of these plants were approaching full expansion, they were transferred

to a growth room at 10³1±5 °C, with light supplied by warm white fluorescent tubes

for 12 h day−" (9.00–21.00 h) at 150 µmol m−# s−" p.f.d. at leaf level. At this time, a

second set of seedlings was planted in a growth room with the same lighting conditions

but at 20³1±5 °C. After 2 weeks plants of both sets had produced fully expanded

second-formed leaves and first leaves were taken for experimentation.

Six first-formed leaves of each oat genotype from each growth room were cut at

9.00 h (the beginning of the 12 h light period) and three were treated with 1±0 m AOPP

while three were treated with water. They were returned for 24 h to their original

growth room to allow chemical uptake, inoculated and then returned to the

appropriate growth room for incubation. Leaves held at 20³1±5 °C, were fixed after

36 h and leaves held at 10³1±5 °C were incubated for 72 h. Preliminary experiments

(Zhang, unpublished) showed that pathogen and germling development was greatly

delayed at 10³1±5 °C necessitating the longer 72 h incubation. It was impractical to

incubate leaves for longer because leaf tips began showing chlorosis at 72 h.

Experiment 5: fifth-formed leaves. Two sets of fifth-formed leaves of similar physiological

age from plants grown under the same lighting environment but acclimated to 10 °Cor 20 °C temperature conditions were required. Therefore, successive sowings of Selma,

Maldwyn and OM1387 were made to produce six plants of each genotype in separate

125 mm pots of John Innes No. 3 compost. All plants were grown in a spore-proof

glasshouse. For the 10 °C requirement, plants from the first sowing were taken when

their fifth-formed leaves were unrolling, and moved to the 10 °C growth room, with

light supplied by warm white fluorescent tubes for 12 h day−" (9.00–21.00 h) at

150 µmol m−# s−" p.f.d. at leaf level. At the same time, plants from the later sowing had

developed unrolling fourth-formed leaves. These plants were moved to a similar

growth room at 20 °C. After 2 weeks, plants of both sets had fully expanded sixth-

formed leaves and their fifth-formed leaves were taken for experimentation. Fifth-


formed leaves were treated with AOPP or water in the same way as first-formed leaves

in Experiment 4.

RESULTS

Conidial germination

Germination rates in Experiment 1 were unaffected by inhibitor treatment, leaf

position or by host genotype. This confirmed previous findings [12, 14–17, 33], and

germination was not scored in subsequent experiments.

The influence of AOPP on adult plant resistance in successi�ely-formed lea�es : Experiments 1

and 2

These experiments compared success of attempted penetration and associated host cell

responses in leaves from ascending positions on the primary shoot of Selma, which has

little adult plant resistance, and of Maldwyn, which has marked adult plant resistance.

To examine the role of plant phenolics in these genotypes, we compared water-treated

with AOPP-treated leaves.

Results from Experiments 1 and 2 were very similar and so only those of Experiment 1

are described in detail. Experiment 2 results are described only when they varied from

Experiment 1.

Percentage penetration (haustorium formation) from appressoria. Overall, a higher percentage of

germlings with appressoria penetrated to form haustoria in the susceptible genotype

Selma than in the adult plant resistant genotype Maldwyn (mean percentages¯ 62±9and 29±2 respectively, P! 0±001, for data pooled over leaf positions and treatments).

Fewer appressoria penetrated (produced haustoria) in the upper leaves of both

genotypes (the main effect of leaf position was significant; P! 0±001). Fig. 1 shows that

this effect was greater in the upper leaves of the adult plant resistant genotype

Maldwyn than in the more susceptible genotype Selma. This led to a significant (P!0±001) host genotype¬leaf position interaction.

Overall, a higher percentage of appressoria formed haustoria in AOPP- than in

water-treated leaves (mean percentages¯ 66±4 and 26±0 respectively, P! 0±001, for

data pooled over oat genotypes and leaf positions). The effect of AOPP was evident in

both oat genotypes (the genotype¬treatment interaction was not significant).

Furthermore, the effect of AOPP on penetration success was evident in leaves from all

positions (the leaf position¬treatment interaction was not significant). It is clear from

Fig. 1 that even with AOPP treatment, upper leaves remained more resistant to

penetration than first-formed leaves. This suggests that PAL activity was not the only

factor contributing to the penetration resistance of upper leaves.

Localized autofluorescent responses of epidermal cells in contact with fungal PGTs and appressoria.

AOPP treatment greatly reduced the percentage of localized autofluorescent responses

to PGTs (Fig. 2). In analyses of variance, the main effect of AOPP treatment was

overwhelmingly important, accounting for more than 90% of the total sums of squares.

Very low percentages of autofluorescent responses to PGTs were noted in AOPP-

treated leaves from all positions. Thus the PAL inhibitor had suppressed PGT-

associated autofluorescence equally effectively in seedling and upper leaves, and this


Leaf 7

100

0Leaf 1

Leaf position

Per

cen

tage

(tr

ansf

orm

ed)

of s

pore

lin

gs w

ith

app

ress

oria

pen

etra

tin

g

Leaf 3

80

60

40

20

SED

Leaf 5

F. 1. Percentages (transformed to arcsines) of E. graminis sporelings with appressoriapenetrating (forming haustoria) in leaves 1 (seedling) 3, 5 and 7 of oat genotypes Selma andMaldwyn treated with water or with 1±0 m AOPP to inhibit PAL. Standard error of differenceof means ( ; d.f.¯ 32) for comparison of treatment means within leaf positions and genotypes,and of leaf positionmeanswithin treatments and genotypes. (+) Selma}water ; (*) Selma}AOPP;(E) Maldwyn}water ; (D) Maldwyn}AOPP.

was consistent in both oat genotypes (no statistical interactions involving treatment

were significant).

AOPP treatment reduced the frequency of localized autofluorescence associated

with appressoria (Fig. 2). Here, the effect was somewhat less dramatic than on the

response associated with PGTs. Again, the effect of AOPP was evident in leaves from

all positions indicating that PAL had been inhibited equally effectively in all leaves.

Analysis of variance showed, once more, that the main effect of inhibitor treatment was

overwhelmingly important, accounting for more than 75% of the total sums of squares.

The effect of AOPP treatment in suppressing autofluorescence associated with

appressoria was consistent in both host genotypes and in leaves from all positions (Fig.

2 ; no interactions involving treatment were significant).

Treatment with AOPP reduced not only the percentage of autofluorescent responses

associated with appressoria, but also the intensity of the autofluorescence in leaves from

all positions. Fig. 3 shows data for first- and seventh-formed leaves to illustrate this

point. In water-treated controls, the majority of responses associated with appressoria

were intense (class 3) or moderate (class 2), and very few had no visible fluorescence

(class 0). By contrast, with AOPP treatment the majority of appressoria were

associated with no fluorescence (class 0) and few with moderate fluorescence (class 2).

Whole-cell autofluorescence. In water-treated leaves of the susceptible genotype Selma, less

than 1% of leaf epidermal cells contacting appressoria showed whole-cell auto-


Leaf 7

100

0Leaf 1

Leaf position

Per

cen

tage

(tr

ansf

orm

ed)

asso

ciat

edw

ith

au

tofl

uor

esce

nce

Leaf 3

80

60

40

20

SED

Leaf 5 Leaf 7Leaf 1 Leaf 3

SED

Leaf 5

PGTs Appressoria

F. 2. Percentages (transformed to arcsines) of E. graminis PGTs and appressoria associatedwith a localized autofluorescent host cell response in leaves 1 (seedling), 3, 5 and 7 of oat genotypesSelma and Maldwyn treated with water or with 1±0 m AOPP to inhibit PAL. Standard errorsof differences ( ; d.f.¯ 32) for comparison of treatment means within leaf positions and of leafposition means within treatments. (+) Selma}water ; (*) Selma}AOPP; (E) Maldwyn}water ;(D) Maldwyn}AOPP.

fluorescence, irrespective of leaf position. In water-treated leaves of adult plant

resistant Maldwyn, about 23% showed whole-cell autofluorescence in both Experi-

ments 1 and 2; analysis of variance showed that this percentage did not vary between

leaves from different positions. In these and all subsequent experiments, when

appressoria were associated with epidermal cells showing whole-cell autofluorescence,

fungal germlings did not develop hyphae on any host genotype. It was impossible to

be sure exactly how fare attempted penetration had proceeded before fungal

development ceased. Most often, no haustoria were visible although it was occasionally

possible to see rudimentary haustoria lacking digitate processes that had ceased

development at a very early stage. Thus whole-cell autofluorescence was associated

with failure of the fungus to produce functional haustoria.

When leaves were treated with AOPP, no epidermal cells showed whole-cell

autofluorescence in either host.

Comparati�e effects of AOPP and OH-PAS on the resistance of fifth-formed lea�es : Experiment

3

This experiment compared the influences of AOPP and OH-PAS on fungal

development and host cell response in fifth-formed leaves of Selma, which has little

adult plant resistance, and of Maldwyn, which has marked adult plant resistance.

Significantly (P! 0±001) more sporelings with appressoria penetrated leaves treated

with AOPP or OH-PAS than penetrated the water-treated controls. The effect of

AOPP was greater than that of OH-PAS (P! 0±05) (Fig. 4). Overall, more appressoria

penetrated leaves of the susceptible Selma than the adult plant resistant Maldwyn

(mean percentages¯ 38±8 and 22±9 respectively, P! 0±001, for data pooled over

treatments). The effects of both AOPP and OH-PAS treatment were consistent in both

host genotypes (inhibitor treatment¬host genotype interaction was not significant).


250

0

Leaf 1

Nu

mbe

rs o

f ap

pres

sori

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200

150

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50

Genotype: MaldwynWater (Control) AOPP (1 mM)

Inhibitor treatment

250

0

200

150

100

50

250

0

Leaf 1

Nu

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Genotype: Selma

250

0

200

150

100

50

0 1 2 3 0 1 2 3Fluorescence intensity score

Leaf 7

Leaf 7

F. 3. Intensities of localized autofluorescent responses in host cells associated with E. graminisappressoria in first- and seventh-formed leaves of two oat genotypes treated with water (controls)or with the PAL inhibitor AOPP (1±0 m aqueous solution). O¯no fluorescence; 1, 2, 3¯ faint,moderate and intense fluorescence respectively. Data based on observation of 300 appressoria ineach case.

Both inhibitors significantly reduced (P! 0±001) the percentage of localized

autofluorescent host cell responses associated with PGTs (Fig. 5) and, again, the effect

of AOPP was greater than that of OH-PAS (P! 0±001). AOPP reduced significantly

(P! 0±001) the percentage of localized autofluorescent host cell responses associated

with appressoria, but OH-PAS had little or no effect. The effects of inhibitor

treatments were consistent in both host genotypes (inhibitor treatment¬host genotype

interactions were not significant).


Per

cen

tage

(tr

ansf

orm

ed)

of s

pore

lin

gs w

ith

appr

esso

ria

pen

etra

tin

g

60

0

40

20

Water OH-PAS AOPPTreatment

SED

F. 4. Percentages (transformed to arcsines) of E. graminis appressoria penetrating (forminghaustoria) in fifth leaves of oat genotypes Selma and Maldwyn treated with water or with 1±0 mOH-PAS to inhibit CAD or 1±0 m AOPP to inhibit PAL. Standard error of difference of means( ; d.f.¯ 12) for comparison of treatment means within genotypes and genotype means withintreatments. (*) Selma; (+) Maldwyn.

Per

cen

tage

(tr

ansf

orm

ed)

asso

ciat

edw

ith

au

tofl

uor

esce

nce

100

0

40

20


SED

60

80


SED(a) (b)

F. 5. Percentages (transformed to arcsines) of E. graminis PGTs (a) and appressoria (b)associated with localized autofluorescent host cell response in fifth leaves of oat genotypes Selmaand Maldwyn treated with water or with 1±0 m OH-PAS to inhibit CAD or 1±0 m AOPP toinhibit PAl. Standard errors of differences of means ( ; d.f.¯ 12) for comparison of treatmentmeans within genotypes and genotypes means within treatments. (*) Selma; (+) Maldwyn.


Treatment with either AOPP or OH-PAS decreased the intensity of localized

autofluorescent host cell responses associated with appressoria. Fig. 6 shows that in

both host genotypes treated with water the majority of appressoria were associated

with intense (class 3) or moderate (class 2) autofluorescent responses. In AOPP-treated

leaves, the majority were associated with faint (class 1) autofluorescence, and few with

intense autofluorescence. With OH-PAS treatment, autofluorescent responses were

generally less intense than in water treated leaves (moderate, class 2 autofluorescent

was most common) but the effect of OH-PAS was less than that of AOPP.

Whole-cell autofluorescence. In water-treated leaves, a mean of 5% and 13±7% respectively

of Selma and Maldwyn epidermal cells showed whole-cell autofluorescence when in

contact with appressoria. When Selma leaves were treated with either inhibitor, whole-

cell autofluorescence was totally prevented by both inhibitors. In Maldwyn, only 0±3%

and 1±7% of cells in leaves treated with AOPP or OH-PAS respectively showed whole-

cell autofluorescence as both inhibitors effectively suppressed this response in this adult

plant resistant genotype.

The influence of AOPP on first- and fifth-formed lea�es of plants acclimated at

10 °C or 20 °C: Experiments 4 and 5

In the previous experiments we examined the effects of adult plant resistance and

inhibitors of phenolic compound biosynthesis at the optimal temperature for E. graminis

development (20 °C). The current experiments were designed to test whether AOPP

treatment and leaf position had consistent effects at a lower temperature, as frequently

encountered in the field. The susceptible genotype Selma, the adult plant resistant

Maldwyn, and the Selma¬Maldwyn hybrid, OM1387, were used for these

experiments.

Experiment 4: first-formed leaves

Haustorium formation by appressoria. Overall, a higher percentage of appressoria penetrated

to form haustoria in AOPP- than in water-treated leaves (mean percentages¯ 69±3and 42±5 respectively,P! 0±001, for data pooled over host genotypes and temperatures).

This AOPP effect was consistent at both temperatures and in all oat genotypes (no

interactions involving treatment were significant).

Fewer appressoria penetrated at 10 °C than 20 °C (mean percentages¯ 46±3 and

65±8 respectively, P! 0±001, for data pooled over oat genotypes and treatments). This

temperature effect was consistent in all host genotypes (the incubation temperature¬host genotype interaction was not significant). Overall, a significantly (P! 0±001)

higher percentage of appressoria formed haustoria in Selma than in either Maldwyn

or OM1387 (mean percentages¯ 73±5, 46±0 and 48±0 respectively, for data pooled over

temperatures and treatments).

Localized autofluorescent host cell responses associated with PGTs and appressoria. As expected,

AOPP treatment reduced significantly (P! 0±001) the percentage of localized

autofluorescence associated with PGTs (mean percentages¯ 0±6 and 78±4 for AOPP

and water treatments respectively for data pooled over oat genotypes and


250

0

Water(Control)

Nu

mbe

rs o

f ap

pres

sori

a

200

150

100

50

Treatment Selma Maldwyn

Host genotype

0 1 2 3 0 1 2 3Fluorescence intensity score

AOPP 250

0

200

150

100

50

250

0

200

150

100

50

OH-PAS

Nu

mbe

rs o

f ap

pres

sori

aN

um

bers

of

appr

esso

ria

F. 6. Intensities of localized autofluorescent responses in host cells associated with E. graminisappressoria on fifth-formed leaves of the oat genotypes Selma and Maldwyn treated with water(controls), with the PAL inhibitor AOPP (1±0 m aqueous solution) or with the CAD inhibitorOH-PAS (1±0 m aqueous solution). 0¯no fluorescence; 1, 2, 3¯ faint, moderate and intensefluorescence respectively. Data based on observation of 300 appressoria in each case.

temperatures). This effect was consistent in all oat genotypes and at both temperatures

(no interactions involving treatment were significant). Temperature had no effect on

the percentage of localized autofluorescent host cell responses associated with PGTs

(mean percentages¯ 31±9 and 28±7 for 10 °C and 20 °C respectively for data pooled

over oat genotypes and treatments).

AOPP treatment also reduced significantly the percentage of localized auto-

fluorescent host cell responses associated with appressoria (mean percentages¯ 6±2 and

95±0 for AOPP and water treatments respectively, P! 0±001, for data pooled over oat

genotypes and temperatures). Overall, temperature had no effect on the percentage of

localized autofluorescent host cell responses associated with appressoria (mean

percentages¯ 52±8 and 49±8 for 10 °C and 20 °C respectively for data pooled over oat


genotypes and treatments). However, AOPP treatment had less effect in reducing

autofluorescence at 10 °C than at 20 °C. This led to a significant (P! 0±001)

treatment¬incubation temperature interaction. Nevertheless, autofluorescence inten-

sities were clearly reduced in AOPP-treated leaves at 10 °C (data not shown), so that

where localized autofluorescence was present, it was invariably faint (class 1)

compared to water-treated leaves that showed moderate or intense (class 2–3)

responses.

Whole-cell autofluorescence. In water-treated leaves of Selma from 20 °C treatment, only

1% of cells showed whole-cell autofluorescence and none did so in the 10 °C treated

plants. In both adult plant resistant genotypes, Maldwyn and OM1387, approximately

15% of cells contacted by appressoria showed whole-cell autofluorescence in water-

treated leaves from both temperatures. No whole-cell autofluorescence was found in

any host genotype following AOPP treatment at either temperature.

In summary, in first-formed leaves AOPP treatment was equally effective at 10 °Cand 20 °C in terms of its effects on penetration success (haustorium formation). Also in

terms of its effect on whole-cell autofluorescence and PGT-associated localized

autofluorescence, AOPP treatment was equally effective at both temperatures. AOPP

treatment was slightly more effective at 20 °C than at 10 °C in suppressing localized

epidermal cell autofluorescence associated with appressoria.

Experiment 5: Fifth-formed leaves

Haustorium formation by appressoria. Overall, a higher percentage of appressoria penetrated

to form haustoria in AOPP- than in water-treated leaves (mean percentages¯ 65±9and 24±8 respectively,P! 0±001, for data pooled over host genotypes and temperatures).

This effect was consistent at both 10 °C and 20 °C.

As in first-formed leaves, in fifth leaves fewer appressoria penetrated to form

haustoria at 10 °C than 20 °C (mean percentages¯ 29±8 and 60±5 respectively, P!0±001, for data pooled over host genotypes and treatments). Overall a significantly (P!0±001) higher percentage of appressoria formed haustoria in the susceptible genotype

Selma than in either of the adult plant resistant genotypes, Maldwyn or OM1387

(mean percentages¯ 68±3, 33±3 and 33±3 respectively, for data pooled over temperatures

and treatments). In all oat genotypes, more appressoria formed haustoria in AOPP-

than water-treated leaves. AOPP treatment had slightly less effect in increasing

penetration in fifth leaves of Selma than the other genotypes (leading to a significant

host genotype¬treatment interaction), but AOPP-treated Selma leaves remained

significantly (P! 0±001) more susceptible than AOPP-treated leaves of Maldwyn and

OM1387. Thus, even when PAL was inhibited, these adult leaves of Maldwyn and

OM1387 remained relatively more resistant to penetration than those of Selma.

Localized autofluorescent host cell responses associated with PGTs. Again, in fifth leaves, AOPP

treatment reduced significantly (P! 0±001) the percentage of localized auto-

fluorescence responses associated with PGTs (mean percentages¯ 10±1 and 84±2 for

AOPP and water treatments respectively for data pooled over oat genotypes and

temperatures). This effect was overwhelmingly important and accounted for

approximately 68% of the total sums of squares in the analysis of variance.


Host genotype had a small but significant overall effect (P! 0±05) on the percentage

of autofluorescent host cell responses associated with PGTs (mean percentages¯ 40±3,

50±6 and 46±3 for Maldwyn, OM1387 and Selma respectively, for data pooled over

temperatures and treatments). However, AOPP treatment reduced significantly the

percentage of localized autofluorescence associated with PGTs in all host genotypes.

Temperature also had a small but significant overall effect and fewer PGTs were

associated with localized autofluorescence at 20 °C than 10 °C (mean percentages¯22±7 and 69±7 respectively, P! 0±001, for data pooled over host genotypes and

treatments). At 20 °C AOPP treatment almost totally suppressed autofluorescence

associated with PGTs (mean percentages¯ 0±1 and 67±3 for AOPP and water

treatments respectively for data pooled over host genotypes), while at 10 °C, AOPP

treatment had slightly less effect (mean percentages¯ 33±3 and 95±7 for AOPP and

water treatments respectively for data pooled over host genotypes). This caused a small

but significant treatment¬temperature interaction (accounting for ! 0±1% of total

sums of squares in analysis of variance).

Localized autofluorescent host cell responses associated with appressoria. AOPP treatment

reduced significantly (P! 0±001) the percentage of localized autofluorescent responses

associated with appressoria (mean percentages¯ 30±8 and 96±7 for AOPP and water

treatments respectively for data pooled over host genotype and temperature). This

effect was overwhelmingly important and accounted for approximately 63% of the

total sums of squares in the analysis of variance.

Host genotype had no effect on the percentage of localized autofluorescent host cell

response associated with appressoria (the main effect of host genotype was not signifi-

cant). AOPP was equally effective in reducing this percentage in all host genotypes.

Temperature had a small but significant overall effect and fewer appressoria were

associated with localized autofluorescence at 20 °C than 10 °C (mean percentages¯88±8 and 46±2 respectively, P! 0±001, for data pooled over host genotypes and

treatments). At 20 °C, AOPP treatment had more effect (mean percentages¯ 3±8 and

92±7 for AOPP and water treatments respectively for data pooled over host genotypes)

than at 10 °C (mean percentages¯ 69±1 and 99±1 for AOPP and water treatments

respectively for data pooled over host genotypes). This led to a significant

treatment¬incubation temperature interaction (accounting for approximately 9% of

total sums of squares in analysis of variance).

AOPP treatment also reduced the intensity of autofluorescence at appressorial

contact sites (data not shown). This was true in all genotypes at both temperatures. In

AOPP-treated leaves autofluorescent responses were almost always faint (class 1) and

there were few moderate (class 2) and no intense (class 3) responses at either incubation

temperature. This contrasted with water-treated leaves where the majority of responses

were moderate or intense (class 2 and 3).

Whole-cell autofluorescence. In water-treated leaves of the susceptible genotype Selma

there was no whole-cell autofluorescence at either incubation temperature. In the adult

plant resistant genotypes Maldwyn and OM1387 approximately 35% of cells in water-

treated leaves showed whole-cell autofluorescence at both temperatures. No whole-cell

autofluorescence was found in any host genotype following AOPP treatment.


In summary, in fifth-formed leaves. AOPP treatment appeared to be equally

effective at 10 °C and 20 °C in terms of its effects on penetration success (haustorium

formation) and in suppressing whole-cell autofluorescence. In terms of its effects in

suppressing localized autofluorescent host cell responses associated with PGTs and

appressoria, AOPP treatment appeared slightly more effective at 20 °C than at 10 °C.

DISCUSSION

The adult plant resistance of Maldwyn oat has proved effective in the field for over four

decades [21, 24, 30, 31]. It has been argued that this durability is due to the fact that

at least six additive Mendelian factors contribute to this resistance [22]. Maldwyn’s

adult plant resistance is quantitative ; while sporulating colonies form on adult leaves,

the percentage of leaf area affected by disease is lower than on its seedling leaves or on

the adult leaves of more susceptible oat genotypes [23]. In part, this is because fewer

E. graminis germling appressoria penetrate successfully [9, 18]. The present findings

corroborate these earlier descriptive studies, showing progressively reduced success of

attempted penetration in successively formed leaves. The hybrid line OM1387 gave

reduced levels of penetration similar to those seen in the resistant progenitor Maldwyn

and significantly lower than in its other progenitor, the susceptible cultivar Selma (Fig.

1).

A main objective was to test the hypothesis that plant phenolic compounds are

involved in adult plant resistance. The finding that treatment with AOPP decreased

penetration resistance of adult leaves as measured by haustorium formation, confirms

phenolic involvement. This finding also indicates that, as in seedling leaves of oat and

barley [12, 14–17], the phenolic compounds are synthesized de no�o in response to E.

graminis attack.

Treatment with OH-PAS to inhibit CAD activity also significantly reduced the

penetration resistance of adult (fifth-formed) leaves. This indicates that cinnamyl

alcohols (the direct product of CAD activity), or later formed products of the lignin

biosynthetic pathway, are involved in adult plant resistance. However, in adult leaves

OH-PAS was slightly, but significantly, less effective in suppressing penetration

resistance than AOPP (Fig. 4). This finding was in contrast to results using nominally

susceptible seedling leaves where the effects of AOPP and OH-PAS on penetration

resistance were indistinguishable [16, 17].

As in previous studies using oat seedlings [12, 14–16], decreased resistance to

penetration following AOPP treatment of adult leaves was correlated to reduction in

the frequency and intensity of localized autofluorescent host cell responses associated

with fungal contact. This clearly suggests that in adult leaves also, autofluorogens

accumulating in contacted epidermal cell wall regions and subtending papillae are

phenolic in nature.

OH-PAS, which was slightly less effective than AOPP in reducing penetration

resistance, did not reduce the frequency of appressorium-associated localized

autofluorescence in adult plant resistant fifth leaves of Maldwyn. However OH-PAS

did reduce the intensity of localized autofluorescence in these fifth leaves (Fig. 3). The

results with Maldwyn contrast to those from the fifth leaves of the susceptible cv. Selma

(Figs 2 and 3) and to oat and barley seedling leaves where OH-PAS always decreased


the frequency and intensity of localized autofluorescent host cell responses [16, 17]. It

may be that differences in leaf architecture prohibited OH-PAS from reaching the

epidermis of Maldwyn’s fifth leaves in sufficient concentration to have full effect.

Alternatively it may be that autofluorogenic compounds other than the cinnamyl

alcohols or later-formed products, also contribute to the adult plant resistance of

Maldwyn.

AOPP and OH-PAS treatments almost totally suppressed whole-cell auto-

fluorescence. In water-treated leaves, the frequency of whole-cell autofluorescence was

variable between experiments. However, it was always infrequent in the susceptible

genotype Selma (associated with ! 5% of appressoria), but was seen quite regularly

in the adult plant resistant Maldwyn (associated with between approx. 14% and 35%

of appressoria). In Experiments 1 and 2, whole-cell autofluorescence was seen at a

similar frequency in seedling and in adult leaves, and so alone cannot account for adult

plant resistance expressed in successively formed leaves.

Preliminary observations showed a negative correlation between occurrence of

whole-cell autofluorescence and the ability of epidermal cells both to take up the vital

dye neutral red and to respond to plasmolysing treatments. This result was comparable

to findings from the barley mildew system [26] which led Koga et al. to suggest that

whole-cell autofluorescence accompanies cell death. We cannot be sure, however, that

treatment with AOPP or OH-PAS, which almost totally prevented whole-cell

autofluorescence, also prevented cell death. It is possible that cell death occurred but

that treatment with inhibitors prevented the accumulation of autofluorogenic phenolics

in dead cells. Nevertheless, cell death was reported as a response to E. graminis in oat

and barley genotypes with no known race-specific resistance [4, 8, 11, 15]. It is

commonly associated with incompatibility conditioned by single gene controlled

resistance in barley true leaves [2, 35] and coleoptiles [2, 6, 7] and in oat, barley and

wheat leaves attacked by inappropriate formae speciales of E. graminis [15]. In all cases,

AOPP treatment reduced the proportion of cells that died in response to attack

[15, 35], and OH-PAS treatment had a similar effect in suppressing cell death when it

was tested in an incompatible E. graminis–barley interaction [35]. Similarly, in the

wheat stem rust-Sr-5 system, OH-PAS treatment reduced hypersensitive mesophyll cell

death due to incompatibility [28]. Taken together, these results strongly suggest that

the cell death response in cereals under attack by obligate biotrophic fungal pathogens

accompanies the engagement of responses which involve the synthesis of cinnamyl

alcohols or later-formed compounds derived from cinnamyl alcohols. Cinnamyl

alcohols provide a substrate for peroxidase in polymerization reactions. Increased

peroxidase activity is implicated in the responses of oat, barley and wheat to E. graminis

attack [5, 20, 25, 29, 34, 36] and peroxidation is associated with the production of free

radicals [20, 25]. Free radicals have been implicated in cell death associated with

several plant diseases [20, 25, 32, 33]. It may be that inhibition of cinnamyl alcohol

production by OH-PAS or AOPP treatment, deprives attacked cells of a substrate

necessary for peroxidase activity, and thus free radical production leading to cell death

does not occur.

Overall, our results indicate that phenolic compound synthesis, while important, was

not the only factor contributing to adult plant resistance in Maldwyn oat. Indeed, the

level of contribution made to resistance by newly synthesized phenolics in upper leaves


appeared to be similar to that in seedling leaves. Thus, analysis of variance of

penetration data showed no significant interaction between leaf position and chemical

treatment in Experiments 1 or 2. If the contribution to resistance of phenolic

compounds varied between leaves, a significant interaction would have been expected.

This suggests that some other, unknown factor(s), either pre-formed or synthesized in

response to pathogen attack also contributed to adult plant resistance. A combination

of this unknown factor(s) together with the effects of phenolic compounds, apparently

conveys the relatively high level of penetration resistance expressed in the upper leaves

of Maldwyn.

All our previous studies using AOPP and OH-PAS [12, 14–17, 35], and the majority

of other studies of resistance to cereal powdery mildew, have used temperatures close

to optimal for powdery mildew development, i.e. 20 °C. Here we also studied plants

acclimated at, and incubated under, relatively low temperature conditions (10 °C).

AOPP treatment substantially increased penetration success at both temperatures

indicating the importance of phenolics at both temperatures. The lack of statistical

interaction between the effects of AOPP-treatment and temperature emphasizes the

similarity of the contribution of phenolics at both temperatures. This was true in

seedling and in adult (fifth) leaves. At both temperatures, AOPP treatment also

effectively reduced the frequency and intensity of localized autofluorescent responses,

and suppressed cell death.

The current results suggest that, overall, fewer haustoria were formed under the

10 °C than the 20 °C condition. Mildew development and epidermal host cell responses

are slower at 10 °C. To compensate for this, we allowed more incubation time for

penetration to be accomplished at 10 °C. However, we were unable to extend

incubation beyond 72 h under the 10 °C condition because the tips of excised leaves

started to show signs of physiological stress (yellowing). In other experiments (Zhang,

unpublished), powdery mildew development at 10 °C and 20 °C was studied on leaves

of intact plants. In these experiments leaves were fixed 96 h after inoculation, and there

was no difference between incubation temperatures in the final proportion of

appressoria that penetrated. Thus, in the current study 72 h may have been insufficient

for some late developing germlings to complete the processes of penetration.

From the present study it seems clear that while phenolic compound synthesis

contributes greatly to the high level of adult plant resistance expressed by Maldwyn,

and its hybrid derivative line OM1387, it is not the only component of adult plant

resistance to penetration. It seems that the processes of phenolic compound synthesis

in response to E. graminis attack have similar effects in adult and in seedling leaves

regardless of eventual adult plant resistance [12, 16, 17]. By plant breeding, it may

prove possible to enhance the production of phenolic compounds produced in response

to disease attack, and thus increase the effectiveness of disease resistance reliant upon

phenolic production. It should then prove feasible to combine this attribute with other,

physiologically independent, resistance mechanisms limiting the success of attempted

infection. These may include the unknown penetration resistance factor(s) in adult

plant resistance, silicon accumulation in papillae and cell walls beneath appressoria

[13] and other papilla or host cell wall characteristics. Such mechanisms may also be

combined with others that limit the growth rate and sporulation capacity of colonies

through limitation of haustorial efficiency and size [10]. The combination of these


mechanisms could provide a complex series of barriers which are unlikely to be

overcome by simple changes in the pathogen genotype and prove extremely durable

in the field.

A cooperative investigation of the Institute of Grassland and Environmental Research,

Plas Gogerddan, Aberystwyth, U.K.; and the Department of Plant Pathology,

University of Minnesota, St Paul, MN, U.S.A. Research supported by MAFF (U.K.),

BBSRC (U.K.) BAGEC initiative, NATO Collaborative Research Grant 00441, and

by Project 22-70 of the Minnesota Agricultural Experiment Station. Published as

Paper u22,227 of the scientific journal series of the Minnesota Agricultural Experiment

Station. Mention of a trademark name or proprietary product does not constitute a

guarantee by the University of Minnesota or by IGER.

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