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SID 5
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Research Project Final Report
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SID 5 (Rev. 3/06)
Project identification
1.
Defra Project code
2.
Project title
AR0712
Biology and genetics of durable resistance to biotrophic
pathogens of cereals
3.
Contractor
organisation(s)
IGER
Plas Gogerddan
Aberystwyth
Ceredigion
SY23 3PD
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 24
£
914,053
start date ................
01 April 2003
end date .................
31 March 2007
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Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
INTRODUCTION AND OBJECTIVES
The Defra Fellowship was awarded to Carver who led studies on powdery mildew resistance and stomatal
dysfunction at IGER and advised on the wheat yellow rust studies led by Boyd at JIC. The work
underpinned development of strategies for sustainable disease management using genetic resistance to
reduce dependence on fungicides. The rapid evolution of virulence has nullified many disease resistance
genes that have been deployed in UK cereal cultivars. Durable resistance is available but it is
acknowledged generally to be partial and under complex physiologic control. Poor understanding of its cell
biological basis, and therefore of the fundamental traits contributing to the resistance, has so far precluded
identification of the controlling genetic factors. This work therefore aimed to increase understanding of the
basis of durable resistance to the obligate biotrophic fungal pathogens that cause cereal powdery mildews
and wheat yellow rust, in order to facilitate its exploitation through plant breeding.
Increased understanding genetic control of durable resistance has been hampered by lack of appropriate
plant material. Two key objectives of the current work were to generate such material for studies of
powdery mildew (Objective 1) and to reassess the potential value of 21 wheat cvs previously identified by
Dr Roy Johnson as having durable resistance (Objective 5).
Evolution of virulence by powdery mildew populations has overcome virtually all single gene resistances
deployed in UK cereal cvs. Plant breeders have, therefore, selected empirically for partial resistance.
Objective 2 examined whether this form of resistance can be overcome by pathogen adaptation.
A form of highly effective, broad spectrum penetration resistance is induced in cereal epidermal cells
subjected to sequential powdery mildew attacks. This induced ‘inaccessibility’ is even more effective than
that conditioned by mlo alleles in barley, and its constitutive expression in cereal epidermis would provide
a novel and extremely efficient form of durable resistance. To achieve this requires detailed understanding
of inaccessibility and Objective 3 aimed to discover its physiological basis and whether it relies on static
or active processes. Chance observations made during this work led to establishment of Objective 7
which examined the effects of resistance expression on stomatal function in attacked leaves.
Earlier MAFF/Defra funded studies at IGER, and very recent collaboration with Würzburg University (Prof.
M. Riederer et al.) indicated that epicuticular leaf wax constitution can affect the ability of powdery mildews
to form infection structures, and this imparts virtual immunity to the abaxial surface of ryegrass leaves.
Objective 4 used introgression lines created at IGER (for a different purpose) with a view to identifying the
basis of this resistance and possibilities of its exploitation.
All of the above studies involving powdery mildew used microscopy to characterise host responses and
study individual resistance mechanisms. To date, however, microscopy of responses to yellow rust attack
have been severely constrained by the fact that under laboratory conditions yellow rust infection
frequencies are extremely low. This impedes data collection and can therefore preclude identification of
SID 5 (Rev. 3/06)
Page 2 of 24
individual resistance mechanisms of quantitative effect. Thus, Objective 6 sought to manipulate
environmental conditions to improve infection frequency as a prelude to comparative work aimed at
identifying resistance characteristics.
APPROACHES AND RESULTS
Objective 1. F1 hybrids were made from crosses between oat cv. Maldwyn (durably resistant) with cv.
Emotion, cv. Millennium and N237/6. F2 plants (200 per cross) and selected F3 descendants were
assessed for powdery mildew resistance in a glasshouse. Juvenile and adult leaves of F4 selections were
assessed microscopically for primary penetration resistance, colony growth and plant epidermal cell death.
This showed considerable variation between lines. Leaf material from each generation was cryo- stored
for future DNA analysis.
Objective 2. Powdery mildew populations collected in 2002 from crops of barley cvs County and Optic
were ‘trained’ by continuous, isolated culture on their source cv. and cv. Golden Promise (susceptible) for
2 years. Each year isolates were tested on County, Optic and Golden Promise and their penetration
efficiency and colony growth was compared microscopically. Mildew from County was slightly more
pathogenic than that from Optic, but both cvs maintained good adult plant resistance. Data indicated no
evidence that training had led to fungal adaptation.
Objective 3. Phosphate scavengers and phenolic biosynthesis inhibitors suppressed induced
inaccessibility in oat, providing evidence that it is energy dependent and relates to lignin biosynthesis.
Tests using cordycepin to inhibit gene transcription by inhibition were inconclusive; cordycepin was
fungitoxic at recommended concentration and ineffective at lower concentration. Cytochalasin E, an
inhibitor of cytoskeletal microfilament polymerisation, increased basal susceptibility but had no effect on
induced inaccessibility suggesting that cytoskeleton reorganisation may not be a key factor.
Objective 4. IGER hybrids between short lived ryegrass species and meadow fescue carrying a ‘stay
green’ gene showed abaxial mildew susceptibility but examination of a derived mapping population
suggested that the susceptibility and stay-greens factor are independent. This work could not be
concluded as the short-lived hybrids died.
Objective 5. Field trials assessed the 21 cvs inoculated with yellow rust isolates representing the raceprofile of the UK yellow rust population. In 2005, 16 were apparently immune, three showed high levels of
partial resistance, but 2 were now fully susceptible. In 2006 the cvs were tested at two sites, Norwich and
Market Rasen. In Market Rasen all varieties supported more yellow rust infection and all previously
immune varieties showed varying degrees of symptoms though most maintained good resistance. In
detailed glasshouse assessments of five selected cvs, adult plant resistance did not affect disease latent
period but fewer pustules formed on the three more resistant cvs, Bersee, Carstens V and Little Joss,
which also produced fewer spores per pustule. CarstensV and Cappelle Desprez have been crossed to
the yellow rust susceptible wheat cv. Lemhi to give F 2 seed. Crosses have also been made between
wheat cvs carrying known sources of durable yellow rust resistance and the susceptible wheat cvs
Brigadier, Robigus and Glasgow and F1 seed is available.
Objective 6. Environmental conditions pre- and post-inoculation were manipulated and, in a susceptible
cv., high light input immediately before inoculation increased both spore germination rates and the
frequency of rust germ tubes entering stomata. Transpiration measurement showed no post-inoculation
differences between leaves due to light pre-treatment, indicating no effect of treatment on stomatal
aperture post-inoculation. This suggests that high light pre-inoculation may stimulate production of volatile
factor(s) acting as germination promotors and germ tube attractants. Comparisons between cvs.
suggested variation in degree of germination/germ tube orientation stimulated by pre-inoculation high light,
indicating a potential novel source of yellow rust resistance.
Ojective 7. Measurement of leaf water conductance and light and cryo-scanning electron microscopy
showed that epidermal cell death associated with three different single resistance genes caused stomata
of powdery mildew-attacked barley to lock open and become unable to close in response to darkness,
drought or application of abscisic acid. Conversely, following brown rust attack, single gene resistance of
wheat and barley caused stomata to become locked shut. By contrast, stomata quickly recovered
functionality where resistance was papilla-based and epidermal cells survived powdery mildew attack.
DISCUSSION
The generation of oat populations, stored genetic material and histological data provides future opportunity
to identify genes for durable powdery mildew resistance: some work will be based on core budget while
other funding options are being considered. Similarly, identification of wheats with durable yellow rust
resistance indicates lines for study and as potential sources of resistance. A constraint to studies of yellow
rust resistance has been its extremely low infection frequency under laboratory conditions. The discovery
that high light input pre-inoculation substantially increases infection opens the way to identify of individual
resistance mechanisms operating throughout the interaction. The hypothesis that the pre-inoculation effect
is due release of volatile(s) that promote germination and germ tube orientation can now be explored and
the finding that conditions affect wheat genotypes differentially may be indicate a novel form of resistance.
Past MAFF/Defra work defined histological methods for analysing powdery mildew resistance, and here
the quantitative resistance expressed in two modern barley cvs proved stable over a period of isolate
‘training’ equating to c. 140 asexual cycles or seven field seasons. Thus, empirical selection by plant
SID 5 (Rev. 3/06)
Page 3 of 24
breeders has delivered durable resistance in the absence of sexual recombination. Confirming previous
studies, induced inaccessibility was shown to convey very effective resistance but the work showed its
requirement for energy and phenolic biosynthesis. That it is expressed strongly in all cereals, irrespective
of their inherent susceptibility, indicates that it is regulation of gene function rather than the presence of
particular genes that controls its expression. Thus, single cell transcript analysis, (collaboration with
Lyngkjær et al.) holds the key to understanding its control. The suggestion that a gene linked to the staygreen factor in meadow fescue influences abaxial leaf surface susceptibility suggests a route to identifying
gene(s) of importance by construction of a long-lived Fescue/Lolium mapping population. An important
physiologic basis for cost of major gene resistance was identified as stomatal dysfunction consequent on
epidermal death. This will have obvious consequences for photosynthesis, respiration, water use
efficiency and respond to abiotic stresses. Papilla-based resistance is therefore more desirable.
The Fellowship facilitated national/international collaborations that added value through progressing
understanding of single cell genetic responses, induced resistance, early host-pathogen interactions and
pea powdery mildew resistance (principally via: Gay, IGER; Gurr, Oxford; Lyngkjær, Denmark; Kunoh,
Japan; Mur, UWA; Riederer, Germany; Prats, Spain; Zeyen, USA). Additionally, Defra’s interest has
fostered relationships between Carver and Pavely (Defra Fellowship AR0511) that led to a proposal for
work to assess costs of disease resistance in field situations, particularly in relation to crop water use
efficiency.
Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
 possible future work; and
 any action resulting from the research (e.g. IP, Knowledge Transfer).
SID 5 (Rev. 3/06)
Page 4 of 24
8.1. Create and assess a range of recombinant inbred lines for oat powdery mildew resistance based on
crosses between the cv. Maldwyn that has proven, durable mildew resistance and modern, susceptible
cvs. (Proposal Objective 1). All milestones were completed.
INTRODUCTION
The quantitative, adult plant resistance of oat cv. Maldwyn provides a rare example of powdery mildew resistance
that has proven durable in the field since 1947. Early studies [1] showed the resistance is controlled by at least six
minor genes with additive effect. To breed for resistance under such complex genetic control is extremely difficult.
Previous MAFF/Defra projects (CE0120 and CE0154) showed that the resistance depends on several
mechanisms: papilla-based resistance and low frequency cell death limit colony establishment, and where
colonies form, reduced efficiency of haustoria limits growth extending disease latent period and reducing
sporulation. This work and studies on the physiological bases of different resistance mechanisms has been
collated [2]. It is totally impractical, however, to screen breeding populations histologically for combinations of
different mechanisms but this might be achieved using marker-assisted selection. Towards this, the current work
aimed to produce plant material in which relevant genetic markers of different resistance genes may be identified.
This involved hybridising Maldwyn with selected, susceptible modern oat cvs with a view to producing progeny in
which various resistance mechanisms limiting colony establishment and development could be identified, with a
view towards providing a resource for future molecular genetic analyses.
MATERIALS AND METHODS
JM Leggett (cyto-geneticist, IGER) hybridised cv. Maldwyn (male) with nine different oat lines including modern
cvs and a high oil experimental line (N327-6). Six hybrids were produced and selfed to produce F2 seed (ripened
in July 04). Three crosses were selected for further study: x cv. Emotion (spring oat, moderately mildew
susceptible); x cv. Millennium (winter oat tolerant of spring sowing, moderately mildew susceptible) and x line
N327-6 (very mildew susceptible).
In August 2004, 200 F2 plants of each of the three F1 hybrids, together with 12 plants of the parental genotypes
for reference, were grown in randomized blocks in a glasshouse. When their fourth-formed leaf was emerging,
segments of their fourth leaf were collected, frozen in liquid nitrogen, and stored at -80ºC for potential future use
for molecular genetic study. Plants were then inoculated with race 5 B. graminis f.sp. avenae, and an epidemic
developed. Each plant was scored at the juvenile stage (5 th leaf unrolling) and again as adults (flag leaf
expanded) for percentage leaf area affected [3]; for juveniles the sum of scores from leaves 3, 2, 1, and for adults
the sum of scores from the flag leaf, flag-1 and flag-2, were summed as an overall indicator of disease. In addition
infection type (IT) was recorded on the uppermost leaf at each stage using a 0-4 scale where: 0 = no visible
symptoms; 1 = colonies just visible, no apparent sporulation; 2 = small colonies, sporulation questionable; 3 =
moderate, obviously sporulating colonies; 4 = large, freely sporulating colonies. Note was taken if colonies were
associated with necrosis. Unfortunately, although the parent cv. Millennium is tolerant of spring sowing, with the
August planting it failed to head. After self pollination, all available F3 seed was collected and stored.
In April 2005, from each of the three hybrid populations, 10 F3 plants were grown from each of five F2 parents
that had shown high resistance (low area affected, low IT), from five that had shown moderate high leaf area
infected but with low IT, from five that had shown low area infected but with high IT, and from five that had shown
high susceptibility (high area affected, high IT). Seed of Millennium and its derivatives were vernalised (2ºC, 2 wk)
and all were planted at the end of April. Plants were assessed as for F2 populations, fourth leaf material was
cryo-preserved and F4 seed was collected.
In May 2006, from each of the three F3 populations, seven plants were selected. Three of these had shown high
resistance as adults (low area affected, low IT), two had shown moderate resistance (one with low IT but high
area infected; the other with high IT but low area infected), and two had shown low resistance (high area affected,
high IT). Five F4 plants of each of the seven lines were grown in a spore-proof glasshouse together with four of
the parental genotypes (Millennium and derivatives were vernalised). Leaf material (from tillers) was taken from
all plants for cryo-storage. On three successive days (one hybrid population per day), the expanded fourth-formed
leaf (juvenile) was excised from all plants and three 3-cm segments were cut from each. These were arranged
floating on 45 mg L-1 aqueous benzimidazole solution [4], inoculated by settling tower (ca 10 conidia mm -2) and
incubated at 20ºC, 12 h light (150 umol m -2 sec-1) until one segment from each leaf was fixed at 42 and another at
72 h for microscopy, the last being inspected daily for symptom development. Plants were allowed to grow on
until the flag leaf had emerged when the flag-1 leaf (adult) was taken, cut into segments used for microscopy and
symptom assessment. Eventually, seed was later collected.
RESULTS
Symptoms appeared on many juvenile F2 plants within 5 d of inoculation and developed fast although there was
great variation between individuals (data not shown). As expected, Maldwyn showed good adult plant resistance
as a relatively small area of its upper three leaves was affected, while adult N327-6 remained extremely
susceptible; the distribution of F2 adult plants derived from this cross was skewed towards susceptibility. Cultivar
Emotion was only slightly more susceptible than cv. Maldwyn (no significant difference) and here the F2
SID 5 (Rev. 3/06)
Page 5 of 24
population data were skewed towards resistance with most behaving similarly to their parents. A similar skew was
evident in the Millennium F2 population), but no data could be obtained from Millennium itself because plants
failed to head under the late summer conditions. (However, data from 2005 indicated that cvs Maldwyn and
Millennium were similarly resistant). In all F2 populations, transgressive segregants were evident as some plants
showing greater resistance than either parent while others were far more susceptible. There was also variation in
IT between plants within F2 populations, although, again, the population derived from N327-6 tended to produce
larger colonies overall (data not shown). Thus it was possible to select plants showing different resistance
phenotypes to provide tester F3 families. Assessment of the F3 selections again segregated for resistance
allowing the selection of resistant, susceptible and intermediate phenotypes for histological tests in the F4
generation.
With advice from Dr R. Sanderson (Statistician, IGER), data for several characteristics describing success of
attempted infection and others reflecting post-infection development, assessed in segments fixed 42 and 72 h
after inoculation, respectively, were examined by various approaches and all data and analyses have been
archived and copies given to interested IGER staff (contact Dr C. Howarth). For brevity, only data from line N3276 is presented (Figs 8.1.1, 8.1.2). Figure 8.1.1 shows data that indicate there was a strong tendency for adult
plant resistance to increase frequencies of effective papilla resistance, to decrease successful penetration but to
have relatively less effect on cell death frequency. To assess post-infection development, data from live colonies
were examined in detail if at least seven of the 10 colonies observed per plant line were associated only with
living epidermal cells. Thus, these data reflected the living plant’s ability to restrict pathogenesis. In lines derived
from N327-6 and Millennium, a large number of lines (>15) fulfilled this criterion. Figure 8.1.2 illustrates data for
per colony numbers of hyphal tips, reflecting growth made prior to fixation, and of secondary appressoria and
haustoria that indicate future growth potential. In cv. Emotion and its derivatives, cell death associated with
established colonies was very common and 26 of 35 showed very frequent post-infection cell death which was,
therefore, a key component of their resistance. Within all populations, individual plants could be identified
showing high, low and intermediate values for each character.
Density
Fig 8.1.1. Kernel densities indicating underlying distributions in papillae formation, cell death and susceptibility to penetration in juvenile (grey
line) and adult (black line) leaves within the F4 population of oat line N327-6 fixed 42 h after inoculation with powdery mildew.
Papillae (%)
Cell death (%)
Penetration (%)
Density
Fig 8.1.2. Kernel densities indicating underlying distributions in per fungal colony numbers of secondary appressoria, haustoria and hyphal
tips in juvenile (grey line) and adult (black line) leaves within the F4 population of oat line N327-6 fixed 72 h after inoculation with powdery
mildew.
Number of
secondary
appressoria
Number of
secondary
haustoria
Number of hyphal
tips
DISCUSSION
The data show variation in susceptibility between plants within and between F2 populations, within and between
families and populations in F3 and F4 generations, and histological analyses of the F4 plants identified individuals
that express different resistance mechanisms with different degrees of efficacy. These include mechanisms acting
to prevent colony establishment and then the growth of colonies that do establish. The former reduce effective
inoculum potential and the latter reduces the rate of colony growth and extends the asexual generation time, so
slowing the rate of epidemic development. Both papilla formation and cell death prevent colony establishment.
Papillae produced by living cells attacked by powdery mildew act as a physical and/or chemical barrier that
prevents penetration of the host and establishment of haustoria (feeding structures), so arresting colony
establishment. In Maldwyn oat and in mlo barley cvs, papilla-based resistance has proved durable. Separate work
reported here (Objective 7) shows that papilla formation has little, and only transient, effect on the function of
stomata close to attack sites, providing a form of resistance that will be particularly useful in sustainable cropping
systems under a changing environment where plants are likely more often to suffer drought and other abiotic
SID 5 (Rev. 3/06)
Page 6 of 24
stresses. By contrast, cell death has the undesirable consequence of causing dysfunction of nearby stomata, so
that this form of resistance may carry a hitherto unsuspected cost to the plant. Where colonies do establish,
restriction of their growth rate would clearly be desirable by limiting direct effects of parasitism as well as indirect
effects in limiting stomatal opening. Again, however, post-infection cell death is likely to affect stomatal function
deleteriously.
The genetic material of all the plants used in this study has been preserved. Given sufficient molecular genetic
markers, it should prove possible in future to relate genetic information to the degree of resistance/susceptibility
expressed by individuals, and from the F4 data to relate genetic information to the individual resistance
mechanisms expressed by particular genotypes. The strength of the relationship between molecular marker and
resistance mechanism can be assessed not only from comparison between individuals within and between
generations but also from comparison between populations arising from the different parents. All data, seed and
cryo-preserved leaf material is now stored at IGER and ongoing discussions will determine how the material will
be used and how the work is to be funded.
REFERENCES
1. Jones IT. 1986. Ann. Appl. Biol. 109, 187-192.
2. Carver TLW. 2000. Powdery mildew disease: studies of fungal biology and host resistance. D.Sc. Thesis,
University of Wales.
3. Large EC, Doling DA. 1962. Plant Pathol. 11, 47-57.
4. Jones IT, Hayes JD. 1971. Ann. Appl. Biol. 68, 31-39.
------------------------------------------------------8.2. Test the durability of powdery mildew resistance mechanisms in modern barley cvs that express
high levels of partial field resistance. (Proposal Objective 2). All milestones were met for 2003-04 when the
work was terminated.
INTRODUCTION
This study was an extension of work started under Defra project CE0154 to identify individual mechanisms
contributing to the partial resistance of two selected cvs and to test their continued efficacy as isolates of B.
graminis f.sp. hordei (Bgh) were grown continuously on them (i.e. 'trained') and so provided the opportunity to
adapt to the resistances.
Current cereal powdery mildew populations in the UK have evolved complex virulence capable of overcoming
virtually all major gene resistances deployed [[1] and previous reports of the UKCPVS]. As a direct consequence
of this pathogen adaptation, breeders have selected empirically for partial forms of resistance and many cvs on
the UK Recommended Lists for cereals show relatively high resistance ratings for powdery mildew (scoring 6-9 in
trials). Although these resistances are quantitative, and are expressed more strongly in adult than seedling or
juvenile plants, it is unclear whether they are durable [2]. To test this possibility two spring barley cultivars, Optic
and County, that appeared on the 2001 Recommended List, were selected for study. Both showed good partial
resistance to powdery mildew (Recommended List resistance ratings 7 and 8, respectively). The approach was to
inoculate juvenile plants of each cultivar with mildew samples collected from them in the field, and attempt to
erode their resistance by continuously culturing the mildew on them in isolation, thus applying pressure for
selection of powdery mildew genotypes better able to grow on their host cv. To provide a control where no
differential selection pressure was applied, isolates were sub-cultured on cv. Golden Promise which is highly
susceptible to all known isolates of Bgh.
MATERIALS AND METHODS
Pathogen material. Samples of Bgh from natural infections on cvs Optic and County were collected by S. Slater
(NIAB) from Cambridge field plots in spring/summer 2002. They were multiplied separately in isolated sporeproof compartments (min. temp. 20ºC) on their donor hosts. When established, both populations were
subdivided. One part, was maintained on its 'own' cv. and the other was transferred to a separate compartment
in which it was inoculated to cv. Golden promise so that four populations were created. Each was then
maintained continuously on juvenile-adult plants of its donor host (cv. Optic or County) or on cv. Golden Promise
by introducing new, healthy plants into the compartments each week. Sporulating colonies developed on these
introductions after ca 5 days. Older plants were destroyed after 2 wks in the culture chambers, except in
September of each year when they were kept until fully mature in an attempt to encourage cleistothecia.
Assessments of fungal development. Histological and macroscopic observations were made in summer 2003 and
2004 to determine whether isolated mildew populations were adapting to their hosts. Healthy adult (5th-leaf
expanded) and juvenile plants (3rd-leaf expanded) were produced for inoculation at the same time in a completely
randomised design with four replicates containing 32 adult and juvenile plants each of cvs Optic, County and
Golden Promise. Leaves of four sets of intact plants were inoculated on four successive days with each of the
four mildew populations. In all cases, the adaxial surface of third leaves of four juvenile plants and of fifth leaves
of four adults of all the cvs was simultaneously inoculated. One set was incubated for 30 h to provide material for
assessment of Bgh penetration success; two other sets were incubated for 72 and 96 h for assessments of
colony development, while a final set was used for macroscopic assessment of latent period (days to symptom
SID 5 (Rev. 3/06)
Page 7 of 24
appearance), final disease severity (percentage leaf area affected; [3]) and disease phenotype a 0-4 scale [4].
Inoculation was by settling tower with ca 10 conidia mm-2. Inoculated plants were incubated at 20 C under 12 h
light (06:00-18:00) at 200 μmol m-2 sec-1, 70% RH. For microscopy leaves were prepared as described previously
[5].
RESULTS
Attempts to generate Bgh sexual recombinants were unsuccessful as no cleistothecia were produced, possibly
because only a single mating type was present or because the environmental conditions within isolation
compartments were unsuitable. The results from assessments of disease development from the clonal
populations cultured in isolation showed no good evidence that trained isolates from Optic or County had adapted
to their host cv. The data obtained from the 2004 tests are presented here in detail.
Penetration success and host responses: Penetration (haustorium formation) into leaves incubated for 30 h
was assessed by examining 100 germlings on each leaf. Percentage data were calculated and transformed to
angles before analysis of variance (ANOVA) using Genstat (inspection of residuals showed data conformed to
normality). Table 8.2.1 shows the mean percentage (transformed) penetration by germlings from each mildew
isolate on cvs Optic, County and Golden Promise. There were significant (P<0.001) main effects of leaf position
and test cv. and, as expected, third leaves were more susceptible than the adult fifth leaves (means = 18.42 and
11.69) while Golden Promise was far more susceptible than Optic or County. Overall, the Bgh populations arising
from County penetrated significantly (P<0.001) more frequently than those from Optic, but their culture on the
highly susceptible Golden promise had no effect on their performance and there was no significant isolate x cv.
interaction indicating that fungal penetration potential was not affected by ‘training’.
Table 8.2.1. Percentage (transformed) penetration by Bgh germlings from populations sourced from cvs Optic and County and then cultured
continuously for 2 yrs on either their source cv. or on the highly susceptible cv. Golden Promise.
Isolate
Source
Culture
cultivar
Test cultivar
Optic
County
Optic
Optic
Golden Promise
5.84
5.58
6.63
8.31
Golden
Promise
21.19
27.4
County
County
Golden Promise
13.02
5.93
13.37
15.17
27.46
31.03
Mean
7.59
10.8
26.78
Mean
17.95
17.39
11.12
13.76
The frequency with which appressoria of all isolates penetrated was significantly (P<0.001) higher in third than
fifth leaves that expressed adult plant resistance. In all cases the great majority of failures was associated with
defensive papillae that were autofluorescent indicating that they contained phenolic compounds [6]. However,
some attacked epidermal cells showed whole-cell autofluorescence indicating that their death as a result of attack
[7]. Cell death can be an important component of durable resistance [8] but here its frequency was significantly
(P< 0.001) higher in third than fifth leaves (means = 8.45 and 4.67, respectively) presumably because papilla
defence was more effective in the later-formed leaf. Nevertheless, in all cases cell death frequency was relatively
rare (ranging from <1%-5%) and even though there were significant differences between isolates (P< 0.05) and
test cv (P<0.01) the biological significance of these small differences is questionable.
Colony development: Counts of secondary haustoria formed by a colony accurately reflect its past growth and
future potential [9]. Ten colonies were examined on every leaf and the number of secondary haustoria counted.
Data subjected to ANOVA (Genstat) were normally distributed and since trends from leaves fixed after 72 and 96
h incubation were similar, Table 8.2.2 presents data only from the older colonies. As expected, colonies formed
significantly (P<0.001) more haustoria in third than fifth leaves (means = 13.46 and 6.80, respectively), and
although most were formed in Golden Promise, the number formed by all isolates was greater in Optic than
County. Overall, the populations sourced from County produced more haustoria than those from Optic, but their
culture on the highly susceptible Golden promise had no effect on their performance. Furthermore, although there
was a significant isolate x cv. interaction (P=0.001) shows that this was largely explained by the very large
colonies formed on Golden Promise by the population sourced from, and cultured on cv. County. Thus, colony
growth potential was not improved by ‘training’.
Table 8.2.2. Numbers of secondary haustoria formed by Bgh colonies arising from populations sourced from cvs Optic and County and then
cultured continuously for 2 yrs on either their source cv. or on the highly susceptible cv. Golden Promise.
Isolate
Source
Culture
cultivar
Test cultivar
Optic
Optic
Golden Promise
8.86
8.34
4.85
6.21
Golden
Promise
13.91
13.21
County
County
Golden Promise
9.75
11.32
4.96
4.36
20.43
15.32
SID 5 (Rev. 3/06)
Optic
County
Page 8 of 24
Mean
11.71
10.34
9.21
9.25
Mean
9.57
5.10
15.72
Macroscopic symptoms: As expected, latent period was overall shorter (P< 0.001) on third than fifth leaves
(means = 4.2 and 5.4 d, respectively), and on Golden Promise than on Optic and County and percentage leaf
area affected and IT were greater for third leaves. The data for percent cover required subjective judgement and
must be treated cautiously, because by 7 d after inoculation, some leaves were senescing. Overall, cv Optic
showed the lowest area affected (Table 8.2.3), and although County gave a value similar to Golden Promise,
colonies on Optic fifth leaves did not appear to be sporulating by 7 d, they were almost invariably associated with
necrosis (indicated by ‘n’), and underlying tissues were chlorotic. Infection type on Optic was superficially similar,
but colonies were sporulating although smaller and more frequently associated with necrosis than those on
Golden Promise.
Table 8.2.3. Latent period (LP), leaf area affected at 7 d (% LA), and infection type [IT] resulting from inoculation with Bgh populations
sourced from cvs Optic and County and then cultured continuously for 2 years on either their source cv. or on the highly susceptible cv.
Golden Promise.
Isolate
Culture
Test cultivar
Source
cultivar
Optic
County
Golden Promise
Mean
LP
%LA
IT
LP
%LA
IT*
LP
%LA
IT
LP
%LA
Optic
County
Optic
(5.1)
2.5
[3n,1n]
(5.5)
9.4 [3n,2n]
(4.0)
11.3
[4,3n]
(4.8)
7.7
Golden Promise
(4.3)
8.3
[3n,2n]
(5.9)
12.5 [3n,2n]
(4.0)
10.6
[4,3n]
(4.7)
10.5
County
(4.9)
3.5
[3n,2n]
(5.8)
13.8 [3n,2n]
(4.0)
15.0
[4,3n]
(4.9)
10.8
Golden Promise
(4.5)
6.4
[2n,3n]
(6.0)
17.0 [3n,2n]
(3.6)
20.1
[4,3n]
(4.7)
14.5
(3.9)
14.3
Mean
(4.7)
5.2
(5.8)
13.2
*NB. Colonies on cv. County fifth leaves were not sporulating by 7 d.
DISCUSSION
After continuous culture on their source cv. since late spring 2002, there was no good evidence to suggest that
either of the original powdery mildew populations has adapted to give increased pathogenicity on either its source
host or the other two cultivars employed in the tests. Throughout, both County and Optic maintained a good level
of adult plant resistance that reduced both penetration success and post-penetration colony development. In this
sense they remained effectively durable.
At the time of the 2004 test, each mildew population is estimated to have been through at least 140 asexual
generations growing at the near optimal conditions provided in the glasshouse. In the field, where environmental
conditions are often far from optimal, it is likely that the fungus goes through no more than an average of four
generations per month over a 5 month growing season for spring barley. Thus, the test applied here equates to
approximately 7 years of continuous culture on the same cultivar under favourable conditions and in the absence
of control measures. In view of the absence of sexual recombination it was impossible to determine whether this
would have allowed adaptation of the pathogen.
REFERENCES
1. Ann. Rep. 2002. Ed. JDS Clarkson. The United Kingdom Cereal Pathogen Virulence Survey Committee,
Cambridge, UK.
2. Brown JKM. 2002. In: The Powdery Mildews: a Comprehensive Treatise. Eds R.R. Belanger, A.J. Dik, W.R.
Bushnell and T.L.W. Carver. American Phytopathological Society, St. Paul, USA. pp. 107-125.
3. Large EC, Doling DA. 1962. Plant Pathol. 11, 47-55.
4. Moseman JG et al. 1965. Trans. Br. Mycol. Soc. 48, 479-489.
5. Carver TLW et al. 1991. Physiol. Mol. Plant Pathol. 39, 269-287.
6. Carver TLW et al. 1994. Physiol. Mol. Plant Pathol. 44, 261-272.
7. Zeyen RJ et al. 1995. Physiol. Mol. Plant Pathol. 47, 119-140.
8. Carver T L W et al. 1999. Physiol. Mol. Plant Pathol. 55, 183-196.
9. Carver TLW, Carr AJH. 1978. Ann. of Appl. Biol. 88, 171-178.
------------------------------------------------------8.3. Investigate the physiologic basis of locally induced resistance (inaccessibility) and susceptibility
(accessibility) to cereal powdery mildew. (Proposal Objective 3). All milestones were completed.
INTRODUCTION
In the field, leaves are repeatedly attacked by pathogens as spores are deposited from aerial pool. A series of
papers arising from a previous Defra project (CE0154, listed in Prats et al. (1) below) showed that cereal cells'
SID 5 (Rev. 3/06)
Page 9 of 24
history of attack by, and response to, powdery mildew attack dramatically affects their subsequent ability to resist
attack, and this has dramatic consequences for field resistance. Where initial attack fails due to papilla defence,
the cell and its near neighbours are 'induced' to show extremely high inaccessibility to subsequent attack. By
contrast, where initial attack succeeds, the infected cell and its near neighbours are induced to show extremely
high accessibility. This is true irrespective of the inherent resistance of the host. The implications of these
observations are profound in terms of exploiting quantitative resistance; there was little understanding of the basis
of these induced changes and this was explored here.
PUBLISHED OUTCOMES: These papers give all details of the relevance of the studies, their context, the
methods used and results and, since they are publicly available, only the summaries are presented.
[NB. Only Carver, Roberts and Thomas were funded by Defra so inputs by others gave value added from other
funding sources.]
[1] This paper shows the effects of energy deprivation and inhibition of phenolic compound synthesis on
expression of induced inaccessibility in oat.
[1] Prats E, Carver TLW, Lyngkjær MF, Roberts PC, Zeyen RJ. (2006). Induced inaccessibility and accessibility
in the oat powdery mildew system: insights gained from use of metabolic inhibitors and silicon nutrition. Mol. Plant
Pathol. 7(1), 47-59.
Fungal-induced inaccessibility in oat to Blumeria graminis requires active cell processes. These are reiterative de
novo cell processes involved in inherent penetration resistance. Therefore, induced inaccessibility may well
involve cellular memory of the initial attack. Phenylpropanoid biosynthesis inhibitors (AOPP and OH-PAS) and
phosphate scavengers (DDG and D-mannose) strongly suppressed induced inaccessibility, but silicon nutrition
had no effect. Induced accessibility was modulated by the presence of fungal haustoria inside cells. Haustoria
actively suppress or reprogram infected plant cells toward a constant state of penetration susceptibility. Neither
inhibitor treatments nor silicon nutrition affected fungal induced accessibility.
[2, 3] These papers show that accessibility to non-pathogens is induced by prior infection with a virulent form but,
conversely that inaccessibility can be rapidly induced by extracellular materials released by non-pathogenic
powdery mildew conidia.
[2] Olesen KL, Carver TLW, Lyngkjaer MF. 2003. Fungal suppression of resistance against inappropriate B.
graminis ff.spp. in barley, oat and wheat. Physiol. Mol. Plant Pathol. 62, 37-50.
When barley, wheat or oat leaf epidermal cells were attacked by their appropriate forma specialis (f.sp.) of
Blumeria graminis DC. Speer (f.sp. hordei, tritici and avenae, respectively), many attempted penetrations
succeeded, functional haustoria were formed and very few plant cells died. When attacked by either of the two
possible inappropriate ff.spp., penetration attempts failed in association with papilla deposition by epidermal cells,
attacked cells died, or, if visible haustoria were formed, the plant cell died very soon afterwards. Double
inoculation experiments were performed where each cereal species was first attacked by its appropriate f.sp., as
inducer, and later by the different ff.spp. as challenger. Infection by the appropriate inducer profoundly affected
cellular responses to challenger attack. Suppression of defensive responses was dramatic within epidermal cells
containing the inducer haustorium, evident to some extent in adjacent cells, but undetectable at two cells
distance. Suppression of penetration resistance allowed most challenger attacks, even by inappropriate ff.spp., to
form a haustorium. Furthermore, death of penetrated epidermal cells was also suppressed so that haustoria of
the inappropriate ff.spp. functioned to support colony development. In oat, delayed epidermal cell death
prevented full colony development by inappropriate ff.spp., but in barley and wheat, no cell death was apparent
by four days after inoculation and colonies of the inappropriate ff.spp. produced extensive hyphae, secondary
haustoria and conidial chains.
[3] Fujita A, Suzuki T, Kunoh H, Carver TLW, Thomas BJ, Gurr SJ , Shiraishi T. (2004). Induced
inaccessibility in barley cells exposed to extracellular material released by non-pathogenic powdery mildew
conidia. Physiol. Mol. Plant Pathol. 64, 169-178.
Before germinating, conidia of Blumeria graminis f. sp. hordei (Bgh) and tritici (Bgt) and Erysiphe pisi (Ep) rapidly
released extracellular material (ECM) onto barley coleoptile cells. More was released if full rather than partial cell
contact was made. Within 5-6.5 h of receiving ECM from Ep or Bgt (non-pathogens), cells showed induced
inaccessibility to challenger Bgh. This effect was greater for Ep than Bgt where full contact was required. Abiotic
particles and ECM from Bgh did not affect cells’ accessibility to subsequent challenger attack by Bgh. This
induced inaccessibility must be due to active component(s) within conidial ECM.
[4, 5]. The possible involvement of nitric oxide (NO) in signalling within and between barley cells expressing
papilla-based and hypersensitive resistance to powdery mildew was demonstrated for the first time. Thus, NO
may be involved induction of accessibility not only in attacked cells but also in their near neighbours. The finding
that NO was generated in stomatal complexes close to attacked cells led directly to the work reported under
Objective 7.
[4] Prats E, Mur AJ, Sanderson R, Carver TLW. (2005). Nitric oxide contributes both to papilla-based resistance
and the hypersensitive response in barley attacked by Blumeria graminis f. sp. hordei. Mol. Plant Pathol. 6, 65-78.
SID 5 (Rev. 3/06)
Page 10 of 24
Blumeria graminis f.sp. hordei (Bgh) attack disrupted stomatal behaviour, and hence leaf water conductance (gl),
in barley genotypes Pallas and Risø-S (susceptible), P01 (with Mla1 conditioning a hypersensitive response, HR),
and P22 and Risø-R (with mlo5 conditioning papilla-based penetration resistance). Inoculation caused some
stomatal closure well before the fungus attempted infection. Coinciding with epidermal cell penetration, stomatal
opening in light was also impeded, although stomata of susceptible and mlo5 lines remained largely able to close
in darkness. Following infection, in susceptible lines stomata closed in darkness, but opening in light was
persistently impeded. In Risø-R, stomata recovered near-full function by around 30 h after inoculation, i.e. after
penetration resistance was accomplished. In P01 stomata became locked open and unable to close in darkness
shortly after epidermal cells died due to HR. In the P22 background, mlo5 penetration resistance was often
followed by consequential death of attacked cells, and here too stomata became locked open, but not until ca 24
h after pathogen attack had ceased. The influence of epidermal cell death was localised, and only affected
stomata within one or two cells distance. These stomata were unable to close not only in darkness but also after
application of abscisic acid and in wilted leaves suffering drought. Thus, resistance to Bgh based on HR or
associated with cell death may have previously unsuspected negative consequences for the physiological health
of apparently ‘disease free’ plants. The results are discussed in relation to the control of stomatal aperture in
barley by epidermal cells.
[5] Mur LAJ, Carver TLW, Prats E. (2006) NO way to live; the various roles of nitric oxide in plant-pathogen
interactions. J. Exp. Bot. 57(3), 489-505.
Consideration of NO in plant-pathogenic interactions usually focuses on its roles in eliciting or confining plant cell
death in the hypersensitive response (HR). We examined NO action in the development of both HR resistance
and susceptible responses in three plant-pathogen interactions; tobacco and Arabidopsis interacting with
pathovars of the bacterium Pseudomonas syringae, and barley (Hordeum vulgare) challenged with the powdery
mildew fungus, Blumeria graminis. We have developed a novel approach to measure in planta NO production in
tobacco and Arabidopsis based on photoacoustic laser detection. In barley, resistance is conditioned not only by
HR but also by prevention of penetration whereby living cells deposit localised wall appositions (papillae). Spatiotemporal NO production elicited by B. graminis was visualised using the NO-specific fluorescent dye, DAF-2DA.
[6, 7] Since epidermal cells of even the most cereal susceptible genotype can be rendered virtually totally
resistant to penetration by powdery mildew, it is clear that all genotypes have the potential to express this form of
resistance and suggests the likelihood that it is gene regulation rather than structure that is key. These papers
address the fact that transcript analysis of entire organs or tissues cannot reveal activity specifically associated
with induced cells, or indeed the genetic basis of inherent resistance or susceptibility to penetration. Work
demonstrating the isolation of transcripts from single epidermal cells of known response type, initiated under a
former Defra project (CE0154), has now been published. This will make possible studies of genetic activity during
expression of not only inherent resistance, but also induced inaccessibility.
[6] Gjetting T, Carver TLW, Skøt L, Lyngkjær MF. (2004).Gene expression profiling of individual barley
epidermal cells attacked by powdery mildew. Mol. Plant-Micr. Inter. 17, 729-738.
Resistance and susceptibility in barley to the powdery mildew fungus (Blumeria graminis f.sp. hordei) is
determined at the single-cell level. Even in genetically compatible interactions, attacked plant epidermal cells
defend themselves against attempted fungal penetration by localised responses leading to papilla deposition and
reinforcement of their cell wall. This conveys a race non-specific form of resistance. However, this defence is not
complete and a proportion of penetration attempts succeed in infection. The resultant mixture of infected and
uninfected leaf cells makes it impossible to relate powdery mildew-induced gene expression in whole-leaves or
even dissected epidermal tissues to resistance or susceptibility. A method for generating transcript profiles from
individual barley epidermal cells was established and proven useful for analysing resistant and successfully
infected cells separately. Contents of single epidermal cells (resistant, infected and unattacked controls) were
collected and, after cDNA synthesis and PCR amplification, the resulting sample was hybridised to dot-blots
spotted with genes including some previously reported to be induced upon pathogen attack. Transcripts of
several genes, e.g. PR1a – encoding a pathogenesis related protein, and GLP4 – encoding a germin-like protein,
accumulated specifically in resistant cells, while GRP94 – encoding a molecular chaperone, accumulated in
infected cells. Thus, the single-cell method allows discrimination of transcript profiles from resistant and infected
cells. The method will be useful for micro-array expression profiling for simultaneous analysis of many genes.
[7] Gjetting T, Hagedorn PH, SchweizerP, Thordal-Christensen H, Carver TLW, Lyngkjær MF. (in press).
Single-cell transcript profiling of barley attacked by the powdery mildew fungus. Mol. Plant-Micr. Inter.
In many plant-pathogen interactions there are several possible outcomes for simultaneous attacks on the same
leaf. For instance, an attack by the powdery mildew fungus on one barley leaf epidermal cell may succeed in
infection and formation of a functional haustorium whereas a neighboring cell attacked at the same time may
resist fungal penetration. To date, the mixed cellular responses seen even in susceptible host leaves have made
it difficult to relate induced changes in gene expression to resistance or susceptibility in bulk leaf samples. By
micro-extraction of cell-specific mRNA and subsequent cDNA array analysis, we have successfully obtained
separate gene expression profiles for specific mildew-resistant and -infected barley cells. Thus, for the first time it
is possible to identify genes that are specifically regulated in infected cells and presumably involved in fungal
establishment. Further, although much is understood about the genetic basis of effective papilla resistance
SID 5 (Rev. 3/06)
Page 11 of 24
associated with mutant mlo barley, we provide here the first evidence for gene regulation associated with effective
papilla-based non-specific resistance expressed in nominally ‘susceptible’, wild-type barley.
ADDITIONAL STUDIES
Cordycepin has been used to inhibit gene transcription in B. graminis-attacked barley [Schweizer P et al. 1996.
Physiol. Mol. Plant Pathol. 49, 103-120]. A range of cordycepin concentrations spanning the published optimum
was tested. It was applied by dipping inoculated, cut leaves into solution. Microscope analyses of fungal
development indicated that cordycepin was fungistatic. Even at 0.2mM, (50% the recommended optimum), only
2% of appressoria penetrated compared to ca 39% in controls. A further experiment therefore used 0.1mM
Cordycepin, and in this case there was no significant effect of treatment. Thus, with its apparent anti-fungal
effects at higher concentration, and its lack of effect at lower concentration, it was concluded that cordycepin was
unsuited for experimentation on induced (in)accessibility.
Cytochalasin E (CytE) inhibits microfilament polymerisation and increases infection in the cowpea rust system
[Skalamera D, Heath MC. 1998. The Plant Journal 16, 191-200]. Preliminary work tested effects on basal
susceptibility: CytE was applied by bathing the mesophyll of leaves (from which the abaxial surface had been
stripped) with various concentrations and 0.2 μg ml-1 gave the greatest increase in successful penetration
compared to controls (treated with 0.02ul ml-1 DMSO). Concentrations of 0.5 and 1.0 μg ml-1 gave less
penetration but significantly greater levels than their respective controls, while results from 0.1 μg ml-1 were
variable. A double inoculation experiment therefore used 0.2 μg ml -1 CytE to test effects on induced
(in)accessibility. The adaxial surface of eight first leaves of intact Pallas barley plants was inoculated with 10
conidia mm-2 as inducer and incubated for 24 h before removal of superficial fungal structures with latex. The
abaxial epidermis was then removed and central segments of four leaves were floated on CytE and four on
control solution, bathing the mesophyll. Non-induced controls were treated identically. Challenger inoculum was
applied to the adaxial surface and incubated for 42 h. In non-induced controls, CytE significantly (P=0.005)
increased basal susceptibility to penetration (ca 46% in CytE; 32% in DMSO). Effects on induced accessibility
and inaccessibility were assessed in double inoculated leaves [1]. This showed that, as expected, control cells
containing an inducer haustorium were highly accessible to the challenger (98% penetrated) while cells with an
inducer papilla were totally resistant to challenger attack (0% penetrated). Interestingly, CytE treatment had no
significant effect on either phenomenon (P>0.45 for both cases). Thus, it affected neither induction of
inaccessibility nor inaccessibility or transmission of the effects to adjacent cells. This suggests that cytoskeleton
reorganisation may not be key to either phenomenon. However, it also supports the view that induced
inaccessibility is a stable and effective form of resistance, likely to be durable, and that breeding lines with high
inherent penetration resistance should enhance their expression of induced inaccessibility. This would be of clear
value in protecting crops produced under both low-input (including organic) and intensive systems.
------------------------------------------------------8.4. Explore the role of Graminaceous epicuticular leaf wax components in disrupting germling
development by the powdery mildew fungus. (Proposal Objective 4)
This study was proposed as a minor component of AR0712. All milestones were achieved apart from gaining
sufficient data to add information regarding the location of a suspected gene controlling a disruptive leaf wax
factor for inclusion in the genetic map of ryegrass. This was because of the death of plants contributing to the
mapping population (see below).
INTRODUCTION
Previous studies (MAFF and Defra projects CE0120 and CE0154) showed that early plant/pathogen interactions
have a profound effect on the germination and germling development that lead to infection structures
(appressoria) formation by powdery mildew fungi [1-4]. There is little evidence that cereals can greatly affect
these processes, but there is clear evidence that failure of appressorium development on the abaxial surface of
ryegrasses (Lolium spp) relates to leaf surface characteristics and probably to surface wax components [5] that
are disruptive. However, in certain Lolium lines carrying a fragment of chromosome introgressed from a genotype
of Festuca pratensis, the abaxial leaf surface supports normal appressorium formation. This characteristic
appears linked to another which interferes with leaf senescence to convey a 'stay green' phenotype. This and
other plant material generated by the IGER Molecular and Applied Genetics group (BBSRC-funded) provided a
resource for the studies.
MATERIALS AND METHODS
A number of genotypes of Lolium perenne (perennial ryegrass), L. multiflorum (Italian ryegrass) and L.
temulentum bearing a segment of a chromosome carrying a stay-green gene from Festuca pratensis (Meadow
fescue) were examined for powdery mildew resistance in glasshouse tests and under controlled conditions. Light
microscopy [1] was used to determine genotypic effects on germling development.
Leaf wax consist of a mix of long-chain (20-40 carbons), typically being mainly a mixture of primary alcohols,
aldehydes, fatty acids, alkanes and esters. In order to identify components that promote or inhibit powdery mildew
germling development, it would be most useful to test fungal behaviour on waxes or their individual components
applied to artificial substrata remote from the influences of other plant factors. To explore this possibility,
SID 5 (Rev. 3/06)
Page 12 of 24
epicuticular waxes were collected from oat leaves, by dipping in chloroform, and by collection with a chloroformsoaked cotton bud, from the adaxial and abaxial surface of L. multiflorum (Bb2364-94) where powdery mildew
germling development is impaired on the abaxial leaf surface, and from an L. temulentum line carrying an F.
pratensis segment conferring the ‘stay-green’ phenotype (cv. Ceres) and where the adaxial leaf surface supports
a relatively high frequency of normal germling development. The chloroform solutions were dried, the different
waxes were redissolved in di-ethyl ether and drops (16 μL, containing ca. 0.9 mg ml-1 of wax) were placed either
on clean glass or on cellulose acetate film laid on 2% agar. The solvent was evaporated and surfaces were
inoculated and incubated in darkness for 17 h at 20ºC, 100% RH before 100 conidia were examined on each
surface.
RESULTS AND DISCUSSION
In planta: Confirming preliminary data, where the F. pratensis segment was present in both Lolium
chromosomes, powdery mildew conidia germinated and frequently formed normal functional appressoria and
successful infections on the abaxial (lower) leaf surface. In contrast, the abaxial surface of wild-type Lolium lines
(lacking the segment) remained mildew free because normal appressoria formed rarely. This supported the view
that the F. pratensis segment carries genetic factor(s) that negate the inherent, durable resistance of Lolium’s
abaxial surface. Until recently, the factor(s) contributing to this mildew susceptibility had been inseparable from
the stay-green factor, raising the question of whether they were the same. However, as part of a separate IGER
programme, progeny of an introgression arose that lacked the stay-green factor but were mildew susceptible on
the abaxial leaf surface. This indicated that the two factors are independent. A mapping population was available
to progress this study, offering the possibility of identifying the plant gene(s) that condition resistance of Lolium
abaxial leaf surfaces.
Nine Lolium multiflorum (cv. Meribel) lines carrying different length fragments of a chromosome segment carrying
the ‘stay-green’ character from F. pratensis genotype Bf993/17 were examined. The L. multiflorum parent was
mildew resistant on its abaxial surface, while the stay-green parent was susceptible. Of the nine lines, four were
susceptible on both surfaces, two showed limited symptoms and three were completely resistant. However, the
data were equivocal because resistance did not relate to the size of the introgressed segment. Unfortunately,
these clones showed many characteristics of L. multiflorum, including a short lifespan. To maintain them
necessitated repeated re-potting of young vegetative tillers and growth under short-day conditions; nevertheless,
plants appeared stressed and ran quickly to head. The poor health of these plants may have affected their ability
to produce leaf surface wax and therefore to influence mildew germling development. Attempts to propagate
further clonal descendents to repeat the study failed because plants died and it was impossible to complete the
planned study and, therefore, to add any reliable information regarding the genetic control of abaxial leaf surfacemildew resistance to the ryegrass genetic map. To progress this work further would require the generation of new
introgression lines based on a parental line with a perennial habit.
A different population derived from L. perenne x F. pratensis (not stay-green), likely to be perennial, and
containing chromosome substitution lines for each of the seven F. pratensis chromosomes, became available.
Unsurprisingly, both parents proved resistant to B. graminis on their abaxial surface because they do not carry
the ‘stay green’ gene which has been associated with abaxial susceptibility. However, in a glasshouse test, the
line carrying chromosome 3 was unique in that it alone developed some powdery mildew on its abaxial surface.
Twelve back-cross (generation 2) lines derived from this substitution line were also tested, each being known to
carry a segment of the F. pratensis chromosome 3 introgressed into the homeologous L. perenne chromosome.
One of these (BX 462/920) also showed some mildew susceptibility on the abaxial leaf surface. Detailed
histological analyses of the chromosome substitution lines were therefore undertaken using replicate leaves
taken from four similar tillers inoculated and incubated under controlled conditions. The data indicated that the
chromosome 3 substitution line did not differ significantly from any other substitution line in that a very low
percentage of fungal germlings formed normal appressoria on the abaxial leaf surface of all lines (range = ca 8%23% [angle-transformed = 16.6-29.1]; chromosome 3 line = 18%); this was significantly (P<0.001) lower than the
percentage of normal appressoria formed on their adaxial surfaces (range = 92%-99% [angle-transformed = 73.984.4]; overall s.e.d. = 5.74). Furthermore, no appressoria penetrated successfully to form haustoria on the abaxial
surface of any line, whereas many did so on the adaxial surface (range 33%-67%). This controlled experiment
therefore indicates no effect of F. pratensis chromosome 3 on resistance of the abaxial leaf surface and suggests
that the earlier observations were perhaps due to leaf waxes being damaged in the glasshouse.
In vitro: When conidia were inoculated onto glass coated with leaf wax, almost all died immediately after forming
a single short germ tube irrespective of the source of wax. A pool of liquid was often evident around the tip of the
germ tube indicating that primary gem tubes had ruptured had ruptured and extruded the spore cytoplasm. Thus,
use of wax on was unsuitable.
SID 5 (Rev. 3/06)
Page 13 of 24
Table 8.4.1. Developmental stages reached by powdery mildew germlings (% angle transformed) incubated on cellulose acetate
(controls) and on epicuticular waxes collected from oat, or from the ad- or abaxial surface of L. temulentum cv. Ceres (stay green)
and L. multiflorum line Bb2364-94. Means based on 100 germinated conidia on each of four replicates per treatment.
Treatment
Two germ tubes
formed
Development of germlings
Long germ tube
formed
Appressorium
differentiated
Control
70.4
55.5
42.5
Oat wax
71.9
65.2
58.5
cv. Ceres adaxial wax
75.2
64.4
59.1
cv. Ceres abaxial wax
62.1
50.8
42.1
L. multiflorum adaxial wax
70.2
66.4
62.3
L. multiflorum abaxial wax
L.S.D. [5%]
L.S.D. [5%]
73.0
59.9
52.6
9.2
6.2
5.4
When conidia germinated on wax deposited on cellulose acetate membrane overlying agar, Table 8.4.1 shows
that a reasonably high proportion formed two germ tubes, and while many of these formed an elongated tube,
less than 63% eventually differentiated an appressorium, even on wax from susceptible surfaces (oat or the
adaxial surface of grasses). Furthermore, the lowest frequency of appressorium differentiation occurred on
abaxial wax from cv. Ceres, the stay green line, whereas in planta observations showed a high proportion of
apparently normal appressoria on its abaxial surface (ca 81%) compared to wild type Lolium (ca 26%). These
contradictory findings suggest that the extraction and deposition of wax from solvents produces unreliable results,
possibly because the chemical components become redistributed unnaturally. The ability to generate an axenic
system for the bioassay of leaf waxes remains a valuable objective, but this apparently cannot be achieved
simply.
ADDITIONAL STUDIES
Published work attracted the attention of Prof. M Riederer (Department of Botany, Universität Würzburg,
Germany) a world authority on leaf wax, and this led to a collaboration studying effects of pea leaf wax
constitution on germling development by the pea powdery mildew fungus. The results were published and the
reference and an abstract are given below.
Gniwotta F, Vogg g, Gartmann V, Carver TLW, Markus Riederer M, Jetter R. 2005. What do microbes
encounter at the plant surface? Chemical composition of Pisum sativum leaf cuticular waxes. Plant Physiol. 139,
519-530.
In the cuticular wax mixtures from leaves of Pisum sativum cvs (cvs) Avanta, Lincoln and Maiperle more than 70
individual compounds were identified. They comprised unbranched C25–C28 alkanes, C24 –C32 primary alcohols,
C24 –C32 aldehydes, C22–C30 fatty acids, and C40–C50 esters containing C14–C24 fatty acids condensed mainly with
C26 primary alcohol. Additionally, small amounts of the secondary alcohols hentriacontan-16-ol, -15-ol and -14-ol
as well as nonacosan-13-ol, -14-ol, and -15-ol were detected. The cuticular wax coverage on the adaxial leaf side
of all three cvs was significantly lower than on the abaxial surface. The adaxial wax was characterized by very
high amounts of primary alcohols (71%), while the abaxial wax consisted mainly of alkanes (73%). An aqueous
glue of gum arabic was employed to selectively sample the epicuticular wax layer on pea leaves, and hence to
analyze the composition of epicuticular crystals exposed at the outermost surface of leaves. The epicuticular layer
was found to contain 74% and 83% of the total wax on adaxial and abaxial surfaces, respectively. The plateletshaped crystals on the adaxial leaf surface consisted of a mixture dominated by hexacosanol, accompanied by
substantial amounts of octacosanol and hentriacontane. In contrast, the ribbon-shaped wax crystals on the
abaxial surface consisted mainly of hentriacontane (63%), with ca. 5% each of hexacosanol and octacosanol
being present. Based on this detailed chemical analysis of the wax exposed at the leaf surface, their importance
for early events in the interaction with host-specific pathogenic fungi can now be evaluated. On adaxial surfaces
approximately 80% of Erysiphe pisi spores germinated, and 70% differentiated appressoria. In contrast,
significantly lower germination efficiencies (57%) and appressoria formation rates (49%) were found for abaxial
surfaces. In conclusion, the influence of the physical structure and the chemical composition of the host surface,
and especially of epicuticular leaf waxes, on the pre-penetration processes of biotrophic fungi is discussed.
Much of the current and past work was reviewed in recent book chapters:
Kunoh H, Carver TLW, Thomas BJ, Fujita K, Meguro A, Wright AJ. 2004. The extracellular matrix of conidia
of powdery mildew fungi: Its functions and involvement in information exchange with host cells. In: Genomic and
Genetic Analysis of Plant Parasitism and Defense. Eds: Tsuyumu, Leach, Shiraishi, Wolpert. APS press, MN,
USA, pp. 150-163.
Carver, TLW & Gurr SJ (2006). Filamentous fungi on plant surfaces. In: Biology of the Plant Cuticle. Annual
Plant Reviews 23. Ed. Riederer. Blackwell, Oxford, pp. 368-397
REFERENCES
1. Carver TLW, Ingerson SM. 1987. Physiol. Mol. Plant Pathol. 30, 359-372.
2. Wright AJ et al. 2000. Physiol. Mol. Plant Pathol. 57, 281-301.
SID 5 (Rev. 3/06)
Page 14 of 24
3. Wright AJ et al. 2002. Physiol. Mol. Plant Pathol. 61, 163-178.
4. Wright AJ et al. 2002. Physiol. Mol. Plant Pathol. 61, 217-226.
5. Carver TLW et al. 1990. Physiol. Mol. Plant Pathol. 39, 573-583.
------------------------------------------------------8.5. Determine consequences of expression of disease resistance for plant stomatal function. (Proposal
Objective 07) All milestones completed.
INTRODUCTION
With Defra’s agreement, this objective was introduced in April 2005 as a result of the observation that nitric oxide
(NO) accumulated in stomatal complexes adjacent to powdery-mildew attacked epidermal cells. Since NO is
known to play a role in causing stomatal closure, this implied that the expression of resistance to powdery mildew
might affect stomatal function with obvious and serious consequences for gas exchange and plant growth.
Initial work [1] has been published and since the paper gives all details of its relevance, context, methods used
and results, only the summary is presented. [NB. Only Carver and Thomas were funded by Defra so inputs by
others gave value added from other funding sources.]
[1] Prats E, Gay AP, Mur LAJ, Thomas BJ, Carver TLW. (2006) Stomatal lock-open, a consequence of
epidermal cell death, follows transient suppression of stomatal opening in barley attacked by Blumeria graminis.
J. Exp. Bot. 57, 2211-2226.
Blumeria graminis f.sp. hordei (Bgh) attack disrupted stomatal behaviour, and hence leaf water conductance (gl),
in barley genotypes Pallas and Risø-S (susceptible), P01 (with Mla1 conditioning a hypersensitive response, HR),
and P22 and Risø-R (with mlo5 conditioning papilla-based penetration resistance). Inoculation caused some
stomatal closure well before the fungus attempted infection. Coinciding with epidermal cell penetration, stomatal
opening in light was also impeded although stomata of susceptible and mlo5 lines remained largely able to close
in darkness. Following infection, in susceptible lines stomata closed in darkness but opening in light was
persistently impeded. In Risø-R, stomata recovered near-full function by around 30 h after inoculation, i.e. after
penetration resistance was accomplished. In P01 stomata became locked open and unable to close in darkness
shortly after epidermal cells died due to HR. In the P22 background, mlo5 penetration resistance was often
followed by consequential death of attacked cells, and here too stomata became locked open, but not until ca 24
h after pathogen attack had ceased. The influence of epidermal cell death was localised, and only affected
stomata within one or two cells distance. These stomata were unable to close not only in darkness but also after
application of abscisic acid and in wilted leaves suffering drought. Thus, resistance to Bgh based on HR or
associated with cell death may have previously unsuspected negative consequences for the physiological health
of apparently ‘disease free’ plants. The results are discussed in relation to the control of stomatal aperture in
barley by epidermal cells.
NB. The Editor-in-Chief of the journal Plant Signaling & Behavior has invited us to write an addendum
(commentary) on this work noting that such contributions are solicited for ‘the most significant recent and
forthcoming papers, published elsewhere, to provide a short summary with additional insights, new interpretations
or speculation on the
relevant topic’.
The finding that HR to B. graminis controlled by the Mla1 allele caused permanent stomatal lock-open raised the
question of the generality of this effect in relation to HR conditioned by other alleles and loci. This was examined
in a series of studies that are in prep. for publication. The approaches were similar to those used in [1] and the
summary of the submitted work is given as reference [2].
[2] Prats E, Gay AP, Roberts PC, Thomas BJ, Sanderson R, Paveley ND, Lyngkjær MF, Carver TLW, Mur
LAJ. (submitted). Temporal and spatial relationships between stomatal dysfunction and cell death due to
different single gene resistances and non-host resistance in barley attacked by Blumeria graminis. J. Exp. Bot.
Early, non-specific responses to Blumeria graminis f.sp. hordei decreased leaf water conductance (gl) in various
barley isolines, and, later, established infection of a suscept reduced light gl although dark gl was unaffected until
leaves senesced. Ultimately, isolines with Mla1, Mla3 or MlLa resistance all showed abnormally high dark gl. This
followed execution of hypersensitive responses (HR) which caused nearby stomata to lock open. Although all
resistant lines eventually gave a high frequency of HR, its spatiotemporal expression was unique to each gene.
Rapid HR in Mla1, mostly limited to single epidermal cells, arrested fungal growth before colonies initiated
secondary attacks. With Mla3, mesophyll HR preceded delayed HR of epidermal cells whose initial survival
supported spreading colonies that produced secondary infections. With MlLa, mesophyll survived but attacked
epidermal cells showed contrasting behaviour: some died immediately while others survived infection supporting
colony growth and secondary infection until they eventually died. The timing and extent of increased dark gl
varied between barley lines according to the timing and frequency of HR and consequential stomatal lock-open.
Lock-open also followed single epidermal cell HR caused by the non-pathogen B. graminis f.sp. avenae, although
here barley genotype affected neither the expression of HR, nor the timing or extent of increased dark gl.
Although chloroplast autofluorescence indicated that guard cells of locked open stomata were alive, they were
unable close in response to either drought or abscisic acid. The data support the view that HR-based resistance
carries a potential cost due to consequential stomatal dysfunction.
SID 5 (Rev. 3/06)
Page 15 of 24
ADDITIONAL STUDIES
Effects of brown rust resistance in barley and wheat
The studies of powdery mildew interactions suggested that major (R) gene resistance associated with HR cell
death has a common consequence of loss of stomatal function that has severe implications even if the attacked
plants appear disease free. It was important to determine if R gene resistance in other cereal-pathogen systems
had similar consequences. Barley and wheat brown rust interactions were considered in separate trials.
Methods. Following experimental designs similar to [1], barley plants were grown to full expansion of their first
leaf under standard conditions of 70% RH and under 12 h light (10:00-22:00 at 450 umol m -2 sec-1) at 20˚C and
12 h dark (22:00-10:00) at 15ºC. In wheat, second leaves were used because first leaves were too narrow for
porometer measurements. Uredospores (100 mm-2) of the barley brown rust fungus, Puccinia hordei (race octal
BRS 273, isolate 03-23), were applied to the first leaf adaxial surface of three barley cvs carrying different R
genes. In prior tests (ERL Jones, pers. comm.) cv. Gold was fully susceptible while resistance was shown by cvs
Estate (IT = 0c; R gene Rph 3) and Cebeda Capa (IT = 0n; R gene Rph 7). Similarly spores of wheat brown rust,
P. triticina (syn. P recondita) isolate WBRS-04-02, were applied to second formed leaves of three wheat isolines
with different R genes. In prior tests (ERL Jones, pers. comm.) cv. Thatcher was fully susceptible, while
resistance was shown by Lr20 (IT = 0c,n) and Lr24 (IT = mainly 0c but with a few small pustules). After
inoculation, all plants, including uninoculated controls, were held for 16 h in darkness under mist at 15˚C before
being returned to standard conditions. Leaf water conductance (gl) was measured by porometer in the middle of
light periods and 1 h before the end of dark periods for several days after inoculation.
Results. Diurnal patterns of change in gl in healthy and inoculated barley and wheat lines are shown in Figures
8.5.1 and 8.5.2, respectively.
Gold
600
NS
400
NS
NS
NS
NS
NS
*
NS
NS
NS
0
(mmol m-2 sec-1)
Leaf water conductance
200
Estate
600
400
200
0
*
Cebeda Capa
600
400
200
NS
0
24
41
48
65
72
89
96
113
120
137
144
161
Hours after inoculation
Fig. 8.5.1. Leaf water conductance in healthy (O) and P. hordei attacked (▲) leaves of barley cvs Gold
(susceptible), Estate and Cebeda Capa in successive light (unshaded) and dark periods (grey shaded) after
inoculation. Comparing within sample times: NS = no significant difference; * = P<0.05; all others P< 0.001.
600
Thatcher
400
Leaf water conductance
(mmol m-2 sec-1)
200
NS
NS
0
Lr20
600
400
**
200
0
*
**
Lr24
600
**
400
200
NS
NS
24
41
48
65
72
89
96
113
120
137
*
*
0
144
161
168
185
*
192
209
216
Hours after inoculation
Fig.8.5.2. Leaf water conductance in healthy (O) and P. triticina attacked (▲) leaves of wheat lines Thatcher
(susceptible), Lr20 and Lr24 in successive light (unshaded) and dark periods (grey shaded) after inoculation.
Comparing within sample times: NS = no significant difference; * = P<0.05; ** = P<0.05; all others P< 0.001.
SID 5 (Rev. 3/06)
Page 16 of 24
All healthy plants showed high gl in light periods when stomata were open, and low gl when stomata had closed in
response to darkness. In the early stages, the gl of inoculated, susceptible plants (Gold barley, Thatcher wheat),
was substantially and significantly (P<0.001) reduced in light, indicating that endophytic infection had impeded
stomatal opening, and slightly, though often not significantly, reduced in darkness. However, when pustules
started to erupt (at 113 h.a.i. in Gold, and 161 h.a.i. in Thatcher), dark gl increased indicating loss of water via
eruption sites. Nevertheless, light gl of infected leaves never exceeded that of the healthy even when pustules
were fully erupted (144 h.a.i. in Gold, 216 h.a.i. in Thatcher), indicating that stomata remained unable to open fully
in response to light.
During the early stages of interaction, both resistant barleys showed inoculation-induced suppression of gl and
little effect on dark gl, as in cv. Gold. The same was true in Lr20 wheat, though here light gl was suppressed more
than in Thatcher. However, in all these resistant lines, where no pustules formed light gl remained low, and there
was no substantial late-stage increase in dark gl. Thus, despite lack of visible disease, expression of resistance
led to a permanent impairment of stomatal opening. A different situation was evident in Lr24. Here, reduction in
light gl was evident but less substantial than in Lr20, dark gl was little affected, and this situation did not change
with time. This indicates that stomata largely retained functionality even though expression of resistance
prevented all but very few, small pustules from forming.
Discussion. As in susceptible barley attacked by powdery mildew [1, 2, 3] and bean attacked by rust [see 3],
infection of susceptible barley and wheat by the respective brown rust fungi impaired stomatal opening, with
obvious deleterious consequences for plant growth and productivity. However, the current studies provide the first
evidence that expression of cereals’ resistance to biotrophic pathogens can also have serious consequences via
effects on stomata. In barley expressing R gene or non-host resistance to powdery mildew, stomata were so
seriously affected that they became permanently locked open and unable to respond to darkness, drought or
application of abscisic acid. By contrast, in barley and wheat attacked by avirulent rusts, the stomata could
become permanently locked shut. The reason for these opposite effects awaits explanation, but the clear
evidence for stomatal dysfunction as a consequence of the expression of all these resistances points to a
previously unsuspected factor contributing to the ‘cost of resistance’ which has been recognised but evaded good
explanation [4, 5]. Thus, the work highlights serious implications of using such resistances in field crops where
stomatal dysfunction would interfere with photosynthesis, respiration, ability to cope with abiotic stresses, and,
importantly in the context of environmental change, water use efficiency. For sustainable production systems
where powdery mildew is a threat, a good alternative is offered by broad spectrum, papilla-based resistance,
which leads to only transient impairment of stomatal opening [1]. The current work showed, however, that some R
gene resistances may cause little stomatal dysfunction. None were yet identified for powdery mildew resistance
[2] but the wheat brown rust resistance of Lr24 is promising. The reason for this remains unknown but deserves
investigation to facilitate breeding for improved resistance phenotype. In addition, more extensive physiological
and field studies are required to explore the implications of HR for costs of resistance. Towards this requirement,
and informed by established collaboration between Carver and ND Pavely (Defra Fellowships AR0712 and
AR0511), a recent proposal to Defra, led by Pavely and involving Dr J. Foulkes (University of Nottingham),
includes work aimed at identifying strategies to minimise the impact of disease on water use efficiency, by
quantifying the trade off between water and dry matter loss due to disease, and the benefits and costs of water
use of disease resistance.
Additional References
3. Ayres PG. 1981. Soc. Exp. Biol. Seminar Series 8 Stomatal Physiology. Eds Jarvis PG, Mansfield TA.
Cambridge University Press, 205-222.
4. Purrington CB. 2000. Curr. Opin. Plant Biol. 3, 305-308.
5. Brown JKM. 2002. Curr. Opin. Plant Biol. 5, 339-344.
------------------------------------------------------8.6. Characterisation of a collection of wheat varieties for durable resistance against current populations
of the wheat yellow rust pathogen carrying complex virulences. (Proposal Objective 5). All milestones were
completed.
INTRODUCTION
Yellow rust of wheat, caused by Puccinia striiformis f.sp. tritici (Pst), is a major disease of UK wheat [1] controlled
through a combination of resistance gene deployment and fungicide application. Many sources of yellow rust
resistance have proved race-specific, and thus ephemeral. To support sustainable wheat production it is
increasingly important to identify and genetically characterise durable sources of resistance [2]. Dr Roy Johnson
identified a number of wheat cvs which appeared to retain good field resistance to yellow rust despite being
grown over extensive acreages and years (Durable Set; Table 8.6.1) and therefore met the criteria he defined [3,
4] for ‘durability’ of resistance. However, the genetic and mechanistic basis of these durable resistances was
never tested. The objective of this study was to retest the Durable Set against modern Pst isolates with complex
virulence under different environmental conditions and to generate materials for a genetic study of durable yellow
rust resistance.
SID 5 (Rev. 3/06)
Page 17 of 24
MATERIALS AND METHODS
Field trials. The wheat Durable Set (Table 8.6.1) included 21 cvs that were field tested for yellow rust resistance
in 2003/2004 at New Found Farm, Norwich, and again in 2004/2005 at Church Farm, Norwich and at Osgodby,
Market Rasen, Lincolnshire. In each trial three replicates were sown in a completely randomised design,
containing 10 plants of each cultivar per row. Yellow rust was introduced to sites on spreader plants of the cv.
Lemhi. These were infected with one of four Pst isolates (Table 8.6.2) collected from wheat crops within the UK in
recent years. Isolates were multiplied under containment, and their virulence genotypes confirmed using the
wheat World and European differential sets for yellow rust virulence identification, and the AvocetS near-isogenic
lines carrying known yellow rust, seedling expressed resistance genes (Yr R-genes; [5]). The tests used standard
protocols [6, 7] and reactions were measured using a qualitative, Infection Type scale [8]. In the field, yellow rust
infection was measured on two dates during May/June using the modified Cobb scale as a quantitative measure
and a qualitative score of necrosis and chlorosis to assess plant response (Table 8.6.1; [7, 9, 10].
Greenhouse tests of adult plant resistance to yellow rust. Five cvs from the Durable Set were selected for a
greenhouse study of the adult plant yellow rust infection phenotypes (Table 8.6.1). Ten plants of each cv. were
grown to first ear emergence under containment conditions and then simultaneously inoculated with a 1:1:1:1
mixture of the four Pst isolates (Table 8.6.2). To assess timing of yellow rust development, plants were examined
daily from 10 days after inoculation (dai) and the time of appearance of fully sporulating pustules on flag leaves
was recorded. To assess urediniospores production per pustule, segments of flag leaves (2cm long and 2
segments per plant) bearing pustules were cut at 20 dai. The approximate number of pustules on the segment
was assessed and urediniospores were collected from each leaf segment and counted [6]. An estimate of the
number of urediniospres produced per pustule was calculated for each leaf segment, and a mean taken to
represent each cultivar. Analysis of variance and general linear modelling were applied using Genstat for
Windows, v.8.0. Comparisons were considered significant at F- and t-value P <0.001.
Crosses made between wheat cvs showing durable yellow rust resistance. Three plants of cvs CarstensV
and Cappelle Desprez were crossed to cv. Lemhi as the pollen parent, to give F1 seed. Ten F1 plants from each
cross were selfed to give F2 seed. The known, durable sources of yellow rust resistance, Yr18 (Avocet S*6/Yr18),
Yr29 (Avocet S*6/Yr29) and Yr30 (Pavon) were crossed to the yellow rust susceptible UK wheat cvs Brigadier,
Glasgow and Robigus to give F1 seed.
RESULTS
Field trials. Of the 21 wheat cvs comprising the Durable Set, five remained immune (Bersee; Elite Lepeuple;
Flinor; Luke and Widgeon), 12 had partial resistance and four were susceptible (Anza; Bezostaya; Bouquet,
Champlain) (Table 8.6.1). Similar yellow rust reactions were seen on all 21 cvs at the two sites near Norwich, in
2004 and 2005 (Table 8.6.1). At the field trial site near Market Rasen, Lincolnshire, all cvs supported more yellow
rust infection than at the sites near Norwich, and previously resistant cvs Bouquet and Champlain were
susceptible (Table 8.6.1).
Yellow rust greenhouse, adult plant tests. The five cvs were selected for greenhouse, adult plant tests on the
basis of yellow rust infection phenotypes seen in field trials (Table 8.6.1) and their susceptibility to the Pst isolates
at the seedling growth stage. Yellow rust pustules developed on all five cvs, although fewer were seen on the
three more resistant cvs, Bersee, Carstens V and Little Joss. There was no significant difference between cvs
with regards to the timing of the first appearance of pustules (18-20 days in all cases). In general, cvs Bersee,
Carstens V and Little Joss produced fewer urediniospores per pustule than Cappelle Desprez and Vilmorin 27.
Crosses made using wheat cvs showing durable yellow rust resistance. CarstensV and Cappelle Desprez
have been crossed to the yellow rust susceptible wheat cv. Lemhi to give F2 seed. Crosses have also been made
between wheat cvs carrying known sources of durable yellow rust resistance, i.e. resistance genes Yr18, Yr29
and Yr30 and the yellow rust susceptible UK commercial wheat cvs Brigadier, Robigus and Glasgow. F1 seed is
available for these crosses.
DISCUSSION
Reassessment of the field resistance to yellow rust in the wheat cultivars of the Durable Set identified four cvs,
Anza, Bezostaya, Bouquet and Champlain, in which yellow rust resistance had been lost. The remaining 17 cvs
retained varying degrees of resistance towards yellow rust, indicating that the durability of the resistance in these
cvs had been maintained. These cvs represent a potential source of useful, durable field resistance for yellow rust
that can be incorporated into modern, commercial UK wheat cvs.
The test of time is the only current means of identifying durable sources of disease resistance, and four cvs had
failed this test. Genetic assessment of the remaining cvs would provide information on the nature and number of
genes responsible for their durable yellow rust resistance, and thereby the ease with which this resistance could
be transferred into new wheat cvs. However, it is clearly important to determine how durable resistance can be
recognised, and studies of the component phenotypes of pathogen infection may identify micro-phenotypes that
provide indicators of the potential durability of a source of resistance.
SID 5 (Rev. 3/06)
Page 18 of 24
Table 8.6.1. Wheat cultivars constituting the Durable Set and their field responses 2003-2005 to a mixture of yellow rust isolates carrying
complex virulence.
Wheat cultivar
2003/04
NF Farm, Norwich
60S
0R
0R
80S
0R
10MR
2004/05
Church Farm, Norwich
50S
0R
0R
70S
0R
5MR
2004/05
Lincolnshire
60S
25MR/MS
1MR/R
25MR
60MS/MR
10MR
Anza
Atou
Berseea
Bezostaya
Bouquet
Cappelle
Despreza,b
Carstens Va
0R
0R
10MR
Champlain
10R
10MR
80S
Elite Lepeuple
0R
0R
2MR
Flander
0R
10MR
3MR
Flinor
5R
5R
1MR
Holdfast
5R
5R
15MR
Hybrid 46
20R
10-20MR
30MS
Huntsman
0R
10MR
3MR
Little Jossa,b
0R
0R
10MR
Luke
5R
5R
very necrotic
Nugaines
50R
80S
20MS
Starke II
25-30MR
20MR
20MR
Vilmorin 27a,b
20MR
20MR
5MR
Widgeon
0R
10R
5MR
Yeoman
5R
10R
15MR
Lemhi
80S
80S
90S
a
cvs selected for greenhouse, adult plant yellow rust infection test.
b
cvs selected to test the effect of light quanta on yellow rust infection efficiency (see objective 6)
R – Resistance; no visible pustule formation
MR- Moderate resistance; pustules surrounded by necrotic tissue
MS – Moderate susceptible; pustules surrounded by chlorotic tissue
S - Susceptible; pustules surrounded by green tissue.
Notes
susceptible
chlorotic flecks
chlorotic flecks
susceptible
chlorotic flecks
partial resistance
susceptible
chlorotic flecks
extensive chlorosis
chlorosis
temp sensitive R
partial resistance
partial resistance
susceptible control
Table 8.6.2. Puccinia striiformis f.sp. tritici isolates used in yellow rust disease tests.
UK Pst isolate
WYR 96/502
WYR 95/12
WYR 93/24
WYR 90/505
Year of first collection
1996
1995
1993
1990
Virulent on Yr R-genes
1,2,3,6,9,17
1,2,3,4,9,13,17
1,2,3,4,6,CV,13,14
1,2,3,4,7,14
Virulent on wheat cvs
Madrigal; Equinox
Brigadier
Hereward
Brock
REFERENCES
1. Boyd LA. 2005. J. Ag. Sci. 143, 233-243
2. Boyd LA. 2006. J. Sci. Food Agric. 86, 2523-2526
3. Johnson R. 1981. Phytopathol. 71, 567-568
4. Johnson R. 1988. Durable resistance to yellow (stripe) rust in wheat and its implications in plant breeding. In:
Breeding Strategies for Resistance to the Rusts of Wheat. Eds. NW Simmonds & S Rajaram, Mexico:CIMMYT,
pp. 63-75..
5 Lewis CM. 2006. The genetic basis of Puccinia striiformis resistance in UK wheat germplasm. PhD thesis,
Faculty of Biological Sciences, University of East Anglia.
6. Boyd LA, Minchin PN. 2001. Euphytica 122, 361-368.
7. Smith PH et al. 2004. Mol. Plant-Micr. Inter. 17, 1242-1249
8. Rodrigues P. 2004. Theor. Appl. Genet.109, 425-432.
9. Boyd LA, et al. 2002. Genome 45, 1035-1040.
10. Ramburan VP, et al. 2004. Theor. Appl. Genet. 108, 1426-1433.
------------------------------------------------------8.7. Define the effects of pre-inoculation lighting treatments on predisposition of wheat to infection by
yellow rust, Puccinia striiformis f.sp. tritici (Pst). (Proposal Objective 6). All milestones were attained.
INTRODUCTION
A principal difficulty in studying Pst infection is the rarity with which germinated urediniospores locate and enter
wheat leaf stomata to establish infection. Some evidence [1] suggested that light quantity received by seedlings
prior to inoculation influenced this phenomenon. Here, therefore, microscopy was used to test rigorously this
suggestion and define the effects of pre-inoculation light input. A subsequent study examined effects of wheat
genetic variation on this light effect which may offer a new and potentially valuable form of resistance.
SID 5 (Rev. 3/06)
Page 19 of 24
MATERIALS AND METHODS
Plant material, inoculation and incubation. According to experiment, the susceptible wheat cv. Lemhi, and the
partial, adult plant resistant cvs CappelleDesprez, Vilmorin 23 and Little Joss, were used as seedling and adult
plants. Seedlings were grown for 10 d and adult plants until first ear emergence, in a light-proof compartment of a
spore-free greenhouse at 18ºC/16 h under sodium lights (300 μmol m -2 s-1) and 15oC/8 h darkness. Plants were
then subjected to various light treatments before inoculation (as published; [2]) with the virulent Pst isolate
WYR75/20 (race 232E137) and incubation in a growth cabinet at 8 oC and 95% humidity for 24 h, in darkness.
They were then returned to the greenhouse under the same conditions in which they were grown and leaves were
checked regularly for the emergence of sporulating pustules. Temperature and humidity were monitored
throughout.
Pre-inoculation light treatments. Three repeat experiments were performed following the light treatments
reported by de Vallavieille-Pope et al. [1]. Accordingly, cv. Lemhi seedlings (GS12) were transferred to a 16/8 h
cycle of low light (45 μmol m-2)/darkness, at 8oC for 3 d and then held in total darkness for 16 h at 8 oC. Seedlings
were then divided into five batches (ca. 40/batch) with one batch receiving the following pre-inoculation quantities
of light for 6 h at 10oC: 1) 0 mol m-2 (total darkness); 2) ca. 6 mol m-2 (269 umol m-2 s-1); 3) ca. 10 mol m-2 (457
umol m-2 s-1); 4) ca. 12 mol m -2 (536 umol m -2 s-1); 5) ca. 17 mol m -2 (780 umol m-2 s-1); as an additional control, a
sixth batch was maintained in the greenhouse under standard growth conditions compartment. After pretreatments, all seedlings were inoculated with Pst and after 24 h under initial incubation conditions (above), all
were removed to a glasshouse where temperature was maintained at between 20 and 22oC, with a relative
humidity of between 40 and 45%. Half the seedlings from each treatment were used to measure leaf water
conductance (gl) by cycling porometer; this reflected the extent to which the stomata were open. Remaining
seedlings were sampled for light microscopy. Porometer readings (12 readings per time point) and samples for
light microscopy (three samples per time point) were taken 2 h before inoculation, and 2, 23, 26, 48-50 and 72-74
h afterwards.
The lighting regimes used above were complex and difficult to interpret, and involved an unrealistically lengthy
dark period before inoculation. Therefore a more realistic, simplified procedure was adopted and a series of eight
experiments were carried out, altering the length of time seedlings were kept in the dark and the amount of preinoculation quanta of light received, until the conditions for a robust, repeatable assay were attained. In this
assay, seedlings of Lemhi were grown under standard greenhouse conditions for 10 d before being subjected to
three light treatments for 18 h immediately before inoculation, viz: 1) total darkness, 2) low light (ca. 13 mol m -2)
or, 3) high light (ca. 29 mol m -2). The three sets of seedlings were inoculated simultaneously with equal densities
of spores and then all three sets of seedlings were subjected to the same light regime. For each set of seedlings
the stomatal status was determined by measuring gl 1 hour before inoculation and at various times up to 74 h
afterwards.
In subsequent experiments using this assay procedure, effects of pre-inoculation light treatments were studied in
seedling and adult plants of three wheat cvs from the Durable Set: Vilmorin 27, Little Joss and Cappelle Despez,
(see Proposal Objective 5). Here again, plants were placed either in 1) total darkness, 2) low light (ca. 13 mol m -2)
or, 3) high light (ca. 29 mol m -2) for up to 18 h before inoculation. From each light treatment 12 plants were used
for porometry and 24 for microscopy. Porometer readings and samples for microscopy (4 samples/time point)
were taken 1 h before inoculation and 2, 23, 26, 48-50 and 72-74 h afterwards.
Light Microscopy. According to experiment, leaf segments (ca. 2 cm) cut from the first leaves (seedling) or flag
and boot leaves (adult plants) were prepared for light microscopy as described by [3]. The following observations
were made on each leaf segment: total numbers of: 1) Pst spores germinated (germlings); 2) germlings where the
germ tube had grown over a stomatal opening; 3) germlings where the germ tube had entered a stomatal
opening; 4) germlings that had entered a stomate and formed sub-stomatal vesicles (SSV). Data were subjected
to analysis of variance and general linear modelling using Genstat for Windows, v.8.0. Comparisons were
considered significant at F- and t-value P<0.001.
RESULTS
Effects of pre-inoculation light treatment on seedlings of susceptible wheat cv. Lemhi. With the preinoculation light treatments used by de Vallavieille-Pope et al. [1], on Lemhi seedlings exposed pre-inoculation to
prolonged darkness, no germlings had entered stomata by 72 h. When exposed to low quanta of light preinoculation, some germlings succeeded in entering stomata SSVs, but the numbers were significantly lower than
where seedlings had been exposed to high quanta of light. Porometry indicated that, in seedlings exposed to
prolonged pre-inoculation darkness, the normal diurnal opening of stomata had been compromised and might
explain the failure of stomatal entry. This may have been because the pre-inoculation dark period used for this
treatment was unrealistically long.
Interestingly, using the simplified light regimes, porometry showed no differences in post-inoculation gl profiles
between seedlings exposed to different pre-inoculation light treatments. Thus, in this more realistic system the
normal diurnal movements of stomata had not been compromised by the different, pre-inoculation light
treatments. Nevertheless, the pre-inoculation light treatments had a significant effect on Pst development and
infection efficiency. This is illustrated in Figure 8.7.1 which shows the numbers of urediospores/germlings that had
attained different developmental stages at each sample time. In general, the higher the quanta of pre-inoculation
light received by a seedling, the larger the proportion of germinated spores observed (Fig.8.7.1; treatment F
value=24.28; P<0.001). By 23 h (when seedlings were still in the incubation chamber at 8 oC in darkness) some
SID 5 (Rev. 3/06)
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urediospores had formed germ tubes on seedlings that had received high, and to a lesser extent low, quanta of
light prior to inoculation and even by this early stage some had entered stomata and differentiated a sub-stomatal
vesicles (SSV). By contrast, on leaves kept in darkness pre-inoculation few spores were present (presumably
indicating loss of ungerminated spores during preparation) and none had germinated. After seedlings had been
returned to the greenhouse, and had been under the normal light regime for 2 or more h (26-74 h samples) a few
urediospores on the dark treatment seedling had germinated, but very few ever entered stomata. By contrast,
both pre-inoculation light treatments led to a number of germlings entered stomates relatively rapidly and formed
SSVs.
Fig. 8.7.1. Temporal development of Puccinia striiformis f.sp tritici germlings on seedlings of wheat cv Lemhi inoculated after pre-inoculation
treatments with darkness (D), low light quanta (L; ca. 13 mol m-2) or high light quanta (H; ca. 29 mol m-2).
Measured as a proportion of total germlings, however, most formed SSVs in seedlings which had received the
highest quanta of light pre-inoculation (Fig. 8.7.1; treatment F value=13.61, P<0.001). This procedure showed,
therefore, that since treatment had not affected stomatal movement some other effect of light must have affected
the ability of Pst to locate and enter a stomatal cavity.
Effects of pre-inoculation light treatment in seedling and adult plants of wheat cvs with field resistance.
The same simplified light regimes were then used to compare seedlings of wheat cvs Vilmorin 27, Little Joss and
Cappelle Desprez, against Lemhi as the susceptible control. Overall, the quanta of pre-inoculation light received
by seedlings of all cvs affected both germination and infection efficiency. As seen previously, high quanta of light
again increased spore germination on Lemhi and also Cappelle Desprez, but no such effect was evident in
Vilmorin 27 or Little Joss. Nevertheless, the overall effect of high light quanta on enhancing stomatal entry and
SSV formation was significant. It was most pronounced on cv Little Joss, followed by Cappelle Desprez and
Lemhi. Importantly, however, no significant effect on stomatal entry or SSV formation was observed on Vilmorin
27.
The results obtained when the pre-inoculation light regimes were applied adult plants provided surprising results
that are hard to interpret. As in seedlings, spore germination was significantly higher on plants exposed to the
highest quanta of light (ca. 29 mol m -2) compared to low light quanta (ca. 13 mol m-2), but the highest levels of
germination were observed on plants receiving the dark treatment and the controls kept in the greenhouse under
normal light conditions throughout. No significant effect of the light treatments was seen on germ tube entry of
stomata and SSV formation. Further work, beyond the scope of the current project, will be required to determine
why the effect of high light quanta seen in seedlings was not observed with adult plants.
DISCUSSION
A unique effect of light quanta on the receptivity of wheat seedlings to infection by the causal agent of yellow rust
has been characterised. Both urediniospore germination, and the ability of the pathogen to establish a successful
infection site are increased on seedlings having received high quanta of light prior to pathogen inoculation. The
effect of light is not due to physiological dysfunction, but appears to bring about a physiological change that
increases the levels of a plant factor used by the fungus to locate and enter stomata. In wheat seedlings the data
indicate that genetic variation influences this effect of light and genotypes that do not show the effect may express
a form of resistance that reduces Pst infection efficiency. Further, the work has demonstrated that by
manipulating pre-inoculation light conditions, it is possible to increase infection efficiency thus increasing the
numbers of germlings that attempt to establish biotrophic endophytic infections. This will greatly facilitate future
studies aimed at identifying resistance mechanisms acting formation of SSVs to limit colony establishment and
growth. Thus, the work undertaken here and in Proposal Objective 5 has set the foundations to identify new and
valuable sources of durable resistance to yellow rust in wheat genotypes that have maintained efficacy over many
years and that continue, therefore, to provide a resource for UK breeders.
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REFERENCES
1. De Vallavieille-Pope C, et al. 2002. Phytopathology 92, 1308-1314.
2. Boyd LA, Minchin PN. 2001. Euphytica 122, 361-368.
3.Melichar JPE. 2007. Genetic and physiological analysis of mutations in wheat showing enhanced adult plant
resistance to yellow rust. PhD thesis, Faculty of Biological Sciences, University of East Anglia.
------------------------------------------------------8.8 OVERVIEW
The reported work aimed to facilitate development of strategies for sustainable disease management using
durable resistance to key biotrophic cereal pathogens, thus allowing reduced dependence on fungicides. The oat
(8.1) and wheat (8.6) germplasm generated and characterised for powdery mildew and yellow rust resistance
provides a resource not only for immediate use by plant breeders but also for future studies aimed at identifying
the mechanistic basis of resistances and their cell biological and molecular genetic basis. Such fundamental
studies will, in turn feed back to further facilitate plant breeding for resistance that is likely to be under complex
physiological and genetic control, and therefore to offer durability. Clearly, studies of models such as the oat or
barley (8.2; 8.3; 8.5) and grass (8.4) powdery mildew systems and the wheat yellow rust (8.6; 8.7) and barley and
wheat brown rust (8.5) systems have direct relevance to efforts aimed at controlling other biotrophic pathogens
not only of the Gramineae but also of dicotyledonous plants (NB collaborations with Riederer et al. [8.4] and
publications arising with Rubiales et al.). Further, the work has generated novel methodologies that are applicable
generally to the study of cell-specific transcriptomics in plant biology (NB collaborations with Lyngkær et al. [8.4
and associated publications]). The breadth of the studies undertaken here was made possible only by the award
of the Defra Fellowship which not only funded directly the association of expertise between Carver and Boyd but
also led to close interaction with Pavely (ADAS) and important components of a research proposal from Pavely
currently under consideration by Defra. Importantly, the Fellowship also allowed continued collaboration between
Carver and many world leaders of plant biological research in the UK, throughout Europe and in the USA,
Canada and Japan.
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References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
Olesen KL, Carver TLW, Lyngkjaer MF. (2003). Fungal suppression of resistance against inappropriate B. graminis
formae speciales in barley, oat and wheat. Physiol. Mol. Plant Pathol. 62, 37-50.
Fujita K, Wright AJ, Meguro A, Kunoh H, Carver TLW. (2004). Rapid pre-germination responses of Erysiphe pisi
conidia to contact and light. J. Gen. Plant Pathol. 70, 75-84.
Gjetting T, Carver TLW, Skøt L, Lyngkjær MF. (2004). Gene expression profiling of individual barley epidermal cells
attacked by powdery mildew. Mol. Plant-Micr. Inter. 17, 729-738.
Fujita A, Suzuki T, Kunoh H, Carver TLW, Thomas BJ, Gurr SJ , Shiraishi T. (2004). Induced inaccessibility in
barley cells exposed to extracellular material released by non-pathogenic powdery mildew conidia. Physiol. Mol.
Plant Pathol. 64, 169-178.
Kunoh H, Carver TLW, Thomas BJ, Fujita F, Meguro A, Wright AJ. (2004). The Extracellular Matrix of Conidia of
Powdery Mildew Fungi: Its Functions and Involvement in Information Exchange with Host Cells. In: Genomic and
Genetic Analysis of Plant Parasitism and Defense. Eds: Shinji Tsuyumu, Jan E. Leach, Tomonori Shiraishi and
Thomas Wolpert. APS press, MN, USA. pp. 150-163.
Prats E, Mur LAJ, Sanderson R, Carver TLW. (2005). Nitric oxide contributes both to papilla-based resistance and
the hypersensitive response in barley attacked by Blumeria graminis f. sp. hordei. Mol. Plant Pathol. 6,65-78.
Gniwotta F, Vogg G, Carver TLW, Riederer M, Jetter R. (2005). What do microbes encounter at the plant surface?
Chemical composition of Pisum sativum leaf cuticular waxes. Plant Physiol. 139, 519-530.
Boyd LA. (2005) Centenary Review: Can Robigus defeat an old enemy? – Yellow rust of wheat. J. Agric. Sci. 143,
233-243.
Zhang Z, Henderson C, Perfect E, Carver TLW, Thomas BJ, Skamnioti P, Gurr SJ. (2005). Of genes and
genomes, needles and haystacks: Blumeria graminis and functionality. Mol. Plant Pathol. 6, 561-575.
Fondevilla S, Carver TLW, Moreno MT, Rubiales D. (2006). Macroscopic and histological characterisation of genes
er1 and er2 for powdery mildew resistance in pea. Eur. J. Plant Pathol. 115, 309-321.
Prats E, Carver TLW, Lyngkjær MF, Roberts PC, Zeyen RJ. (2006). Induced inaccessibility and accessibility in the
oat powdery mildew system: insights gained from use of metabolic inhibitors and silicon nutrition. Mol. Plant Pathol. 7,
47-59.
Mur LAJ, Carver TLW, Prats E. (2006) NO way to live; the various roles of nitric oxide in plant-pathogen interactions.
J. Exp. Bot. 57, 489-505
Prats E, Gay AP, Mur LAJ, Thomas BJ, Carver TLW. (2006). Stomatal lock-open, a consequence of epidermal cell
death, follows transient suppression of stomatal opening in barley attacked by Blumeria graminis. J. Exp. Bot. 57,
2211-2226.
Carver TLW, Gurr SJ (2006). Filamentous fungi on plant surfaces. In: Biology of the Plant Cuticle. Ed. Markus
Riederer. Annual Plant Reviews 23, Blackwell, Oxford. pp. 368-397.
Fondevilla S, Carver TLW, Moreno MT, Rubiales D. (Published Online, Dec 21 2006). Identification and
characterisation of sources of resistance to Erysiphe pisi Syd. in Pisum spp. Plant Breeding.
Gjetting T, Hagedorn PH, Schweizer P, Thordal-Christensen H, Carver TLW, Lyngkjær MF. (In press). SingleCell Transcript Profiling of Barley Attacked by the Powdery Mildew Fungus. Mol. Plant Micr. Inter..
Prats E, Carver TLW, Fondevilla S, Rubiales D. (2006). Cellular bases of resistance to different formae speciales of
Blumeria graminis in Hordeum chilense, wheat, and the amphiploids tritordeum and agroticum Can. J. Plant Pathol.
28, 577-587.
Boyd LA. (2006). Perspective: Can the durability of resistance be predicted? J. Sci. Food Agric. 86,2523-2526.
Olesen KL, Carver TLW, Lyngkjær MF. (submitted). Brown rust attack affects accessibility of barley epidermal cells
to powdery mildew. Mol. Plant Pathol.
Prats E, Gay AP, Roberts PC, Thomas BJ, Sanderson R, Paveley ND, Lyngkjær MF, Carver TLW, Mur LAJ. (in
prep.). Temporal and spatial relationships between stomatal dysfunction and cell death due to different single gene
resistances and non-host resistance in barley attacked by Blumeria graminis. J. Exp. Bot.
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