Promotor
:dr.ir. J. Sneep, emeritus hoogleraar indeleervan
deplantenveredeling.
Co-promotor :dr.J.C. Zadoks,hoogleraar indefytopathologie.
Ditproefschrift isbewerktonderleidingvandr.ir.J.E.Parlevliet,
wetenschappelijkhoofdmedewerker aandevakgroep Plantenveredeling.
Studies onthehistology ofpartial resistance inbarley toleaf
rust, Puccinia hordei
CENTRALELANOBOUWCATALOQUS
000000164364
^oVtfA^SL
R.E. Niks
Studies on the histology of partial resistance
in barley to leafrust, Puccinia horde/
Proefschrift
terverkrijgingvandegraadvan
doctor inde landbouwwetenschappen,
opgezagvanderectormagnificus,
dr.C.C.Oosterlee,
hoogleraar indeveeteeltwetenschap,
inhetopenbaarteverdedigen
opvrijdag18maart1983
desnamiddagstevieruurindeaula
vandeLandbouwhogeschool teWageningen.
\S\^
\8\dl-°l
Groot zijn de werken desHEREN,
na te speuren door allen die er behagen in hebben.
Majesteit en luister is zijn doen,
en zijn gerechtigheid
houdt eeuwig stand.
Psalm 111 :2,3
AMJ0??Of , 9 32STELLINGEN
1. Het feit dat compatibiliteit uitzondering is inplant-schimmel
combinaties wordt door Ward en Stoessl ten onrechte gebruikt
als argument dat niet de overgevoeligheidsreactie, maar de
compatibele reactie van eenwaardplant op een herkenningsreactie
berust.
Ward,E.W.B.£A.Stoessl (1976),Phytopathology66:940-941.
2. Parlevliet en Zadoks baseren hun bewering dat waard-pathogeen
systemen die berusten op een gen-om-gen basis in de natuur uiteindelijk een stabiel evenwicht bereiken ten onrechte op het
model van Mode.
Mode, C.J. (1958),Evolution 12: 158-165.
Parlevliet, J.E. £J.C. Zadoks (1977),Euphytica 26: 5-21.
3. Schaarste aan gegevens in de Middeleeuwse literatuur over desastreuze epidemieën in gewassen kan wijzen op een gunstig evenwicht tussen de toen geteelde landrassen en depathogeen populaties.
Orlob, G.B. (1971). Ann. Rev. Phytopath. 9: 7-20.
4. Het ontbreken van een statistisch significante cultivar x
isolaat interactie in grootheden die gebruikt worden om de mate
van resistentie te schatten, impliceert geen duurzaamheid van
de resistentievorm.
Kuhn,R.C.,H.W.OhmeG.E.Shaner (1978),Phytopathology68:651-656.
5. De opvatting dat avirulentie bij pathogenen als meeldauw en
roestschimmels een "altruïstische eigenschap" is, die de coexistentie van het pathogeen met de waard regelt, is waarschijnlijk onjuist als de waardsoort een autogaam gewas is.
Eshel, I. (1977), Theor. Popul. Biol. 11: 410-424.
Parlevliet, J.E. (1981). Proc. Plant Breeding Symp. II,Iowa State
Univ.: 309-364.
6. Bij taxonomisch onderzoek in Puccinia en Uromyces spp. dient
ook de morfologie van het mycelium, zoals de vorm van het substomatale blaasje, betrokken te worden.
Ditproefschrift.
7. Baker's conclusie dat panmixie in deF_ generatie van een zelfbevruchtend gewas de selectiemogelijkheden in de F duidelijk
vergroot, is onjuist. De resultaten van het werk waarop hij
zijn conclusie baseert, tonen aan dat panmixie op korte termijn
weinig voordeel biedt.
Baker,R.J. (1968),Crop Sei. 8: 547-550.
.v:-»;,Ï*J.Tij»;*ï
OER
8. Debewering vanLewis,datde fylogenievan hetsegmentatiepatroon van de vlieg redelijk goedbegrepen is,isniette
verifiëren.
Lewis,E.B. (1978),Nature 276:565-570.
9. Iniederpsychiatrischziekenhuisbehoorteenpsychogeriatrisch
circuit tebestaan,waarvan,naasteenkliniek,ookeenpolikliniekdeeluitmaakt.
10.Trimmeniseenhondebaan.
StellingenbijhetproefschriftvanR.E.Niks,getiteld "Studieson
thehistologyofpartialresistance inbarleytoleafrust (Puccinia
hordei)",teverdedigenop18maart1983indeAulavandeLandbouwhogeschool,Wageningen.
Woord vooraf
Bij devoltooiing van ditproefschrift wil ikgraagmijndankbaarheid totuitdrukking brengen aan allen diehebbenbijgedragen
aandetotstandkomingervan.
Inde eerste plaats ben ik dank verschuldigd aanmijnouders,
die mij op zo royale wijze in de gelegenheid hebben gesteld een
studie aandeLandbouwhogeschool tevolgen.
Mijn promotor,prof.dr.ir.J. Sneep,benikerkentelijkvoorde
mijgebodengelegenheid ditproefschrift aanhet IvPtebewerkenen
voorzijnbelangstellingvoormijnonderzoek.
Metveelgenoegendenk ikterugaandebesprekingenmetmijncopromotor,prof.dr.J.C.Zadoks. Zijn critische commentaar op mijn
rapportenenmanuscriptenwasbijzondervormend enstimulerend.
Mijnbegeleider,dr.ir.J.E.Parlevliet,dankikvoordevrijheid
diehijme liethetonderzoeknaareigeninzichtuittevoeren.Zijn
kennismaaktehemtoteenwaardevollevraagbaak enikbenhemerkentelijk voor zijn hulp bij het gereedmakenvanditmanuscriptvoor
deoffset.
ZonderdehulpvanHenkKuiperzouditwerknietalleennietmogelijk,maarookveelminderplezieriggeweestzijn.Ikhebheterg
gewaardeerd dat hijmetenthousiasme eengroteverscheidenheid aan
werkzaamheden,waaronderhonderdenurenmicroscopie,heeftverricht.
Inhet kadervanhundoctoraalstudie hebbenPhilippine Goettsch
en Erik Jongedijk aan het onderzoek meegewerkt.Hun enthousiasme
voorhetwerkdeedmedeugd.
Dr.M.S.Ramanna dank ikvoor zijn stimulerende belangstelling
en nuttige wenken m.b.t.de tekstvanenkelevandemanuscripten.
Ik mocht profiteren van zijn rijke ervaring in het cytologische
werk. Ir.lesBos gafwaardevollehulpbijdestatistischeverwerkingvandegegevens.
WoutHoogkamerenCorrieGeerdtsdankikvoorhetbeherenvande
isolaten. Tony van Ommeren verschafte gegevens over de gebruikte
isolaten.LuukSuursenJanMolenveldvoorzagenmevandebenodigde
chemicaliën, waarvoor mijn oprechtedank.DeherenA.Florissenen
B.Navestbenikerkentelijkvoordetechnischeverwerkelijkingvan
ideeëndietotdoelhaddendegebruiktemethodenteperfectioneren.
Annie Marchai ben ik dankbaar voor het uitstekend typen, niet
alleenvandemanuscripten,maarookvandetussentijdserapporten.
Ook naar Rinia Baldew van de afdelingTekstverwerking, diededefinitieve versie van hetproefschriftheeftgetypt,gaatmijndank
uit.
DeheerJ.S.deBlockcorrigeerde demanuscripten ophetEngels.
DeheerH.W. Sengersheeftopuitstekendewijze eeneindelozereeks
foto'safgedrukt.Degrafiekenwerdengetekend doordeheerW.C.T.
Middelplaats (p.23)en doordeheerW.D.J.vanderVliet (p.35).
De heer A.M.Vroegop en collega's van de offsetdrukkerij van de
Landbouwhogeschool verzorgdendeproduktievan "deboekjes".
Ookdeoverige,nietmetnamegenoemde collega'svanhet IvPwil
ik inmijn dank betrekken, daar zeeensfeercreëerdenwaarinhet
prettigwerkenwas.
Tenslotte wil ikmijn vrouw, Marlien, hartelijk bedanken voor
haar levendige belangstelling inhet verloop vanhetonderzoeken
voor haar waardevolle adviezen. Hiervoor, en voor nog veelmeer,
benikhaarzeerdankbaar.
Contents
ARTICLES
1.Appressorium formationofPuccini a hordei on
partially resistantbarley andtwonon-host
species.
Netherlands
Journal of Plant
Pathology
(1981)87,201-207.
1
2.Early abortionofcoloniesofleafrust,
Puccinia hordei, inpartially resistant
barleyseedlings.
Canadian Journal of Botany (1982)60,714-723.
9
3.Histologyoftherelationbetweenminorand
majorgenes forresistanceofbarleytoleaf
rust.
Phytopathology
(1982)72:000-000.
29
4.Comparativehistologyofpartial resistance
andnon-hostreactioninbarley seedlings
toleafrusts.
Phytopathology
(1982)72:000-000.
44
5.Haustorium formationofPuccinia hordei in
leavesofhypersensitive,partially resistant
andnon-hostplantgenotypes.
Phytopathology
(1982)72:000-000.
57
GENERALDISCUSSION
SAMENVATTING
CURRICULUMVITAE
65
70
73
1. Appressorium formation of Pucciniahordei
on partially resistant barley and two
non-host species
by
R.E.Niks
InstituteofPlantBreeding,Agricultural University,
Wageningen,theNetherlands.
Accepted 30July1981
ABSTRACT
One of the components of partial resistance ofbarley toleaf
rust, Puccinia
hordei, isareduced infectibility. Itwasinvestigatedwhether thislowinfectibilitymayrestonahamperedappressorium formation of the leaf rust fungus.Theappressorium formation on the primary leaves of 11barley genotypes with anintermediate-to-low infectibility was compared with that onthehighly
infectible L94.Thenumber ofstomatapercm2 leafareaoccupiedby
appressoria of P. hordei was determined per genotype by means of
fluorescence microscopy. No consistentdifferences couldbedetected, indicating that themechanisms causing alowinfectibility of
partially resistant barley seedlings actataphase laterthanthe
formation of the appressoria. On the non-hostwheatnot fewerappressoriawere formed thanonL94,butnoappressoriawere foundon
a lettuce genotype. The latter probably lacks the stimuli that
enablethe fungusto findstomata.
Additional
tance.
keywords:
Hordeum vulgare,
leafrust,horizontalresis-
INTRODUCTION
Little isknownaboutthemechanisms thatgiveplantshorizontal
resistance sensuVan der Plank (1968)tofungalpathogens,butit
is supposed thatvarious mechanisms,both activeandpassive,may
underliehorizontal resistance (Robinson, 1976).
Partial resistance (PR)ofbarley (Hordeum vulgare L.)toleaf
rust (Puccinia hordei Otth)canberegarded asacaseofhorizontal
resistance: it is largely race non-specific and is controlled by
polygenes (Parlevliet, 1977, 1978a).PRismanifested inareduced
rate of epidemic build-up inspiteofasusceptible infectiontype
(Parlevliet, 1978b). One ofthe factorsresponsible ~orthereductionoftheepidemicbuild-up isthe lowinfectibility ofpartially
resistantgenotypes (Parlevliet andKuiper, 1977).
The aim of thisstudywastoinvestigatewhetherlowinfectibilityiscausedbyahampered appressorium formationoftheleafrust
fungus.
MATERIALSANDMETHODS
Plant material.
The pure lines from barleycompositecrossXXI
(Suneson and Wiebe, 1962)show a largevariation in infectibility
to leaf rust (NiksandParlevliet, 1979).Nine linesoflowinfectibility were used to investigate appressorium formation. These
lines are designated C-lines.Thebarley cultivarJulia,whichhas
anintermediate levelofinfectibilitywasalsoused.L94andVada,
barley cultivarshaving averyhighandvery lowinfectibility respectively,wereused asreferences.Thegenotypesweredivided into
two groups, since two flats (37x39cm)wererequired togrowsix
seedlings per genotype.Three consecutive series with the twelve
genotypesweregrown.
In a second experiment the appressorium formationonnon-hosts
was studied.Thewheat (Triticum
aestivum L.)cultivarsDuri,Adonis
and Saratovskaja 210 (S210), representatives of the graminaceous
non-hosts, and a genotype of lettuce (Lactuca sativa L.), adicotyledonous non-hostspecies,werecomparedwithbarleycultivarL94.
In the first tworeplications eachgenotypewasrepresentedbysix
plants, grown in a flat. Inthethird and fourthreplicationsthe
lettuce was omitted, and the number ofplantsofthewheatcultivars andL94was increased toca.12perentry.
Inoculation.
The primary leaves of the barley and wheat seedlings and the first true leaves of lettuce were inoculated when
they had reached their final size.The lettuce was sownapproximatelytwoweeksbefore thebarley andwheat.The leaveswerepinned
to the soil in the flats into ahorizontal position with their
adaxial sidesup.Per flat,eightgreased slideswere addedtocheck
thedensity anddistribution oftheinoculum.Thisinoculum,urediospores of leaf rust isolate 121A,wasapplied inasettling tower
(Eyal et al., 1968). After inoculation the flatswere transferred
to a greenhouse compartment.Theurediosporeswereallowedtogerminate and to form appressoria under natural darkness,while the
relativehumiditywaskeptatsaturationpointbymeansofanelectrichumidifier.As freewater affectstheregularity oftheappressorium distribution on the leaves, aproperly adjusted hygrostat
and time-switch were connected to the humidifier. In the morning
theleaveswerecoveredwithdroplets asifwithdew.
Sampling,
staining
and observation.
Approximately36hafterthe
onset of the infectionprocess,asegment fromthecentralpartof
each of the leaveswascollected.Theleafsegmentswereprocessed
as described by Rohringer et al. (1977), but Blancophor BA267%
(Bayer,Leverkusen)wasused instead ofCalcofluor.Per leafsegment
theoccupied stomataper25randommicroscope fields (1.54mm 2 each)
werecounted at*125magnification,usingtheZeiss epifluorescence
equipmentNXL (Rohringeret al., 1977). Thenumbersofoccupied stomata per 25 microscope fieldsweretheexperimental units forstatisticalanalysis.
Stomatal densities were determinedby screening fivemicroscope
fields (2.75mm 2 each)per leafatx60magnification,using anormalwhite lightmicroscope.
RESULTS
Appressorium
formation
on host genotypes. Thenumberofoccupied
stomataper25microscope fieldswasaveraged overthesix leafsegmentspergenotype andconverted intodensitiespercm2.About2-5%
of the occupied stomata carried two appressoria andonestomawas
f o u n d t o h a v e t h r e e . The d i s t r i b u t i o n of t h e i n o c u l u m was e v e n : t h e
c o e f f i c i e n t o f v a r i a t i o n w a s l e s s t h a n 10%. T h e i n o c u l u m d e n s i t y
a v e r a g e d 250 t o 450 u r e d i o s p o r e s p e r cm2.
Table 1. R e l a t i v e number of stomata per cm2 leaf area occupied by a p p r e s s o r i a
of Puccinia hordei on the primary leaves of 12 b a r l e y genotypes. Per s e r i e s
the grand mean has been s e t a t 100%.
Series
Barley
genotype
C-29
L94
C-92
Vada
C-17
C-41
C-123
Meannumber
ofoccupied
stomataper
cm2 (=100%)
Genotype
effect
(P<0.05,
ANOVA)
Series
I
II
III
X
Barley
genotype
145
127
81
89
93
78
87
82
100
116
106
98
104
97
99
98
107
102
99
101
94
109
108
101
99
97
94
93
C-70
Vada
L94
C-118
C-120
C-197
Julia
I
II
III
X
111
102
104
107
95
111
72
108
114
106
81
106
86
99
95
95
94
114
97
99
111
105
104
101
101
99
99
94
164 129 283
194 104 287
yes no no
yes no no
The percentages of urediospores giving rise to an appressorium
were approximately 55,40 and 63% for the respective replications.
The average numbers of occupied stomata are presented as relative values to the grand means in Table 1. The genotype effect was
significant in the first series only (ANOVA, P < 0.05). This effect
might have been caused by fortuitous factors, since in the second
and third series not even a tendency towards a ranking of the genotypes similar to that in the first series was found. To investigate
whether the genotypic differences in occupation of the stomata in
the first series was attributable to differences in stomatal density, the number of stomata per m m 2 leaf area was determined. The
stomatal densities in this series differed significantly between
the genotypes indeed (ANOVA, P < 0.01), ranging from 25.3 (C-70) up
t o 32.0 (C-118) stomata per mm2 l e a f a r e a . The c o r r e l a t i o n with t h e
number of occupied stomata per cm 2 , however, was not s i g n i f i c a n t
(r = -0.15).
Appressorium formation on non-host genotypes. In the four r e p l i c a t i o n s with t h e non-host genotypes, approximately 17, 25, 28 and
33% of t h e u r e d i o s p o r e s produced an appressorium on t h e graminaceous genotypes.
Tabel 2 . R e l a t i v e number of stomata per cm2 leaf area occupied by a p p r e s s o r i a
of Puccinia hordei on the primary leaves of one b a r l e y and t h r e e wheat c u l t i v a r s and on t h e f i r s t t r u e leaves of a l e t t u c e genotype.
Per s e r i e s the mean number of occupied stomata per cm2 on L94 has been s e t
a t 100%.
Series
Species
Cultivar
I
II
III
IV
ioo a
100b
130,
100b
72a
1
C
Barley(host)
L94
ioo a
Wheat(non-host)
Duri
Adonis
S210
132a
112a
102a
64b
45a
38a
127a
138a
118a
0
0
-
84
88
112
Lettuce (non-host)
Meannumberofoccupied
L94
stomatapercm2on
(=100%)
100
127
1 Percolumndifferentlettersindicateasignificantdifference
(P<0.05)accordingtoDuncan'smultiplerangetest.
Theappressorium formationonthethreenon-hostwheatcultivars
wasnotconsistently lesssuccessful thanonthehighly infectible
barley cultivar L94(Table 2). Onlettuce,however,noappressoria
were found.Onthetypically jigsaw-puzzle-like epidermis ofthe
dicotyledonous lettuce,thegermtubesgrewrandomly overtheleaf
surface,without findingstomata.Onwheat,thegermtubesgrewdirectionally towardsthestomataasonbarley.Thegraminaceous speciesequaleachotherintheir structureoftheleafsurface.Wheat
cultivarS210,however,hasadenselyhairedepidermis.Inspiteof
this, the appressorium formationonthiscultivarwashardly lower
thanontheothergenotypes.Thewheatandbarleygenotypes differed
little in the number of stomata per mm 2 leaf area.Inthe fourth
series, the densities ranged from28.4 (Adonis)to31.6 (L94)stomatapermm 2 .
DISCUSSION
Inthe search forpossibly durable typesofresistance torust
fungi, it is usefultoinvestigate thedifferentphasesoftheinfectionprocess inordertoelucidatetheresistancemechanisms that
are operative (Zadoks, 1972). Here, the roleofpossiblebarriers
duringthegermination andappressorium formationby P. hordei were
studied.
Theremay alreadybeabiochemical interactionbetweentheplant
and the germinating rust fungus (Ehrlich and Ehrlich, 1971).
Grambow and Riedel (1977)demonstrated that in vitro differentiation of appressoria of P. graminis f.sp. tritici
canbe stimulated
withcertain substancesextracted fromwheatleaves.Itisconceivable that cultivar differences in the qualityorquantityofsuch
compoundsmayresultinhostgenotypicdifferences inthedegreeof
appressorium formation by rusts.There are few accounts, indeed,
thatreportondifferences inappressorium formationbycerealrusts
that are not based on apparent morphological differences.Thereported differences,however, are small and erratic (Brown,1968;
Russell, 1976)or (Stubbs and Plotnikova, 1972)not reproducible
(H.D. Frinking,pers.comm.).
Other studies indicate that the success ofthegerminationand
appressorium formationbyrustfungimerelydependsonmorphological
stimuli (Dickinson, 1970;Wynn, 1976). As aconsequence,anormal
germination and appressorium formation should occur on non-host
species with a leaf surface structure similartothatofthehost
and little appressorium formation when the epidermal structureis
different. This was actually reported (Heath, 1974;Wynn,1976;
Tani etal., 1978). Within ahost species a reduced appressorium
formation due to anaberrantepidermal structure (Mlodzianowskiet
al., 1978)orareduced leafwettability (Cook,1980)mayoccur.
Thepresent results show an irregular host genotype effect on
theappressorium formationby P. hordei (Table 1). Thedatasuggest
that the host effectswere rathercausedbychancethanbygenetic
differences for characters thatinfluencetheurediosporegermination and appressorium formation.Therefore,thereducedinfectibility ofpartially resistant barley ismostlikelycausedbyaresistance mechanism that is effective after the appressoria have
been formed. Even the density of available stomatawasnotcorrelatedwiththedegreeofappressorium formation.Thisisunderstandable, since the number of available stomata was at least five
times as high asthenumberofdeposited urediospores andthusdid
not formalimitingfactor.
Neither wheat nor lettuce show symptoms afterinoculationwith
P. hordei. From the results (Table2)itisclearthatthe 'resistance' ofboth species arises fromdifferentmechanisms,asanormal appressorium formation was found onwheat and no appressoria
were found on lettuce.Thenon-infectibilityofwheatto P. hordei
apparently restsonamechanism thatiseffectiveaftertheappressorium formation. These results agreewiththe findingsofTaniet
al. (1978). They reported that most of the rusts pathogenic on
Gramineae are able to form appressoria on graminaceous non-host
species, but fail todosoondicotyldonous non-hosts.Apparently,
the graminaceous hosts andnon-hostsprovidestimulithatenablea
good appressorium formation,whereasonthedicotyledonous non-hosts
appressorium formation isprevented, probably because thestimuli
are lacking. The similarity of the epidermal structure ofgraminaceous leaves,which differs fromthestructureofdicotyledonous
leaves, and the reported sensibility ofrustgermtubestomorphological stimuli suggest thatthesuccessofappressorium formation
ofbarley leaf rust on different species dependsonthe structure
oftheleafsurface.Lessradicalmorphological differences suchas
hairiness (wheat cultivar S210)hardly affectthe formationofappressoria.
ACKNOWLEDGEMENTS
TheauthorwishestothankMrH.J.Kuiper forhisassistanceand
DrJ.E.Parlevliet and Profs J. Sneep and J.C.Zadoks for their
valuablecriticism ofthemanuscript.
REFERENCES
Brown, J.F. 1968. Histological studies of the factors affecting infection of
wheat by the stem rust fungus. Proc.Cereal Rusts Conf., Oeiras,Portugal:
129-131.
Cook, M. 1980. Peanut leaf wettability and susceptibility to infection by
Puccinia arachidis. Phytopathology 70:826-830.
Dickinson, S. 1970. Studies in the physiology of obligate parasitism. VII. The
effect of a curved thigmotropic stimulus.Phytopath.Z.69:115-124.
Ehrlich, M.A. &Ehrlich, H.G. 1971.Fine structure of the host-parasite interfaces inmycoparasitism.A.Rev.Phytopath. 9:155-184.
Eyal, Z., Clifford, B.C. & Caldwell, R.M. 1968. A settling tower for quantitative inoculation of leaf blades of mature small grain plants with urediospores. Phytopathology 58:530-531.
Grambow,H.J. &Riedel, S. 1977. The effect of morphogenically active factors
from host and nonhost plants on the invitro differentiation of infection
structures ofPuccinia graminis f•sp.tritici• Physiol.PI. Path.11:213-224.
Heath, M.C. 1974. Light and electron microscope studies of the interactions of
host and non-host plants with cowpea rust-Uromycesphaseoli var.vignae.
Physiol.PI.Path. 4:403-414.
Mlodzianowski,F., Werner,A. & Siwecki,R. 1978. Germination of Melampsora
larici-populina uredospores onpoplar leaves.Eur.J.For.Path. 8:119-125.
Niks,R.E.&Parlevliet,J.E. 1979.Variation forpartial resistance to Puccinia
hordei in thebarley composite XXI.Cereal Rusts Bull. 6:3-10.
Parlevliet, J.E. 1977. Evidence of differential interaction in the polygenic
Hordeumvulgare -Puccinia hordei relation during epidemic development.
Phytopathology 67:776-778.
Parlevliet, J.E. 1978a. Further evidence of polygenic inheritance of partial
resistance inbarley to leaf rust,Puccinia hordei.Euphytica 27:369-379.
Parlevliet,J.E. 1978b. Race-specific aspects of polygenic resistance ofbarley
to leaf rust,Puccinia hordei.Neth.J.PI.Path.84:121-126.
Parlevliet,J.E. & Kuiper,H.J. 1977.Partial resistance ofbarley to leaf rust,
Puccinia hordei. IV. Effect of cultivar and development stage on infection
frequency.Euphytica 26:249-255.
Robinson, R.A. 1976. Plant pathosystems. Springer Verlag, Berlin, Heidelberg,
NewYork.
Rohi'.nger,R., Kim,W.K., Samborski, D.J. & Howes, N.K. 1977. Calcofluor: an
optical brightener for fluorescence microscopy of fungal plantparasites in
leaves. Phytopathology 67:808-810.
Russell,G.E. 1976. Germination ofPuccinia striiformis uredospores on leaves of
adultwinterwheatplants.Ann.appl.Biol.82:71-78.
Stubbs,R.W. & Plotnikova,J.M. 1972.Uredospore germination and germ tubepenetration of Puccinia striiformis in seedling leaves of resistant and susceptible wheat varieties.Neth.J.PI.Path.78:258-264.
Suneson,C.A. & Wiebe,G.A. 1962.A 'PaulBunyan'plantbreeding enterprise with
barley. Crop Sei. 2:347-348.
Tani, T. Yamamoto,H., Ohasa,Y. &Yamashita, Y. 1978. Non-host response of
oat leaves against rust infection.Ann.phytopath.Soc.Japan44:325-333.
Van derPlank,J.E. 1968.Disease resistance inplants.Academic Press,New York
London.
Wynn, W.K. 1976. Appressorium formation over stomates by the bean rust fungus:
response toa surface contact stimulus.Phytopathology 66:136-146.
Zadoks, J.C. 1972. Modern concepts of disease resistance in cereals. In:
F.G.H. Lupton, G. Jenkins &R. Johnson (Eds.), The way ahead inplantbreeding. Proc. 6th.Congr.Eucarpia,Cambridge: 89-98.
2. Early abortion of colonies of leaf rust,
Puccmiahorde/,in partially resistant barley
seedlings
by
R.E.Niks*
Institute ofPlantBreeding (I.v.P.)/ Agricultural University,
Wageningen,TheNetherlands.
Accepted 15October 1981
ABSTRACT
Partial resistance (PR)inbarley to leaf rust is assumedlya
case of durable resistance. PR is characterized byareduced rate
of epidemic development in spite of asusceptible infectiontype.
One of the components ofPR, lowinfectibility,wasstudiedhistologicallybymeansoffluorescencemicroscopy.
Quantitative analyses ofthephasesoftheinfectionprocessbeyond appressorium formation showed that the reduced infectibility
of partially resistant barley seedlings rests on a significant
'earlyabortion'ofcolonies.Thistypeofabortionoccurs atabout
the moment of the formationofthe firsthaustoria,whentheyoung
colonies have formed up to five or six haustorial mothercells.
Early abortionisonly incidentally associatedwiththecollapseof
host cells.Not only thevariation in infectibility among barley
genotypes but also the variation in infectivity among leaf rust
isolates is based mainly on differences in the degree of early
abortion. The occurrence of a high degree of early abortion in
several unrelated barley genotypes indicatesthatthePRgenesare
partofagenerallyoccurring system.
*Presentaddress:ICARDA,P.O.B. 5466,Aleppo,Syria.
10
INTRODUCTION
Hypersensitive resistance to biotrophicpathogenshasbeenused
widely by breeders because,owing to its simple inheritance and
ease with which itisrecognized, thecharactercanbemanipulated
very well in breeding programmes. The important disadvantage of
thistypeofresistance isthatitisusually shortlived. Itisnot
surprising therefore that theattention isshifting fromhypersensitive resistance towards other,moredurabletypesofresistance.
This shift,however, has not yet had a significant impactinthe
actualbreeding programmes.Genotypeswithnon-hypersensitive types
of resistance are less easy to select in the field andlittleis
known about gene action and inheritance ofthepertinentgenesof
the host/parasite system. Adeeper insightintotheprinciplesunderlying durable types of resistance may stimulate and facilitate
theuseofthem inresistancebreeding.
It is the aimofthepresentstudytohelpenlarge insightinto
thenatureofnon-hypersensitive orpartialresistance.Thebarley/
leaf rust (Puccinia
hordei Otth)relationship hasbeenchosenasa
model. The term partial resistance (PR)isused in the sense of
Parlevliet (1978): "PRischaracterized byareduced rateofepidemic development in spite of a susceptible infectiontype":7-9 on
the scale ofMcNeal et al. (1971). The reduced rate of epidemic
buildup results fromthecombined effectofseveralcomponents:low
infectibility, long latent period, lowsporulation rate,and short
infectious period. PR appears to be a durable type ofresistance
sincemanyWest-Europeanbarley cultivarshavegoodPRtoleafrust
althoughthegeneshavenotbeenconsciously introduced orselected
forinrecenttimes (Parlevlietetal. 1980;Parlevliet 1981).
The present study is confined to one component of PR:reduced
infectibility. This aspect has been studied by Parlevliet and
Kuiper (1977) at amacroscopic level.They measured the infectibility of several cultivarsbydetermining relative infectiondensities on the leaves.Theyusedtheterm 'infectionfrequency'for
infectibility.Heretheterm infectibility isused, asitindicates
the ability of aplant tobecome infectedby apathogen.Infectibility says that a character of the plant is atissue,whilein-
11
fection frequency is a result of infectibility and density and
quality ofthe inoculum.
Little isknownaboutthehistologyoflow infectibility. Ithas
been suggested that there may exist several mechanisms which can
blocktheinfectionprocess indifferentphases (Hayes 1973;Russell
1976; Zadoks and Schein 1979). If thesemechanismshave agenetic
basis, itshouldbepossibletolowerthelevelofinfectibilityby
genetic recombination (Zadoks and Schein 1979). To determine in
what stage of the infectionprocess thedefence system ofthehost
becomes effective, a detailed histological analysis is required.
Zadoks (1972)proposed toresolve theresistance intocomponentsby
a quantitative analysis ofphases in theinfectioncycle.Sofar,
comprehensive and successful studies with such an approach have
rarely (ZadoksandSchein 1979), ifever,beenreported forcerealrustrelationships.
Thispaperreportswhenandinwhatproportions thecolonies are
arrested by PR beforetheyreachthereproductivephase.Thestudy
hasbeencarriedoutusingthecomponents analysis approachsuggestedbyZadoks (1972).
MATERIALSANDMETHODS
Host
genotypes
To be able to generalize on the histology of thelowinfectibility component of PR, it is essential to study anumberofunrelatedbarley lineswith alowinfectibilitybutwithasusceptible
infectiontype.Former studies (Parlevliet 1976a;Niks and Parlevliet
1979)showed thatthebarley compositeXXIcontains such lines.The
composite originates from the 6200 spring barleys intheAmerican
World Collection,gathered fromallparts oftheworld (Sunesonand
Wiebe 1962). Since the composite has beenmultiplied in isolation
formore than ten years, itcanbeconsidered amixtureoffairly
homozygous lines.No artificial selectionhasbeenapplied.Anumber of lines that were assumed to have an infectibility aboutas
low as the partially resistantcontrolcultivar (cv.) 'Vada' (Niks
andParlevliet1979)wereusedinthepresentexperiments.Theyare
designated as C-lines. Their genetics regarding hypersensitivity
genes andPRgenes isunknown,butalllinesusedhadasusceptible
12
infectiontypewithisolate 121A,unless statedotherwise.The cultivars 'L94', 'Julia' and 'Vada',which show respectively avery
high, intermediate, and lowlevelofinfectibility (Parlevlietand
Kuiper 1977), were also included. All experiments concerned seedlings.
Pathogen
isolates
In all experiments the isolate 121A wasused,whichisamonospore culture derived from isolate 1-2 (Parlevliet 1976b). Inexperiment3, eight isolates were tested; their countriesoforigin
andtheirputative alleles ofavirulence arelisted inTable1.
Table 1•The assumed virulence/avirulence factors of theeight leaf rust isolates inexperiment 3regarding the known Pa resistance genes ofbarley.
Isolate
Country of orgin
18
A
201
22
9
121A
F
5
Virulence/avirulence factors*
Holland
United Kingdom
Israel
France
Kenya
Holland
United Kingdom
Israel
1,4/2,3,5,6,7,8,9
1,4/2,3,5,6,7,8,9
2,4,6,8/1,3,5,7,9
2,4,6,8/1,3,5,7,9
2,3,4,5,8/1,6,7,9
1,2,4,5,6,8/3,7,9
1,2,4,5,6,8/3,7,9
1,2,3,4,5,6,7,8/9
* Parlevliet and Van Ommeren, unpublished.
Isolate 5 is amonospore culture from the Israelian isolateT-46,
whichcarriesvirulence towards almostallknownhypersensitiveresistancegenes (Golanet al• 1978). Theisolatesweremultiplied on
adult plants of cv. 'L98'.The vialswith inoculumwere stored in
liquidnitrogen atapproximately -160°C.
Inoculation
and
incubation
The plants were sownin37x39cm flats intwoorthreeparallelrows.Afterthecompleteunfolding oftheprimary leaves (about
14days after sowing), the leaves were pinned to the soil inthe
flats, to bring them into ahorizontal position with theadaxial
13
side facingupward soastoobtainanequal sporedistributionover
the leaves.Eight greased slides were placed among the leavesof
each flat.With these the inoculum density andspreadweredetermined by means ofamicroscope.Aweighedquantity ofurediospores
was applied to each flat,using asettling tower,constructedaccording to the device of Eyal et al. (1968).Theinoculumwasappliedbymeansofa 'Kabierske'powderblowermounted onatubeinserted 50cm under the top of thecylinderofthesettlingtower.
The other end of the tube with itsfunnel shapedopeningdirected
down just reached the axis of thetower.The inoculum,mixedwith
Lycopodium spores, was gently blown down in the cylinder. After
about10minthetowerwasopened andtheflatwasremoved.
After inoculation the flats were transferred to a greenhouse
compartment.The firstnighttherelativehumidity (RH)waskeptat
saturation pointby means of an electric humidifier. A hygrostat
and a time-switch were adjusted soastopreventanexcessivecondensation which preliminary testshadshowntogiveerraticgermination. After the first natural dark period (at least 10h)the
pins were removed fromtheleaves,andtheplantswerekeptinthe
greenhouse at temperatures notexceeding 30°C.TheRHwaskeptfar
below saturationpoint,sonogerminationofurediospores couldoccurafterthe firstnight.
Staining,
preparation,
and
observation
Middle segments generally of 1 to 3cm2 of inoculated leaves
were collected 2 to 4 days after theemergenceofurediaandprepared aswholemounts forfluorescencemicroscopy (Rohringer etal.
1977). Instead ofCalcofluor,Blancophor BA267%(Bayer,Leverkusen)
was used. Both are optical brighteners that stain the cell wall
rather than the cytoplasm offungi.Thepreparationswere examined
with a Zeiss epifluorescence equipmentNXL (Rohringeret al. 1977)
under x200 magnification, sometimes at x500 for observation of
details. The infection units inthesegmentswere scored andclassified according to their stage of development.Aninfectionunit
isdefined asthemycelial structurethatoriginates fromaurediospore (Zadoks and Schein 1979),butgermtubesthatfailedtoform
14
an appressorium are notincludedhere.Somestructuresbecamemore
visiblewhentheregularwhitetransmitted lightofthesamemicroscope was switched on. Replacement of filter BP 390-440 by H436
facilitated observation on necrosis of host cells,which display
agoldenyellowautofluorescence.
Macroscopically
periment
1)
and microscopically
determined
infectibility
(ex-
An experiment was conducted toinvestigate therelationshipbetween themacroscopically determined infection density, i.e., the
number of uredia per cm2 leaf area, and the microscopically observed colony abortion in stages after appressorium formationand
to quantify abortion intherespectivephasesofdevelopment.Four
host genotypes were studied: 'L94'and 'Vada'ascontrols andC-41
and C-118 as lines which were previously showntobe considerably
less infectible than 'Vada' (NiksandParlevliet 1979). Twoflats,
onewith7or8seedlings andtheotherwith about20seedlingsper
genotype were planted. The plants in the first flat wereusedto
determine the infectibility of the genotypes microscopically. The
plants in the second flatwere inoculated without the useofthe
settling tower while the primary leaves were inuprightposition.
These plants were usedtodetermine theinfectibility ofthegenotypes macroscopically, using the method of Parlevliet and Kuiper
(1977). Per leaf theinfectiondensitywasdetermined inasegment
of at least 3cm in length. Theexperimentwasconducted inthree
consecutiveseries.
Qualification
and quantification
genotypes
(experiment
2)
of colony
abortion
in seven
Thegeneralvalidity oftheresults fromexperiment 1wasinvestigated by performing components analyses on six more C-lines:
C-92, C-29, C-70, C-123, C-120 and C-197, which were thought to
have low infectibility. Two flats were planted eachwiththreeof
the lines and with 'Vada'ascontrol.Each linewasrepresentedby
eight leaves. Before sampling the leaf segments, the number of
urediapercm2 ofleafareawasdetermined.Afterpreparation,each
barley
15
oftheeightleafsegmentsperlinewasscanned for20to25infection units.These were classified according totheirstageofdevelopment.Theexperimentwasconducted intwoconsecutiveseries.
Qualification
rust isolates
and quantification
(experiment
3)
of colony
abortion
of eight
leaf
To verify whether results with other isolateswouldbeconsistentwiththoseobtainedwithisolate 121A,anexperimentwithseven additional isolates of diverse origins (Table1)was setup.
Four flatswereplantedwith 'Julia'and 'Vada'lackinghypersensitivity genes to anyofthe isolatesunderstudy.Each flatwasdivided intwo,onehalfreceivingtheinoculum ofoneisolate,while
the other was coveredby asheetofpaper.Afterthe appearance of
uredia five leaf segments per cultivar/isolate combination were
collected and processed as usual.Each segment was scanned as a
whole; all infection unitswere scored andclassified accordingto
their stage of development. The experiment was conducted without
replication.
The infection
cycle
of Puccinia
hordei
in
barley
For a good understanding of the results, successive phases of
fungal development are distinguished, asrecognizedbythemorphological structures that are formed (Table 2). Infectionunitscan
be arrested inanyphaseofdevelopment,sothatdifferenttypesof
abortioncanberecognized (Table2,Figs.1-5).
RESULTS
Macroscopically
periment
1)
and microscopically
determined
infectibility
The average numbers ofurediapercm2 ofleafareaofthelines
andthemicroscopically determinedproportions ofsuccessfulinfectionunitswereconverted torelativevalueswithcorrespondinginfectiondataon 'L94'setat100%.Thisenabled acomparisonofthe
results between the series and the two methods. The experimental
(ex-
16
Table 2. The phases ofthe infection process of leaf rust inbarley, the
mycelial structures that are formed therein and thedesignation of the
types of abortion if the corresponding structure is the last one that is formed.
Phase of development
Successive mycelial
structures
Germination
Germ t u b e
Appressorium
formation
Appressorium
o v e r stoma
Non-penetration
Stoma penetration
and substomatal
vesicle
formation
Infection peg,
substomatal
v e s i c l e (SSV)
SSV a b o r t i o n
Establishment
First infection
hyphae,
haustorial
mother c e l l s
(HMC)
Colonization
Hyphae and p o s s i b l y
sporogenic t i s s u e
Reproduction
Mature u r e d i o s p o r e s
(successful
infection)
Designation of the
types of abortion
Early abortion
Late abortion
u n i t s f o r t h e s t a t i s t i c a l a n a l y s e s were t h e i n f e c t i o n d e n s i t y and
t h e p r o p o r t i o n of s u c c e s s f u l i n f e c t i o n u n i t s p e r l e a f . G e n o t y p i c
d i f f e r e n c e s were t e s t e d f o r s i g n i f i c a n c e u s i n g n o n - p a r a m e t r i c t e s t s
(Siegel 1956).
R e s u l t s of t h e m a c r o s c o p i c a l l y and m i c r o s c o p i c a l l y o b t a i n e d i n f e c t i b i l i t y a s s e s s m e n t s ( T a b l e 3) a r e s i m i l a r w i t h r e g a r d t o t h e
r a n k i n g of t h e g e n o t y p e s : ' L 9 4 ' and C-41 a r e h i g h l y i n f e c t i b l e and
' V a d a ' and C-118 a r e of a b o u t e q u a l l y low i n f e c t i b i l i t y . As t o t h e
s i z e of t h e g e n o t y p i c d i f f e r e n c e s , t h e two methods a r e n o t e q u i v a l e n t : t h e d i f f e r e n c e s between t h e g e n o t y p e s f o r r e l a t i v e i n f e c t i o n d e n s i t y a r e l a r g e r t h a n f o r r e l a t i v e p r o p o r t i o n of s u c c e s s f u l
infection units.
Qualification
and quantification
of colony
resistant
barley (experiments
1 and 2).
abortion
in
partially
The a b o r t e d c o l o n i e s i n e x p e r i m e n t 1 were c l a s s i f i e d a c c o r d i n g
t o t h e p h a s e of development i n which t h e i r growth s t o p p e d ( T a b l e 4 ) .
17
Table 3.Relative infectibilities of four barley genotypes with leaf rust
isolate 121A,the infectibility of 'L94' being set at 100%,determined macroscopically and microscopically (experiment1 ) .
Infectibility
measured as:
Barley genotype
Infection
densityt
'L94' s
C-41
'Vada'
C-118
'L94'§
C-41
'Vada'
C-118
Proportion
successful
infection units
()
Series
II
III
100
76 a
49
39
100
6la
33
19
100°
iooa
iooa
71
77
100
ioo a
97
75
75
72
5 0
Mean
\
h
44 b
a
68
h
74 b
100
70
44
34
100
99
71
75
* Per subcolumn different letters indicate a significant difference (P< 0.02)
according to the non-parametric Mann-Whitney test inpairs.
About 80 seedlings per flat,inoculated without settling tower.
About 32 seedlings per flat,inoculated ina settling tower.
§
On 'L94'the average infection density amounted to 11.8,6.7, and 10.8
uredia/cm2 inthe respective series.
§§ On'L94'theaverageproportionofsuccessfulinfectionunitsamountedto0.90,
0.89,and0.94intherespectiveseries.
Theproportionofinfectionunits aborted inoneofthe fourphases
ofdevelopment (Tables4,5;Fig.6)hasbeencalculated asfollows.
The number of infectionunitsaborted inagivenphasewasdivided
bythetotalnumber ofinfectionunitsthatenteredthatphase.
The non-penetrating appressoria (Fig.1)wereeasily recognized
by the absence of a substomatalvesicle (SSV).Agenotypic effect
onthedegreeofnon-penetrationwasnotdetected.
Only a low numberof SSVs,sometimes surroundedbynecrotic and
collapsed host cells, failed todevelop infectionhyphae (Fig.2 ) .
Thus SSV abortion does not show significance forthe levelofinfectibility.
Themostpronounced genotypic effectconcerns thedegreeofearlyabortion.Acolonywasconsidered tobe 'earlyaborted' ifupto
five or six haustorial mother cells (HMC)were formed andifthe
infection hyphae were not or little branched (Figs.3,5).Such
aborted colonies were rarely associated with thecollapse ofhost
cells.Oftentheinfectionhyphaewerehighly emaciated. Inthelow
infectible genotypes, 'Vada' andC-118,aboutaquarter ofthein-
18
Table4.Proportionsofarrestedinfectionunitsofleafrustisolate121Ain
fourbarleygenotypes (experiment 1). Theinfectionunitsareclassified infour
typesofabortion.Pergenotypeperseries7or8leaveswerescreenedwithat
least200infectionunitsinall.
Type of
abortion
Non-penetration
Barley
genotype
'L94'
'L94'
C-41
'Vada'
C-118
SSVabortion
'L94'
'L94'
C-41
'Vada'
C-118
Earlyabortion
'L94'
'L94'
C-41
'Vada'
C-118
Lateabortion
'L94'
'L94'
C-41
'Vada'
C-118
I
.08.08
Series
II
.08
.01
.01 .01
.02
.06
.08
.00
.01
.01
.00
.01
.01
.03
.23
.21
.00
.23
.17
.01 .01
.04
.03
.05
.00
.00
.01
.01
.00
.02
Significance of effects§
Genotype
Series
.04
.09
.11
.08
.06
.11
.09
.00 .00
III
.02
.02
.32
.23
.01
.03
.02
.06
.02
.04
.04
§AccordingtotheKruskal-Wallistest:
notsignificant
* significant (P<0.05)
**significant (P<0.01)
fection units was stopped by this type of abortion (Table4 ) .
Once established, few colonies aborted. Established colonies
that had not reached the reproductive phase at the day of sampling
were classified as 'late aborted' colonies. This category contained
really aborted infection units and possibly also some infection units that eventually would have formed uredia, although the sampling was carried out several days after the appearance of the majority of the uredia. The late aborted colonies were strongly branched and bore more HMCs than the early aborted colonies (Fig.4 ) ,
but generally no sporogenic tissue had been formed. Late abortion
was not necessarily associated with host cell necrosis. The cultivars showed no large differences in this late abortion (Table4 ) .
19
Inexperiment2 infection densitiesofthesixlinesand 'Vada'
showed a significant genotype effect, although allgenotypeswere
assumed to be of low infectibility (Table 5). As in experiment1
the differences in infection density are explained mainlybydifferences in the degreeofearly abortion.Again,inthehostgenotypeswith lowinfectibility early aborted colonieswere rarelyassociatedwithautofluorescentorcollapsed hostcells (Table 6). If
so, there usually wereoneortwonecrotic cells.Inthegenotypes
with little early abortiontheassociationwithhostcellnecrosis
wasstronger.
No significant genotype effects for the degree ofnon-penetrationandSSVabortionwere found.
Regarding the degree ofabortionduringthecolonizationphase,
thebehaviourofC-120attracts attention:itslowinfectibility is
to ahigh degree due to late abortion (Table 5). Often sucharrestedcolonieswereassociatedwith autofluorescenthostcells.It
must be mentioned, however,thatC-120 didnotmeetthedefinition
of PR in this experiment: the uredia were associated with some
chlorosis (infection type 6 on the scale of McNeal et al. 1971).
Theothergenotypes,which showed fully susceptible infectiontypes,
did not differ significantly among themselves for the degree of
lateabortion.
Qualification
rust isolates
and quantification
(experiment 3)
of colony abortion
of eight
The results of thecomponents analysisoftheeightisolateson
'Vada' and 'Julia' arepresented inFig.6.As aresultofunknown
causes, only3 to 13%of the urediospores gaverisetoanappressorium, so that the conclusions about some of the isolates were
based on a small number of infectionunits.Theresultswithisolates F,5 and 22arebased on60infectionunitsorlesspercultivar, those with isolates 121A, 18,and 201 on atleast100per
cultivar.
According to the Kruskal-Wallis test (Siegel 1956) 'Vada'and
'Julia' differed only for the degree of early abortion.Alsothe
isolate effect regarding thistypeofabortionprovedtobesignificant. The Everyman's Contingency Table Analysis (Everitt 1977)
leaf
20
** » I
21
Table 5. Infection densities inuredia per cm2 and proportions of succesful and
aborted infection units of leaf rust isolate 121A in 'Vada' and sixC-lines
(experiment 2 ) . Each entry is themean of eight seedlings.The classificationof
the aborted colonies is given (n= 170 - 190 infection units per genotype per
series)
Series Barley Infection ProporProportions of infection units arrested by
genotype density tionsuccessful Non-pene- SSV abor-Early
Late
infec- tration tion
abortion abortion
tions
II
II
'Vada'
C-92
C-29
C-70
19 9
20
37 1
40 3
.56
ab
.62
.78
.80
.11
.04
.07
.06
.01
.03
.05
.01
.35°
.3la
.08
.13
.02
.04
.03
.03
'Vada'
C-92
C-29
C-70
27
35
58
56
2
9
5
7
.40
.53a
.72
.74
.03
.05
.03
.06
.01
.01
.01
.00
.54
b
.35
be
.17
c
.15
.08
.14
.09
.07
'Vada'
C-123
C-120
C-197
35
36
41
55
4
3
4
1
.65°
.66a
.70a
.76a
.06
.02
.03
.06
.01
.02
.01
.04
.26°
.26a
.09
.09
.05
.01
.25
.06
'Vada'
C-123
C-120
C-197
31
23 3
45 5
57 9
.38
.42a
.49a
.81
.03
.05
.06
.04
.00
.01
.02
.02
.59
.56°
.17
.12
.03
.00
.36
.02
Per subcolumn different letters indicate a significant difference (P< 0.01)
according toDuncan's multiple range test.
Figs. 1-5. Infection units ofPuccinia hordei,aborted in different
phases of development.
Fig. 1.Non-penetrating appressorium. x415.
Fig. 2.Aborted substomal vesicle (SSV). Note the collapse of
surrounding host cells,x510.
Fig. 3.Early aborted infection unit.The substomatal vesicle
(SSV)has formed infection hyphae (IH)and a few haustorialmother cells (HMC). x470.
Fig. 4. Late aborted infection unit.Note the excessively branched hyphae. x 400.
Fig. 5. Part of a sporulating colony and an early aborted colony
(arrow), x375.
Figs. 1,3,4,and 5 are fluorescence micrographs.Fig. 2 isawhite
lightmicrograph.
22
Table6. Proportions of early aborted infection units of leaf rust in seven
barley genotypes, which were not associated with host cell necrosis. The data
are obtained from the second replication of experiment 2.
Barley
genotype
Vada'
C-123
C-92
C-120
C-29
C-197
C-70
Proportion of early aborted
infection units without necrosis
.95
.93
.87
.67
.33
.27
.27
Number of infection
units studied
88
83
61
43
79
40
59
does not indicate a significant interaction betweencultivarsand
isolates for early abortion (critical level:0.36). Absenceofinteraction between cultivars and isolates indiseasedevelopmentis
bydefinition (VanderPlank1968)characteristic ofhorizontalresistance.
The isolate effect for non-penetration was notsignificant.Again,thedegreeofSSVabortionwassolowthatitwasepidemiologically insignificant, despite astatistically significantisolate
effect.Generally,theleveloflateabortionwaslow.
Variation in infectivity among isolates apparently is based
mainly on differences inearly abortion,asisvariation ininfectibilityduetoPRamongbarley lines.Thedegreeofearly abortion
isnotrelated tothenumber and identityoftheavirulence alleles
ofthe isolates (Table1 ) .
DISCUSSION
There is a large variation in infectibility toleafrustamong
seedlings ofbarley genotypes despite the factthatthe infection
typesindicate susceptibility (ParlevlietandKuiper1977;Niksand
Parlevliet 1979).Thevariation ininfectiondensity isatleastin
part explained by variation in the degree of colony abortion.In
experiment 1thegenotype differences inrelative infectiondensity
were larger than those for the relative proportion of successful
infection units.Also the formerlyobserved lowinfectibilities of
some of the lines,e.g., C-41 andC-70 (NiksandParlevliet 1979)
were not confirmed by present results. Possibly differences in
23
TYPE OFABORTION
non-penetrant
F
5 22
Fig.6.Proportions of aborted infection units of eight isolates of leaf
rust in seedlings of thebarley cultivars 'Vada'and 'Julia'.Four types of
abortion aredistinguished according to the stages of development of
the infection units.
24
inoculation andincubationtechniques areresponsible forthese discrepancies.
In the histology of low infectibility owing to PR of barley
seedlings to leaf rust, early abortion turned out to bethemost
important factor.Genes forPRdonotappeartoaffect appressorium
formation on seedlings (Niks 1981). The present results did not
show a relationship betweenthelevelofPR andthedegreeofnonpenetration andSSVabortion.
Early abortion probably occurs when the firsthaustoria areabout tobe formed, i.e., approximately 20haftertheonsetofthe
infection process.Unfortunately, the fluorescence stainingmethod
does not permit observation of the haustoria. Accordingly, it is
not clear whether theinfectionunits arearrestedbefore orafter
the formation of the firsthaustoria.Early abortionseldom isaccompanied by host cell necrosis.Low infectibility owingtoearly
abortion does not appear apeculiarity ofaspecialhostgenotype:
a high degree ofearly abortionoccurred inseveralunrelatedbarley genotypes ('Vada',C-118,C-123, C-92). Similarly, differences
in infectivity of isolates are explained by differences in early
abortion. It is not clear whether thevariation ininfectivity is
genetic orphysiological innature.Possibly,the isolatesusedin
experiment3 differ in their environmental requirements. Theisolate effects may also have been causedbydifferences ininoculum
quality:thesporeswerecollected indifferentperiods.
Early abortion has notbeen recognized before as the primary
factor which determines infectibility ofbarley toleafrust.AccordingtoClifford (1972), the low infectibility of 'Vada'toleaf
rust should, at least inpart,be due to asignificant SSVabortion. In a recent study (Clifford and Roderick 1981)also with
'Vada', no colony abortion in the earlyinfectionphaseswasmentioned. Studying the histology of the infection process on some
othercultivarsClifford andRoderick (1978)stated: "Thebiological
processes that result in aproportion of the colonies failingto
sporulate is not clear fromhistology".Thepresentresults,which
show a consistently low incidence of SSV abortion, seem to contradictClifford's (1972)earlierresults.Possibly, inhisstudies
the colonies thatwere arrestedearlywereoverlooked.Likealmost
all workers on the histology of resistance, he used dyes (cotton
25
blue and acid fuchsine) that stain the fungal cytoplasm. Early
aborted colonies tend to become unstainable quickly with these
typesofdyes.
The occurrence of abortion at the timeofthefirsthaustorium
formation,which isnotaccompaniedbyhostcellnecrosis,hasalso
notbeenreported forothercereal/rustsystems.Fromahistological
studyofthepostpenetrationphenomena insomewheatcultivarswith
a 'low receptivity' to infection by P. graminis f.sp.
tritici,
Ashagari and Rowell (1980)concluded thatthequantitativedifferences in the receptivity ofthecultivars apparently resulted from
therapidity and frequencyofthenecrotic reactionofthehost.In
a similar investigation, Martin etal. (1977) indicated 'slow
rusting' could be relatedwithhostcellnecrosiswhilethe fungal
growth did not ceasewithin24h.Recently,CartwrightandRussell
(1980)showed thatthemajorityoftheinfectionunitsof P.
striiformis in 'LittleJoss' (awheatcultivar supposed toexhibitadurable resistance against this pathogen) are arrested during or
shortly after the SSV formation.Here again,aseriouscollapseof
host cells accompanied colony abortion. It isnot clear whether
these andotherreports dealwithPR sensu Parlevliet (1978);often
the infection types are not mentioned or they are notofafully
susceptibletype.
Probably theonlyaccountofearlyabortiononahostwithasusceptible reaction has been givenby Heath (1977).Describingnonhost reactions, shemade apassing reference to the cessationof
fungalgrowthof23%oftheinfections of P. helianthi
on sunflower
(cv. Sunrise) after the formation of the first haustorium. This
abortionwasnotnecessarily associatedwithacollapseofthesurrounding host cells.Remarkably, the early abortion of leaf rust
colonies inpartially resistantbarley seedlingsresemblesthesocalled non-host reaction: onaplantofanon-hostspecies theinfection units usually are arrested justbetweentheHMC formation
andthe firsthaustoria formation andgenerallynosubstantial collapseofhostcellsoccurs (Heath 1977).
Withthehypersensitive typeofresistance,thecoloniesusually
stopwhenseveralhaustoria andafairamountofmycelium havebeen
formed (e.g.,Hilu1965;Zimmer 1965;Littlefield andAronson1969;
Sood and Sackston 1970;Mendgen 1978;Rohringer etal. 1979). In
26
a l l instances the interaction i s associated with host c e l l necrosis.
P o s s i b l y t h e r e a c t i o n o f l i n e C-120 ( T a b l e 5) s h o u l d b e a t t r i b u t e d
t o an i n c o m p l e t e l y e x p r e s s e d gene f o r h y p e r s e n s i t i v i t y : t h i s
line
i n o c u l a t e d w i t h i s o l a t e 121A s h o w e d a n i n t e r m e d i a t e i n f e c t i o n t y p e ,
w h i l e a c o n s i d e r a b l e p r o p o r t i o n of i n f e c t i o n u n i t s was a r r e s t e d
af-
t e r e s t a b l i s h m e n t . Often such c o l o n i e s were s u r r o u n d e d by s l i g h t l y
a u t o f l u o r e s c e n t c e l l s . The low p r o p o r t i o n s o f l a t e a b o r t e d c o l o n i e s
i n ' V a d a ' , C - 1 1 8 , C - 1 2 3 a n d C - 9 2 ( T a b l e s 4 a n d 5 ) s u g g e s t t h a t PR
genes h a r d l y cause t h e b l o c k i n g of i n f e c t i o n u n i t s during t h e c o l o nization stage in barley seedlings.
I n t h e l i t e r a t u r e two s y s t e m s a r e p r o p o s e d t o b e o p e r a t i v e i n
h o s t - p a t h o g e n i n t e r a c t i o n s : b a s i c c o m p a t i b i l i t y which c o n t r o l s t h e
e s t a b l i s h m e n t of t h e p a t h o g e n , and h y p e r s e n s i t i v i t y presumed t o be
s u p e r i m p o s e d on b a s i c c o m p a t i b i l i t y
(e.g.,
Day 1 9 7 6 ;
Loegering
1 9 7 8 ) . The s y s t e m o f h y p e r s e n s i t i v i t y h a s b e e n i n v e s t i g a t e d
thor-
o u g h l y i n many r e p o r t s , b u t r e s e a r c h o n t h e s y s t e m o f b a s i c c o m p a t i b i l i t y h a s h a r d l y s t a r t e d ( e . g . , G a b r i e l e t a l . 1 9 7 9 ) . The h i s t o l o g i c a l resemblance of e a r l y a b o r t i o n of l e a f r u s t c o l o n i e s
in
p a r t i a l l y r e s i s t a n t barley with the non-host reaction suggests
that
t h e s e genes a r e p a r t of t h e system of b a s i c c o m p a t i b i l i t y .
ACKNOWLEDGEMENTS
I would l i k e t o thank H . J . Kuiper f o r h i s a s s i s t a n c e t h r o u g h o u t t h e
e x p e r i m e n t a l work. Thanks a r e a l s o due t o Dr. J . E . P a r l e v l i e t ,
and
t o P r o f s . J . Sneep and J . C . Zadoks f o r t h e i r v a l u a b l e c r i t i c i s m of
and h e l p w i t h t h e m a n u s c r i p t .
REFERENCES
Ashagari, D . , and J . B . Rowell. 1980. P o s t p e n e t r a t i o n phenomena in wheat c u l t i vars with low r e c e p t i v i t y to i n f e c t i o n by Puccinia graminis f . s p . t r i t i c i .
Phytopathology 70:624-627.
Cartwright, D.W., and G.E. R u s s e l l . 1980. H i s t o l o g i c a l and biochemical n a t u r e of
' d u r a b l e ' r e s i s t a n c e t o yellow r u s t in wheat. Proc. 5th E u r . M e d i t e r r .
Cereal Rusts Conf. (Bari and Rome), pp. 23-26.
C l i f f o r d , B.C. 1972. The h i s t o l o g y of race n o n - s p e c i f i c r e s i s t a n c e t o Puccinia
hordei Otth. in b a r l e y . Proc. 3rd E u r . M e d i t e r r . C e r e a l Rusts Conf. (Prague).
Vol. 1. pp. 75-79.
C l i f f o r d , B.C., and H.W. Roderick. 1978. A comparative h i s t o l o g y of some b a r l e y
brown r u s t i n t e r a c t i o n s . Ann.Appl.Biol. 89:295-298.
27
Clifford, B.C. and H.W.Roderick. 1981. Detection of cryptic resistance ofbarley toPuccinia hordei. Trans.Br.Mycol.Soc.76:17-24.
Day, P.R. 1976.Gene functions inhost-parasite systems.InSpecificity inplant
diseases. Editedby R.K.S. Wood and A. Graniti. Plenum Press, New York,
London,pp.65-73.
Everitt, B.S. 1977. The analysis of contingency tables. Chapman and Hall Ltd.,
London.Appendix B.
Eyal, Z., B.C. Clifford,andR.M.Caldwell,1968.A settling tower forquantitative inoculation of leaf blades of mature small grain plants with urediospores. Phytopathology 58:530-531.
Gabriel,D.W., A.H.Ellingboe, and E.C.Rossman. 1979.Mutations affectionvirulence inPhyllosticta maydis. Can.J.Bot.57:2639-2643.
Golan, T., Y.Anikster,J.G.Moseman, and I.Wahl. 1978.A new virulent strain
ofPuccinia hordei.Euphytica 27:185-189.
Hayes, J.D. 1973. Prospects for controlling cereal disease by breeding for increased levels of resistance.Ann.Appl.Biol.75:140-144.
Heath, M.C. 1977.A comparative study of non-host interactions with rustfungi.
Physiol. Plant Pathol.10:73-88.
Hilu,H.M. 1965. Host-pathogen relationships of Puccinia sorghi in nearly isogenic resistant and susceptible seedling corn.Phytopathology 55:563-569.
Littlefield, L.J., and S.J. Aronson. 1969. Histological studies of Melampsora
liniresistance in flax.Can.J.Bot.47:1713-1717.
Loegering, W.Q. 1978. Current concepts in interorganismal genetics. Annu.Rev.
Phytopathol.16:309-320.
Martin, C D . , L.J. Littlefield, and J.D. Miller. 1977.Development of Puccinia
graminis f.sp. tritici in seedling plants of slow-rusting wheats.Trans.Br.
Mycol.Soc.68:161-166.
McNeal, F.H.,C F . Konzak,E.P. Smith,W.S.Tate,and T.S.Russell. 1971.A uniform system for recording andprocessing cereal research data.USDA,Agric.
Res.Serv.,Washington,D.C.ARS34-121.
Mendgen, K. 1978.Der Infektionsverlauf vonUromycesphaseolibei anfälligenund
resistenten Bohnensorten. Phytopathol.Z.93:295-313.
Niks,R.E. 1981.Appressorium formation ofPuccinia hordei onpartially resistant
barley and twonon-host species.Neth.J. Plant Pathol.87:201-207.
Niks, R.E., and J.E. Parlevliet. 1979. Variation for partial resistance to
Puccinia hordei inthebarley composite XXI.Cereal Rusts Bull. 6:3-10.
Parlevliet, J.E. 1976a. Screening for partial resistance in barley to Puccinia
hordei Otth. Proc. 4th Eur.Mediterr. Cereal Rusts Conf. (Interlaken).pp.
153-155.
Parlevliet, J.E. 1976b. Evaluation of the concept of horizontal resistance in
the barley/Puccinia hordei host-pathogen relationship. Phytopathology
66:494-497.
Parlevliet, J.E. 1978. Race-specific aspects of polygenic resistance of barley
to leaf rust,Puccinia hordei.Neth.J.PlantPathol.84:121-126.
Parlevliet, J.E. 1981. Race-non-specific disease resistance. In Strategies for
the control of cereal disease. Edited by J.F. Jenkyn and R.T.Plumb.
Blackwell Scient.Publ. Ltd.Oxford,Edinburgh,pp. 47-54.
Parlevliet, J.E., and H.J. Kuiper. 1977. Partial resistance of barley to leaf
rust, Puccinia hordei. IV. Effect of cultivar and development stageoninfection frequency.Euphytica 26:249-255.
Parlevliet, J.E., W.H.Lindhout,A.vanOmmeren,andH.J. Kuiper. 1980.Level of
partial resistance to leaf rust, Puccinia hordei, in West-European barley
and how to select for it.Euphytica 29:1-8.
Rohringer,R., W.K.Kim, D.J. Samborski, and N.K.Howes. 1977. Calcofluor: an
optical brightener for fluorescence microscopy of fungalplantparasites in
leaves. Phytopathology 67:808-810.
Rohringer, R., W.K. Kim, and D.J. Samborski. 1979. A histological study'of interactions between avirulent racesof stem rustandwheat containing resistance genes Sr5,Sr6,Sr8or Sr22. Can.J.Bot.57:324-331.
28
Russell,G.E. 1976. Characterization of adultplant resistance toyellow rust in
wheat. Proc. 4thEur.Mediterr.CerealRusts Conf. (Interlaken).pp.21-23.
Siegel,S. 1956.Nonparametic statistics for thebehavioral sciences. McGraw-Hill
NewYork,NY.
Sood,P.N., and W.E. Sackston. 1970. Studies on sunflower rust.VI. Penetration
and infection of sunflowers susceptible and resistant toPuccinia helianthi
race 1.Can.J.Bot.48:2179-2181.
Suneson, C.A., and G.A. Wiebe. 1962.A 'Paul Bunyan' plant breeding enterprise
withbarley. Crop Sei. 2:347-348.
Van derPlank,J.E. 1968.Disease resistance inplants.Academic Press,NewYork,
London.
Zadoks, J.C. 1972.Modern concepts of disease resistance in cereals.InTheway
ahead inplantbreeding.Edited byF.G.H. Lupton,G. Jenkins,andR. Johnson
Proc. 6th Congr.Eucarpia (Cambridge),pp. 89-98.
Zadoks, J . C , and R.D. Schein. 1979. Epidemiology and plant disease management
Oxford Univ. Press,NewYork,Oxford.
Zimmer, D.E. 1965.Rust infection and histological response of susceptible and
resistance safflower.Phytopathology55:296-301.
29
3. Histology of the relation between minor
and major genes for resistance of
barley to leaf rust
by
R.E.Niks andH.J. Kuiper
Institute of Plant Breeding, Agricultural University, Lawickse
Allee166,6709 DB Wageningen,TheNetherlands.Presentaddressof
seniorauthor:ICARDA,P.O.B. 5466,Aleppo,Syria.
We are grateful to J.E.Parlevliet, J. Sneep and J.C.Zadoks for
critically readingofthemanuscript.
Accepted 21May1982
ABSTRACT
Pa7 and Pa3, twomajorgeneswhichconferhypersensitiveresistanceinbarleytoleafrust, Puccinia hordei, were introduced into
three genetic backgrounds with different levels ofpartialresistance (PR)to study the interaction between both types ofresistance. The growth rate and the degreeofabortionofthecolonies
in the genotypes were determined by fluorescence microscopy. The
degree of host cell necrosis was recorded. The PR genes affected
the success of colony establishment in the host and reduced the
growth and development rate of colonies afterestablishment.This
effectwas also apparent inpresence ofPa7,theeffectsofwhich
were seen relatively lateintheinfectionprocess.Apparently,PR
genes and Pa7 acted independently and consecutively. Pa3 acted
shortly after the establishment of the colonies and largely obscured the effect of the PR genes.YetthelevelofPRcanbeassessed inthepresenceofPa3bydetermining theproportionofearlyabortedcoloniesnotassociatedwithhostcellnecrosis.
30
INTRODUCTION
Since Van der Plank (24)introduced theconceptofvertical and
horizontal resistance (VR and HR), these two types of resistance
have been compared in many publications. One host genotype may
possess alleles for both HR and VR,butmany authors surmisethat
the expression ofHR isobscured ifthehostalsopossesseseffective alleles forVR (eg,20).As aconsequence,selection forHR,
which isdesirablebecauseofitsassumeddurability,wouldbepossibleonlyintheabsenceofVR.
Inthebarley/leaf rust (Puccinia hordei Otth)relationship,the
major genesconferring ahypersensitive reactionwith avirulentraces are considered VR genes.These genes are designated Pa-genes
(13). The minor genes conferring partial resistance (PR)arepresumedtobelongtothetypeofHRgenes (8,14).PRischaracterized
by a reduced epidemic build-up, despite a susceptible infection
type (IT) (15).Bothtypesofresistance canoccurinonegenotype
(17).
Inthispaper,ahistological studyispresented ontherelation
betweenbothtypesofresistance.
MATERIALSANDMETHODS
A back-crossing procedure was used to introduce the dominant
alleles Pa7 of CebadaCapaandPa3ofRikax (RikaxBaladi)(see
13)intothreerecipientcultivarsofbarley:L94,Zephyr andVada.
These have undetectable,moderate and high PR,respectively(19).
From each of the six major gene/recipientcombinations onehypersensitive plantwas singled out after the fourth backcross(B4).
After seifing,eachB4plantproduced apopulationwhich segregated
for the major gene.These populations were screened for ITwith
isolate 121A,whichisavirulenttoPa7andPa3.Theseedlingswith
thelowest ITwereassumedtobehomozygous forthedominantallele
of themajor gene.The progenies fromtwoselfedveryhypersensitiveandtwoselfed fullysusceptibleplants (ie,second generation
after B4) for eachmajor gene/recipient combination were used in
this study. The two donor cultivars, indicated respectively asCC
and Rika,were alsostudied.Thelinesaredesignated accordingto
31
the presence (P)or absence (p)of the dominant alleleofthePa
gene (7or 3). Thegeneticbackground oftheline,whichresembles
that of the recipients (L94,ZephyrorVada)isindicatedbyL,Ze
orVa (seealsoTable1 ) .
The 24 lines were grown in 12 flats.Each flat contained one
line derived from ahypersensitiveplantandone fully susceptible
sisterline.Eachlinewasrepresentedby 16seedlings.CCandRika
were grown in an additional flat.The primary leaves wereinoculated with urediospores of isolate 121A, using a settling tower.
Between80and 190sporespercm2were applied,ofwhich50%formed
an appressorium over a stoma. Central segmentsoftheleaveswere
sampled 2^, 4^, 6%and14*sdayspostinoculation (d.p.i.),butfor
the lines with aVadabackground the finalsamplingwas16*5d.p.i.
because of the reduced rate of uredialdevelopment.Foursegments
per line were collected per sampling day. The leafpieces were
stained (22),withBlancophor BA267%(Bayer,Leverkusen)replacing
theCalcofluor.Theobservationswerecarried outwithaZeissepifluorescence equipment NXL. Details of the foregoing procedures
weregivenelsewhere(10).
Infection units (each unit representing the thallus developing
from a single urediospore [25])wereclassified accordingtotheir
developmental stage andbythemeasurementofcolony lengths.Here,
germtubeswerenotobserved,norweregermination and appressorium
formation studied. The developmental phases of theinfectionprocess and the designations ofthecorresponding infectionunitsare
given in Table2 (seealso10).Eachleafwas screened forasmany
infection units aswerenecessaryto find20measurable andestablished colonies which were either aborted lateorsuccessful.The
sizeofthecolonieswasassessedwithaneyepiecemicrometer,measuring the length oftheprojection ofthecolonyonthelongaxis
of the leaf. Intertwined colonies were considered immeasurable.
Theywere,however,recorded todeterminetheproportions ofinfectionunitsperdevelopmentalphase.
Host cells were classified as necrotic if they showed one or
moreofthreequalities:browningofthecell contents,collapseof
cellwalls andautofluorescence.The lattercriterionhasbeenused
also by Samborski etal (23).The number of autofluorescent host
32
Table 1.Average colony length (in|jm)ofPuccinia hordei in26different barley
lines at fourmeasuring times.
Host
genotype
Time of sampling
U\
(>\
143*
16%
v
P7-L -1
P7-L -2
p7-L -1
p7-L -2
174g
178g
172g
330
326
419
385
f
f
gh
8
429
447
768
773
f*
8
j
j
973 f
940 f
1640 .
i
1436 i
P3-L-1
P3-L -2
p3-L -1
p3-L -2
134 def Z
133de
171g
178g
174
187
430
400
bc
bed
h
gh
230
262
816
761
cd
d
j
j
446
490
1490
1433
c
c
i
i
P7-Ze-1
P7-Ze-2
p7-Ze-l
p7-Ze-2
141 ef
142ef
143ef
141ef
257
255
328
314
e
e
f
f
379
330
572
585
ef
e
h
h
780
772
1188
1194
e
e
Rh
gh
P3-Ze-1
P3-Ze-2
p3-Ze-l
p3-Ze-2
94a
117bc
151f
144ef
123
125
310
273
a
a
f
e
126
133
651
553
a
ab
i
h
220
169
1279
1290
ab
a
h
h
P7-Va-1
P7-Va-2
p7-Va-l
p7-Va-2
143ef
137ef
128ede
135def
206
200
217
209
bed
bed
d
bed
241
255
379
341
d
d
ef
e
P3-Va-1
P3-Va-2
p3-Va-l
p3-Va-2
109b
120bed
141ef
135 ef
116 a
211 cd
209 bed
116
128
401
405
a
a
f8
fg
_
-
Cebada Capa"
Rika"
174V
-
135def
194bed
182be
105ab
113a
112a
656
740
1118
1229
d
de
8
gh
299
168
1210
1185
b
a
8h
gh
287b
211 ab
Expressed indays after inoculation.
P indicates presence,p absence of the dominant allele of themajor gene;
7 and 3 refer toPa7 and Pa3,respectively; L, Ze and Va indicate the repicient genotypes,respectively L94,Zephyr andVada.
Cebada Capa is the donor of thePa7 allele,Rika ((=Rika x (Rika x Baladi)))
isdonor of the Pa3allele.
Each entry is themean length of 80 established colonies in four primary
leaves unless indicated otherwise.
Per column,entrieswith a common letter are not significantly different
(P<0.05) according toDuncan's multiple range test.
Underlined entries are averages of the lengths of early aborted and ofestablished colonies,since the latter could notbe recognized unambiguously.
33
Table 2. Designation and definition of the infection units of Puccinia hordei
inbarley,according to their stage ofdevelopment.
Developmental
phase
Designation of the
infection unit
Definition
Pre-penetration
Non-penetrant
Appressorium over stoma
without formation ofa
substomatal vesicle
Substomatal vesicle
(SSV) formation
Aborted SSV
SSVwithout hyphae
Establishment
Early aborted colony
SSVwith primary infectionhyphae and up to six
haustorial mother cells
Colonization
Established colony
or
Late aborten colony
Branched hyphae,six or
more
haustorial mother cells;
sporogenic tissue may be
present
Reproduction
z
Successful colony
At l e a s t some u r e d i o spores are formed
Arrested in the r e s p e c t i v e developmental phase
c e l l s p e r i n f e c t i o n u n i t was c o u n t e d o r a s s e s s e d . W i t h l a r g e c o l o n i e s t h e p r o p o r t i o n of t h e c o l o n y a r e a w i t h c e l l b r o w n i n g was e s t i mated.
In t h e s t a t i s t i c a l a n a l y s e s , t h e e x p e r i m e n t a l u n i t s were
means o f ) t h e r e s p o n s e s p e r l e a f .
RESULTS
OneoftheP3-Va linessegregated forhypersensitiveresistance.
Theobservations onthis linewerediscarded asfortheleavescollected at4*5d.p.i., since the results suggested that theleaves
sampled onthatdaybelonged tosusceptible segregants.Nosegregation for hypersensitivity occurred in the otherlines,indicating
that these lines descended from parents thatwere homozygous for
thischaracter.
Colony growth. The average lengths of theestablished colonies
at the four timesofsampling arepresented inTable 1.Theeffect
of Pa3 on the colony growth was pronounced: a significant lagin
colony length was apparent as early as 2\ d.p.i. The growthre-
(the
34
tarding effect of Pa7 was less serious.At2^d.p.i.thecolonies
in the P7 lines did not provetobesmaller thaninthep7lines.
The effect of Pa7 became only detectablewhenthecolonies inthe
susceptiblecounterparts averaged atleast300pm.
Four susceptible lines perrecipientgenotypewere availableto
compare the effect of the minor genes forPRoncolonygrowth.No
systematic differences between p3 andp7lineswere foundwithina
recipientgenotype. Inaccordancewiththe levelofPRoftherecipients, the colonies in the susceptible L-lineshadahigherrate
of growth than those in the susceptibleVa-lines.Thecoloniesin
theZe-linesgrewatanintermediaterate.
The effect of the PR genes on colony growth inthelineswith
Pa7 was evident.At6*sd.p.i. therewasasignificant interaction
effectbetween the Pa7alleleandthePR-background (ANOVA,critical level P <0.01), indicating thatthelargerthe average colony
length in the susceptible line,thelargertherelative arrearsin
the hypersensitive counterpart. In the lines carrying the Pa3
allele, the colonies in the L-lines reached the largest size,as
expected. The colony lengths in Ze- and Va- lines showednosignificant differences; in both types of lines the average colony
length hardly increased with time.The growthretardingeffectof
PRgeneswasobscuredbythestrongexpressionofPa3.
Colony abortion.
The proportion of infection units blocked as
non-penetrants and as aborted substomatalvesicles (SSV)were calculated from the data obtained from all 16leaves per line.The
mean proportion of non-penetrantswas6%,withoutconspicuousdifferences between the lines (Kruskal-Wallis non-parametric test,
P =0.14). The differences in SSV-abortion were not significant
either (Kruskal-Wallis, P =0.10) and too low to be of interest
(averageproportion 0.9%).
Forthedegreeofearly abortion,pronounced differences between
the lines were found (Table3,Fig.1 ) .InalltheVa-lines andin
the P3-L and P3-Ze lines ahigh proportion of colonieswas found
that fitted the (morphological) definition of early abortion as
giveninTable 2 (Table 3). Manyothercolonies inthePa3carrying
lineswereblocked littlebeyondtheestablishmentstage:thesepossessed more branched hyphae suggesting asuccessfulestablishment,
but had only five to eight haustorial mother cells. As aconse-
35
P7-L
n.170
4030
20
10
H^F^
P 7 - Z e n=187
40
30j
20
10
rflMJ .
p3.7-Va
P3~Va "-271
=643
40
I
200
40
P7-CC
20
O
,0
*
o
ij
200 400
•hwfTT-.
1200 1600 2000
P3-Rika
n=116
H
l u 30
fc
JTk
400 800
EARLY ABORTED COLONIES
I
I LATE ABORTED COLONIES
^
SUCCESSFUL COLONIES
GENETCAL BACKGROUND:
l=L94
Ze=ZEPHYR
40
n=81
30
20
Va=VA0A
CC=CEBADACAPA
800
1200
COLONY LENGTHlym)
Rlka=RIKAX(BALAOI XRIKA)
P 7=PA7PA7
P 3 :Pa 3 PA3
P7:p*7M7
P3=P»3P»3
200
400
800 1200
COLONY LENGTHfpm)
Fig 1- Frequency distribution of the colony length of Puccinia hordei in
seedlings of barley genotypes differing in genetical background and in the
presence (P) or absence (p) of a major gene (Pa7 or Pa3) for resistance. The
leaves were collected 16% (Vada background) or 14% (the others) days after
inoculation. Each graph is based on the measurements of n colonies. The three
classes of colonies are defined inTable 2.Note the change of scale at 400u.m.
36
Table 3. Percentage ofearly aborted colonies of Puccinia hordei inbarley seedlings with different genotypic backgrounds and different alleles ofmajor genes
for resistance.
y
Genotypic
background
Line
Alleles of themajor genes
Pa7
pa7
Pa3
L94
1
2
10 bc Z
2a
4 ab
3a
32 de
33ef
4 ab
2a
2
7 abc
12c
3a
4 ab
50gh
22d
5 ab
3a
2
52h
37ef
Zephyr
Vada
1
1
Cebada Capa
Rika
55hi
42 fg
pa3
54hi
33 def
42 fg
43 fg
31de
63i
y
Pa7 and Pa3 cause ahypersensitive reaction,pa7 and pa3 a susceptible
reaction.
z
Entries with a common letter are not significantly different (P< 0.05)
based onDuncan's multiple range test.
Table 4. Percentage of late aborted colonies of Puccinia hordei inbarley seedlings with different genotypic backgrounds and different alleles ofmajor genes
for resistance.
Alleles of themajor genes
Genotypic
background
Line
L94
1
Zephyr
Vada
Pa7
pa7
Pa3
pa3
2
88 y de Z
99 e
0a
2a
99 e
91 de
1a
2a
2
97 e
86 d
2a
3 ab
00 e
00 e
2a
2a
64c
56c
5 ab
8 ab
96 de
00 e
14 b
10ab
1
1
2
Cebada Capa
100
e
Rika
100 e
x Pa7 and Pa3 cause a hypersensitive reaction, pa7 and pa3 a susceptible reaction.
y
Entries are the average percentages of established colonies that failed to
form urediospores. The data are obtained from leaf segments collected 16%
(Vada-background)or 14% (the others)days after inoculation.
Entries with a common letter arenot significantly different (P<0.05) based
onDuncan's multiple rangetest.
37
quence, adistinction between established and early abortedcoloniesoftenwasimpossiblewiththese lines.ThepresenceofthePa3
allele did not raisethe levelofearlyabortion intheVadabackground. In contrastwith Pa3,the Pa7allelehad scarcely anyeffectonthelevelofearlyabortion.
The degree of lateabortionwasassessed fromtheleafsegments
of the final sampling date.The coloniesthathadnotreachedthe
reproduction phase at this date were classified as late aborted
colonies. Itmust beborne inmind thattheleveloflateabortion
maydependonthedateofsampling.Almostallestablished colonies
inthegenotypeswithoutadominantallele forhypersensitivity had
reachedthereproductionphaseatthe final sampling date (Table4,
Fig.1 ) .Those that hadnot,werenotlikelytohavethepotential
to become reproductive. The susceptible Va-lines showed abarely
higher leveloflateabortionthantheL-andZe-lines.
Of the Pa7 containing genotypes the leveloflate abortionwas
highest in Cebada Capa.No reproduction of the pathogen wasobserved inthiscultivar (Table4)andthecolonygrowthceased ata
rather small colony size (Table1,Fig.1 ) .Intheother genotypic
backgrounds the Pa7 gene did notpreventreproductioncompletely.
Especially intheP7-Va-lines asubstantialproportion oftheestablished colonies succeeded in the formation ofatleastafewurediospores (Fig.1 ) .
With the genotypes carryingPa3,lateabortiongenerallyoccurred atsmallercolony sizesthanwithPa7.Except fortheP-L-lines,
themajorityofthelateaborted colonieswerearrested justbeyond
the establishing phase (see above).Ofthecoloniesthatcontinued
growth, only a fewhadreached thereproductionphaseatthe final
sampling date. In the P„-L lines relatively numerous colonies
reached lengthsofover400)jm(Table1,Fig.1 ) .
Host cell necrosis.
Generally, small colonies were associated
with the necrosis associatedwithautofluorescence,large colonies
withbrowned andcollapsed cellswhichwerenon-autofluorescent.
In the genotypes with a high levelofearlyabortionduetoPR
(Va-lines and CC) (Table3)theearlyabortedcolonieswererarely
associated with host cell necrosis (Table 5 ) .The early abortion
due to Pa3 (Table3)onthecontrary,wasalmostalways associated
withnecrotic hostcells (Table5 ) .
38
Table 5. Percentage ofearly aborted coloniesofPuccinia hordei associated with
at leastoneautofluorescent host cell inbarley seedlings with different genotypic backgrounds anddifferent allelesofmajor genesforresistance.
„
..
Allelesofthemaior genes
J
pa7
Pa3 °
Genotypic
background
Pa7
L94
Zephyr
Vada
32
10
5
Cebada Capa
11
Rika
23
14
3
88
92
29
pa3
16
10
3
88
z Pa7 and Pa3 causeahypersensitive reaction,pa7andpa3 asusceptiblereaction.
Pa7 and Pa3 differed in the degree to which hostcellsbecame
necrotic inestablishedcolonies.At2%d.p.i.theestablished colonies inPa7 carrying lines were associatedwithcellnecrosisas
much as or little more than the susceptible lines (Table 6). From
4*sd.p.i. on, the Pa7 gene induced abrowningofhostcellswhich
progressed until at thefinal sampling dateinthelargercolonies
cell browning occurred over 25-50%, and in CC up to 100%of the
colony area.These colonies were macroscopically visible assmall
dark spotsontheleaves.
Pa3 induced cellnecrosis aroundthemajority ofthecoloniesin
theP3-L,P3-Ze andRikagenotypes asearly as 2\ d.p.i. (Table6 ) .
InP3-Va the degree of necrosis appeared less, probably owingto
the early abortion causedbyPRgenes,whichseldom goeswithhost
cellnecrosis.Thecolonies seemedtobe 'knockeddown'byPa3 just
at or shortly after establishment. The cell necrosis mostly concerned the cells thatwere associated with the haustorial mother
cellsoftheyoungcolony.
In all genotypes carrying Pa3,especially in the P3-Llines,
part of the colonies seemed to recover from the early inhibitory
effects of Pa3. Their mycelium passed by the necrotic hostcells
andcontinued growthwithoutprovoking furthercellnecrosis.These
larger colonies were associatedwithonly afewbrownedhostcells
39
Table6.AveragepercentageofestablishedcoloniesofPucciniahordeiassociatedwithatleastonenecrotic (=autofluorescent,brownedorcollapsed)host
cell, 2\ daysafterinoculation.
„ . .
Genotypic
background
Allelesofthemajorgenes
J
Pa7
L94
1
2
48 (1.4)z49(1.9) 84(1.6) 40(1.3)
55(1.3) 21(1.2) 95 (2.1) 51(1,5)
Zephyr
1
2
33(1.5) 21(1.4) 88(2.4) 20(1.1)
36(1.4) 35(1.3) 98(2.2) 20(1.6)
Vada
1
28(2.0) 6(1.3) 25(1.3) 10(1.3)
2
CebadaCapa
Rika
pa7
Pa3
"
Line
pa3
29(1.4) 20(1.2) 35(1.4) 5(1.5)
15(1.2)
75(1.7)
Pa7andPa3causeahypersensitivereaction,pa7andpa3asusceptiblereaction.
y
InthelinescarryingPa3theestablished coloniescouldnotberecognized
unambiguouslyfromtheearlyabortedcolonies.Thedataconcernallcolonies
whichformedatleastonehaustorialmothercell.
z Withinbracketstheaveragenumberofnecroticcellsassociatedwiththecoloniesconcernedaregiven.
in the centre of the colony. The browning seldom occupied more than
25% of the colony area. Since Pa3 causes amacroscopically visible
chlorosis and necrosis of the host tissue around the infection
sites, it appears that the larger colonies also were associated
with host cell alterations that were not detectable with the methods used here.
In the susceptible lines, particularly the L- and Ze-lines, many
established colonies were associated with autofluorescent cells
(Table 6 ) ,but the number of these cells per colony was small and
the amount of necrosis was negligible in relation to the colony
area.
40
DISCUSSION
As is known from macroscopical investigations, theminorgenes
for PR to leaf rust inbarleycause areducedrateofdevelopment
of the colonies (8,12).Inthepresentexperiment,theurediaon
the susceptible lines withthepartially resistantVadabackground
appeared a few days later than on the lines with L94 and Zephyr
background. Inconnectionwiththereduced rateofdevelopment,the
growth rate of thecolonies isaffectedbythePRgenes (Table1 ) ,
ashasbeenreportedbefore (2,4,5 ) .Inaddition,minorgenes for
PR can reduce thenumber ofurediapercm2 leafarea (18)by causing a substantial early abortionofcolonieswhichseldom isassociated with host cell necrosis (10) (see the data collected from
thep7-Vaandp3-Va lines inTables 3and5 ) .
The dominant alleles of the two major genes for resistanceto
leaf rust considered (Pa7 and Pa3)clearlydiffered intheirmode
of action, irrespective of the genetic background. The action of
Pa7 became manifest several days after the establishment of the
fungusby agradualgrowthretardation associatedwithanextensive
cell browning. Incontrast, Pa3 caused an early cessation ofthe
fungal growth associated withhostcellnecrosis.Partofthecolonies continued growth without provoking further necrosisofhost
cells.Similardifferences inactionbetweenmajorgenes forresistancewere reported,eg,forflaxand flaxrust (9),wheatandstem
rust (21)and wheatandpowderymildew (7).Thegeneticbackground
may modify themodeofactionofthehypersensitivity alleles,although the general characteristics of the resistance reactionare
notlost (6,21).Inthepresentstudy,thegeneticbackground also
influenced the expression ofthemajorgenes.WithPa7,relatively
many established colonies reached the reproductive phase in the
Vada background, whereas inthedonorcultivarCebadaCapanosuccessful colonies were found (Fig.1 ) .Since both Cebada Capa and
Vada possess ahigh gene dose for PR (17),itislikelythatthe
mitigation of the action of the Pa7allele intheVadabackground
is attributable to other than PR genes.Apparently, the PRgenes
andPa7actindependently andconsecutively:onlythecoloniesthat
are not arrested byearly abortionduetoPRhavetocopewiththe
barrier raised by Pa7. These results confirm the findings of
41
Parlevliet (16)who demonstrated thatthemoreminorgenes forlow
infectibility arepresent, thefewernecrotic flecksappearinthe
presenceofPa7.Clifford (3)also found agoodcorrelationbetween
thelevelofPRinthegeneticbackground andthenumberofnecrotic
spotsduetohypersensitivity.Heinvestigated cultivarswhichwere
recessive for Pa7,but dominant for at least Pa2. Thegrowthretarding effect of PR genes remained unimpeded in the presenceof
Pa7 (Table 1), inaccordancewiththeslower appearance ofnecrotic
flecks in lines with Pa7 in a high PR background found by
Parlevliet(16).
The effects of the PR genes were lesseasily recognized inthe
presence of Pa3, since this major geneacts aboutasearlyasthe
PR genes.Differences in growth rate duetoPRgeneswere largely
obscured by the substantial abortion of coloniesduetoPa3ator
shortly afterestablishment.Theresults suggestthatPa3 actseven
more intensely in theZephyrbackground thaninthedonorcultivar
Rika.Despitethelowdegreeofreproduction intheP3-lines atthe
finalsampling date,itcannotbeexcluded thatmorecolonieswould
have come to reproduction if the sampling of the leaveshadbeen
postponed some days. J.E.Parlevliet (unpublished)observed afair
degreeofsporulation inthethreePa3carryingrecipients.
Itcould be demonstrated that the early abortionbyPa3occurs
shortly after the formationofthe firsthaustoria (11).Thecells
inwhich these haustoria are formedbecomenecrotic.Thisexplains
the high association of early abortion due to Pa3 withhostcell
necrosis in genotypes with little PR (Table 5 ) .Considering the
formationofthe firsthaustoria assuccessful establishment,itis
moreappropriate tospeakofearlyhypersensitivity. Early abortion
by PR genes occurs before the formationofhaustoria asafailure
of establishment (11).This means that thePRgenes andPa3 again
actconsecutivelybutwith ashorttimeinterval.Onlythecolonies
not arrested by PRgenes (withoutnecrosis)maybe arrestedbyPa3
(with necrosis). The levelofPR ingenotypeswith anearly acting
allele for hypersensitivity can be assessed by thedegreeofhost
cell necrosis. This implicates that Rika does not have anappreciable level of PR.A similar difference inthedegreeofhost
cell necrosis between race non-specific and race-specific resistancewasreported foroatsandpowderymildew(1).
42
In the presence
level
than
of
a late
a c t i n g major gene for r e s i s t a n c e
o f PR i n t h e g e n e t i c b a c k g r o u n d c a n b e e s t i m a t e d m u c h
in the presence
suggest
dently
t h a t minor
of
and major
and c o n s t i t u t e d
hypersensitive
colonies
an e a r l y
lacking host cell
material,
resistance
because
genes
different
resistance
with
for
gene.
resistance
The
acted
necrosis
indepen-
of small
should be promising
a high l e v e l of p o s s i b l y
easier
results
defence systems. Plants
a high proportion
t h e y may c a r r y
in their genetic
a c t i n g major
the
combining
aborted
parental
durable
background.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Carver, T.L.W., and A.J.H. Carr. 1980. Some e f f e c t s of host r e s i s t a n c e on
the development of oat mildew. Ann. Appl. B i o l . 94: 290-293.
C l i f f o r d , B.C. 1972. The h i s t o l o g y of race n o n - s p e c i f i c r e s i s t a n c e to
Puccinia hordei Otth. in b a r l e y . Proc. 3rd Eur. Mediterr. Cereal Rusts Conf.
Prague, I : 75-79.
C l i f f o r d , B.C. 1974. Relation between compatible and incompatible i n f e c t i o n
s i t e s of Puccinia hordei on b a r l e y . Trans. Br. Mycol. Soc. 63: 215-220.
C l i f f o r d , B.C., and H.W. Roderick. 1978. A comparitive h i s t o l o g y of some b a r ley brown r u s t i n t e r a c t i o n s . Ann. Appl. B i o l . 89: 295-298.
C l i f f o r d , B.C., and H.W. Roderick. 1981. Detection of c r y p t i c r e s i s t a n c e of
b a r l e y to Puccinia h o r d e i . Trans. Br. Mycol. Soc. 76: 17-24.
Dyck, P . L . , and D.J. Samborski. 1968. Genetics of r e s i s t a n c e to l e a f r u s t in
the common wheat v a r i e t i e s Webster, Loros, B r e v i t , Carina, Malakof and
Centenario. Can. J . Genet. Cytol. 10: 7-17.
Haywood, M.J., and A.H. E l l i n g b o e . 1979. Genetic c o n t r o l of primary h a u s t o r i a l development of Erysiphe graminis on wheat. Phytopathology 69: 48-53.
Johnson, D.A., and R.D. Wilcoxson. 1979. I n h e r i t a n c e of slow r u s t i n g of b a r ley i n f e c t e d with Puccinia hordei and s e l e c t i o n of l a t e n t period and number
of u r e d i a . Phytopathology 69: 145-151.
L i t t l e f i e l d , L . J . 1973. H i s t o l o g i c a l evidence for d i v e r s e mechanisms of r e s i s t a n c e to flax r u s t , Melampsora l i n i . P h y s i o l . Plant P a t h o l . 3: 241-247.
Niks, R.E. 1982. Early abortion of colonies of leaf r u s t , Puccinia h o r d e i ,
in p a r t i a l l y r e s i s t a n t b a r l e y s e e d l i n g s . Can. J . Bot. 60: 714-723.
Niks, R.E. 1982. Haustorium formation of Puccinia hordei in leaves of hypers e n s i t i v e , p a r t i a l l y r e s i s t a n t and non-host p l a n t genotypes. Phytopathology
72: (This i s s u e ) .
P a r l e v l i e t , J . E . 1975. P a r t i a l r e s i s t a n c e of b a r l e y to leaf r u s t , Puccinia
h o r d e i . I . Effect of c u l t i v a r and development s t a g e on l a t e n t p e r i o d .
Euphytica 24: 21-27.
P a r l e v l i e t , J . E . 1976. The g e n e t i c s of s e e d l i n g r e s i s t a n c e to l e a f r u s t ,
Puccinia hordei Otth. in some s p r i n g b a r l e y c u l t i v a r s . Euphytica 25: 249-254
P a r l e v l i e t , J . E . 1978. F u r t h e r evidence of polygenic i n h e r i t a n c e of p a r t i a l
r e s i s t a n c e in b a r l e y to leaf r u s t , Puccinia h o r d e i . Euphytica 27: 369-379.
P a r l e v l i e t , J . E . 1978. R a c e - s p e c i f i c a s p e c t s of polygenic r e s i s t a n c e of b a r ley to leaf r u s t , Puccinia h o r d e i . Neth. J . Plant P a t h o l . 84: 121-126.
43
16. Parlevliet, J.E. 1980. Minor genes for partial resistance epistatic to the
Pa7 gene for hypersensitivity in the barley- Puccinia hordei relationship.
Proc. 5thEur.Mediterr. CerealRusts Conf.,Bari and Rome: 53-57.
17. Parlevliet, J.E., and H.J. Kuiper. 1977.Resistance of some barley cultivars
to leaf rust,Puccinia hordei;polygenic,partial resistance hidden bymonogenic hypersensitivity. Neth.J. Plant Pathol.83:85-89.
18. Parlevliet, J.E., and H.J. Kuiper. 1977. Partial resistance of barley to
leaf rust, Puccinia hordei. IV.Effect of cultivar and development stage on
infection frequency. Euphytica 26:249-255.
19. Parlevliet, J.E., and A. van Ommeren. 1975. Partial resistance ofbarley to
leaf rust, Puccinia hordei. II. Relationship between field trials, micro
plot tests and latent period.Euphytica 24:293-303.
20. Robinson,R.A. 1973.Horizontal resistance.Rev. Plant Pathol.52:483-501.
21. Rohringer,R., W.K.Kim, and D.J. Samborski. 1979. A histological study of
interactions between avirulent races of stem rust and wheat containing resistance genes Sr5, Sr6,Sr8,or Sr22. Can.J. Bot.57:324-331.
22. Rohringer,R., W.K. Kim,D.J. Samborski,and H.K. Howes. 1977.Calcofluor: an
optical brightener for fluorescence microscopy of fungalplant parasites in
leaves. Phytopathology 67:808-810.
23. Samborski, D.J., W.K.Kim, R. Rohringer, H.K. Howes, and R.J. Baker. 1977.
Histological studies on host cell necrosis conditioned by the Sr6 gene for
resistance inwheat to stem rust.Can.J. Bot.55:1445-1452.
24. Van der Plank, J.E. 1963. Plant diseases: epidemics and control. Academic
Press,NewYork/London. 349pp.
25. Zadoks, J.C., and R.D. Schein. 1979. Epidemiology and plant diseasemanagement. Oxford Univ. Press,NewYork,Oxford. 427pp.
44
4. Comparative histology of partial
resistance and non-host reaction to leaf
rusts in barley and wheat seedlings
by
R.E.Niks
Institute of Plant Breeding, Agricultural University, Lawickse
Allee166, 6709 DB Wageningen, The Netherlands.Presentaddress:
ICARDA,P.O.B. 5466,Aleppo,Syria.
Theauthorexpresseshisgratitude toH.J.Kuiper forhistechnical
assistance,andtoJ.E.Parlevliet,J. Sneep andJ.C.Zadoks,AgriculturalUniversity ofWageningen,TheNetherlands,fortheirvaluablecriticism onthemanuscript.
Accepted 21May1982
ABSTRACT
The histological responses involved inpartial resistance (PR)
andnon-hostreactionwerecompared inthreebarley andthreewheat
genotypes inoculated with Puccinia hordei and P. recondita
tritici,
the leaf rustpathogens ofbarley and wheat.Non-host andPRreactionstotheleafrustpathogenswerecharacterizedbyahighpro
portion of colonies thatwere arrested early, (i.e., immediately
after the formation ofthe firsthaustorialmothercells)andwere
associatedwithlittleornoplantcellnecrosis.Eightbarleygeno
types, fourwithalowand fourwithahighlevelofPRto P. horde
were inoculated with theleafrusts P. hordei,
P.recondita
secalis
and P.recondita
tritici.
The latterpathogenproduced reproductive
structures on all four barley genotypes withalowlevelofPRto
P. hordei, suggesting thatalleles forlowPRto P. hordei alsoreducedtheeffectivity ofthereactionto P. recondita
tritici.
45
INTRODUCTION
Apart from the hypersensitive resistance,another and lessconspicuous type of resistance isknowninthebarley/leaf rust (Puccinia hordei) relation, which is characterized by a slow rateof
epidemic development despite the presence of asusceptibleinfectiontype.Thisresistance hasbeendesignated 'partialresistance'
(PR)(15).Although theepidemiological andgenetical aspectsofPR
arewell documentated (6,16),little isknownabouttheunderlying
mechanisms. Inahistological studyoftheinfectionprocessofthe
leaf rust fungus inbarley seedlings,developmentofalargeproportion of leaf rustcolonies inpartially resistantgenotypeswas
arrested justafterthe firstinfectionhyphae andafewhaustorial
mother cells were formed (11).This 'earlyabortion'resemblesthe
non-hostreactions describedbyHeath(5).
The present study compares the histological responses involved
in PR to barley leaf rustwith non-host reactions to other leaf
rustpathogens andinvestigateswhetherthereisarelationshipbetweenlevelofPRandnature ofthenon-hostreaction.
MATERIALSANDMETHODS
Inthe firstexperimentthehistology ofeffects associatedwith
PR and non-host reactionwerecompared.Twoplantboxeswiththree
barley andthreewheatgenotypesweregrown.Oneboxwas inoculated
withmonospore culture 121Aof P.hordei, theotherwith amonospore
culture of P. recondita
tritici.
The barley genotypes wereL94,
Vada and 139-4. L94 is highly infectible to P. hordei, Vada and
139-4have a high level of PR. The wheat cultivars,Saratovskaja
210 (S210), Adonis, andDuri, arefullysusceptible totheisolate
of P. recondita
tritici
used,buttheir levelofPRtothispathogen
was unknown. The experimentwascarried outintwoconsecutiveseries. In each genotype/pathogen combination, six orsevenprimary
leaves were inoculated with urediospores utilizing a settling
tower. Segments of the cental parts of the leaves were collected
200 (inthe firstseries)or225 (inthesecond series)hourspost
inoculation (h.p.i.). They were cleared and stained for fluorescence microscopy (11).The infection units in the leaves were
46
classified according to their phase of development at the dayof
sampling. The classes were: non-penetrant, aborted substomatal
vesicle,early abortedcolony andestablished colony (13).Theproportion of abortion in eachphase was calculated by dividingthe
number of infection units arrested inthegivenphasebythetotal
number of infection units that entered thatphase.Thenumbersof
colonies in the fourclasseswererecorded foreachleaf.Alsothe
presence of host cell necrosis, recognized byautofluorescenceor
cell browning, was noted. Generally, the experimental units for
statistical analyseswere (theaveragesof)theresponsesperleaf,
but sometimes the results from two or three leafpieceswithfew
infectionunitswerecombined to formone largersample.
Inthesecondexperiment thereactionsofeightbarley genotypes
to the leaf rustpathogens ofwheat, P.recondita
tritici,
andof
rye, P. recondita
secalis,
were compared with the reactions to
P. hordei. Particularly, the relationship between the levelofPR
to P.hordei and the nature of thereactionstothetwoother leaf
rustpathogens was studied. Four barley genotypeswith ahighinfectibility (19,20),L94, L92, L98,andAkka,and fourwith alow
infectibility (due to earlyabortion, 11),Vada,C-118,C-123,and
C-92, were grown. Eachwasinoculatedwithurediospores ofamonospore culture of one of the three leaf rust pathogens.The leaf
pieces were collected at 275 h.p.i. for those inoculated with
P. hordei. Plants inoculated with the other leaf rust pathogens
were sampled 2 days later.Theexperimental process andthenature
of the observations were the same asinthe firstexperiment,but
in addition the colony lengthsof15established coloniesperleaf
were assessed, if available,with aneyepiecemicrometer.Thisexperimentalsocomprised oftwoconsecutiveseries.
RESULTS
Thethree leafrustpathogens couldbedistinguished bythemorphology of their substomatal vesicles (SSV) (Fig.1 ) .Generally,
the SSV of P. hordei was cigar-shaped with atransverse septum in
the middle. From the SSV two infection hyphae developed, growing
parallel to the long axis of the leaf. The SSV of P. recondita
47
Fig. 1. Typical shape of early aborted colonies of Puccinia hordei (A),
P. recondita t r i t i c i (B), and P. recondita secalis (C) in primary leaves of barley. Indicated are substomatal vesicles (SSV), infection hyphae (IH), and haust o r i a l mother cells (HMC). Part of the walls of barley mesophyll cells are drawn.
The bar represents 10 |jm.
secalis
was more s l e n d e r and t h e septum was e i t h e r a b s e n t o r h a r d l y
v i s i b l e . The SSV of P. recondita
tritici
was e g g - s h a p e d and d e v e l o p e d
one i n f e c t i o n hypha, which t e n d e d t o grow t r a n s v e r s e l y t o t h e l o n g
a x i s of t h e l e a f . The t h i c k n e s s of t h e hyphae of P. hordei was a b o u t
h a l f of t h a t of t h e two o t h e r l e a f r u s t s ( c a . 2 . 5 and 5.0 pm d i a meter, r e s p e c t i v e l y ) .
In t h e f i r s t e x p e r i m e n t , u r e d i a of a low i n f e c t i o n t y p e (IT 5 on
t h e s c a l e of McNeal e t a l [ 8 ] ) a p p e a r e d on b a r l e y g e n o t y p e L94 i n -
48
oculated with P. recondita
tritici.
The colonies hadhyphaeofca.
5.0 urndiameter.Urediospores fromtheseurediaweretransferred to
a fewplantsofbarley (cv.L98)andwheat (cv.Kaspar)bymeansof
a brush. After incubation of these plants, only Kaspar showed a
susceptible reaction (IT 9). P. recondita
tritici
didnotgivevisible symptoms in Vada and 139-4, nor did P.hordei in the three
wheatgenotypes.
There were no consistent differences among theplant/rust speciescombinations intheproportions ofpenetrations ofappressoria
through the stomata (Table 1). There was atendency towards areduced stoma penetration on non-hosts (e.g., Duri, Vada), but the
opposite also occurred (S210,series 2). Often,theproportions of
non-penetrating appressoria of eitherpathogenic ornon-pathogenic
rustspeciesonaplantgenotypehardly differed (e.g.,139-4).The
degree ofnon-penetration onthepartially resistantgenotypesVada
and 139-4wasnothigherthanonthehighly infectible L94.
There was little SSV abortion in either the host andnon-host
combinations.
InTable2theproportions ofearlyabortedcolonies arepresented. Inthe combinationswhichwere considerednon-pathogenic,the
degree of early abortionwasclearlyhigherthaninthepathogenic
combinations. In the wheat/P.hordei combination practically all
colonies were arrested early. The degree of early abortion of
P. hordei in the host genotypes Vada and139-4washigher thanin
L94 reflecting differences inlevelofPR. Inthethreewheatcultivars fewcoloniesof P. recondita
tritici
aborted early.Thethree
cultivars did not differsignificantlyindegreeofearly abortion
(Kruskal-Wallis test,P£ 0.05). Inthecombinations (hostaswell
as non-host) with a high degree of early abortion, usually less
than 50%of the early aborted colonieswere associatedwithplant
cell necrosis,but inwheat the degree of necrosis was notwell
reproducible (Table2 ) .
The leaf segments were sampled before the latent period had
elapsed. Consequently, the percentage of colonies that formed
urediacouldnotbe assessed.Ascanbeconcluded fromTable 2,the
proportions of established colonies (i.e., 1-the proportion of
early aborted colonies)washigh inthehost/pathogen combinations
with L94, S210, Adonis,andDuri andmoderate inthepartiallyre-
49
Table 1.Average proportions ofnon-penetrating appressoria when the primary
leaves of three barley and threewheat genotypes were inoculated withurediospores of Puccinia hordei and P.recondita tritici.
Proportion of non-penetrating appressoria
Species
Genotype Series 1
Series 2
inoculated
Barley
Wheat
L94
Vada
139-4
S210
Adonis
Duri
Average
P.hordei
P.recondita P.hordei
tritici
P.recondita
tritici
0.47
0.14
0.24
0.46
0.24
0.26
0.13
0.08
0.13
0.28
0.12
0.15
P.recondita
tritici
P.hordei
P.recondita P.hordei.
tritici
0.26
0.64
0.26
0.31
0.74
0.69
0.24
0.29
0.13
0.08
0.27
0.15
0.34
0.45
0.17
0.18
Each entry isbased on observations of six or seven leaves.
Table 2.Average proportions of early aborted colonies of Puccinia hordei and
P. recondita tritici in the primary leaves of three barley and three wheatgenotypes and the degree of cellnecrosis,associated with early abortion.
Species
Genotype
inoculated
Barley
Wheat
L94
Vada
139-4
S210
Adonis
Duri
Proportion of early
aborted colonies
Proportion of early
aborted colonies associated
. . .
-.n
- h
with
host cell
necrosis
Series
Series
1
2
1
2
1
2
1
2
P.hordei
P.recondita
tritici
P.hordei
P.recondita
tritici
0.08 0.02
0.47 0.45
0.43 0.46
0.78
0.90
0.99
0.45 0.50
0.01 0.04
0.00 0.05
0.18 0.17
0.02 0.03
0.06 0.03
0.83
0.97
0.97
P.recondita P.hordei
tritici
P.recondita P.hordei
tritici
0.06 0.00
0.08 0.02
0.11 0.03
0.17
0.00 0.50
0.00 0.33
1.00
1.00
1.00
0.99
1.00
1.00
Calculated asproportion to the total number of colonies that formed
at least one haustorial mother cell.
Calculated asproportion to the number of early aborted colonies.
0.41 0.54
0.16 0.48
0.12 0.35
50
Table 3.Average proportions of non-penetrating appressoria and aborted substomatal vesicles (SSV)of three leaf rustpathogens in seedlings of eight barley
genotypes.
Proportion of
non-penetrating
appressoria
Rust pathogen
Series
1
P. hordei
P.recondita secalis
P.recondita tritici
0.10
0.04
0.08
Proportion of
aborted SSVs
Series
0.05
0.04
0.05
0.01
0.01
0.01
0.03
0.03
0.04
sistant Vada and 139-4with P. hordei. Of the combinations considered as non-host combinations,L94/P. recondita
tritici
gavethe
highestproportion ofestablishedcolonies.
In the second experiment the eight barley genotypes showed no
reproducible differences indegreeofnon-penetration andSSVabortion in any of the leaf rustpathogens. Therefore,perleafrust
pathogen the proportions of these types ofabortionwere averaged
over the genotypes (Table 3). The degrees of non-penetration and
SSVabortionofthenon-pathogenic P.recondita
tritici
and P.recondita secalis werenothigherthanofthepathogenic
P.hordei.
The eight genotypes represented awide range inlevelofPRto
P. hordei, aswasmanifested bythelargedifferences inproportion
of early abortion and the average lengthoftheestablishedcolonies of this pathogen (Table 4 ) . In L98,which showed alowlevel
of PR inprevious studies (18,20),ahighproportionof P. hordei
colonies abortedearly.
All the colonies of P. recondita
secalis were arrested before
theyhadproducedvisible symptoms inbarley.Arbitrarily, colonies
wereconsidered 'established'whenatleasteighthaustorialmother
cells (HMC)were observed.Suchcoloniesusuallyweremorebranched
than shown in Fig.1C;Theyweremuchsmallerthantheestablished
colonies of P. recondita
tritici
inbarley andmostlywithoutcell
necrosis. Colonies with seven or lessHMCswereconsidered 'early
aborted'. The proportion of early aborted coloniesof
P.recondita
secalis was high in all the barley genotypes (Table 4). Inthe
firstseries,thegenotypic differences forearlyabortionwerein-
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52
significant, in the second series significant according to the
Kruskal-Wallis test (P<0.01). The majority of theearly aborted
colonies of P. recondita
secalis induced amarkedthickeningofthe
mesophyll cellwall attheplaceofcontactwiththeHMC (Fig.2A).
In the preparations for fluorescence microscopy, these reactions
were only visible whenwhite lightwasused.Thedepositsprobably
contained callose, since they showed abrightyellow fluorescence
after staining with aniline blue in 0.07 MK 2 HP0 4 ,pH8.9 (see4)
(Fig.2B).Such a reaction was absent ormuch less pronounced to
that associated with early abortion of P. hordei due to PR.With
P. recondita
secalis,
no reproducible differencesbetweenthegenotypes were found fortheaveragenumberofHMCspercolony,colony
length or the degree of cell necrosis associated with thecolony
abortion.
The colonies of P. recondita
tritici
couldbeclassifiedunambiguously intothetwogroups 'earlyaborted'and 'established'.Also
with this fungus, ahighproportionofthecolonies abortedearly,
i.e., after the formation of one to fourHMCs (Table 4 ) . Itcould
notbe established whether asimilardepositofcallose containing
material occurred aswith P. recondita
secalis,
sincethe infection
hyphaeof P.recondita
tritici
grew intothedeepermesophylllayers.
In all genotypes less than 20%oftheearlyaborted colonieswere
associated with necrosis ofmesophyll cells.Established colonies
were more branched than early aborted ones,andusuallytheywere
associated with cell browning. In four of the genotypes, one or
more established colonies of P. recondita
tritici
producedurediospores.Suchurediawere of IT5orlower (Table4)andhadthicker
hyphae than the few P. hordei colonies thatappeared,duetocontamination.
OnAkkaonlysomeurediawereproduced inthe firstseries.ParticularlyonL92 P. recondita
tritici
succeeded inproducinguredia:
6 (series1)and10%(series2)oftheinfectionunits thatdeveloped beyond the SSV stage produced urediospores.The barley genotypesonwhich P. recondita
tritici
formedurediawerethe fourthat
were chosen as representative of genotypes with alowlevelofPR
to P.
hordei.
53
F i g . 2. Deposit of c a l l o s e c o n t a i n i n g m a t e r i a l (arrow) in b a r l e y s e e d l i n g adjacent to a h a u s t o r i a l mother c e l l (HMC) of Puccinia recondita s e c a l i s . A, White
l i g h t micrograph in whole mount p r e p a r a t i o n prepared for fluorescence microscopy; B, Fluorescence micrograph a f t e r s t a i n i n g in 0.005% a n i l i n e blue in 0.07 M
K 2 HP0 4 , pH 8 . 9 . The fluorescence of the d e p o s i t i s c l e a r l y v i s i b l e .
Both f i g u r e s x 1560.
DISCUSSION
There i s a remarkable resemblance between the h i s t o l o g i c a l r e s ponses involved i n PR of b a r l e y (to P. hordei) and the non-host r e a c t i o n s of wheat (to P. hordei) and b a r l e y (to P.recondita
secalis
and P.recondita tritici).
Neither PR of b a r l e y nor non-host r e a c t i o n of wheat to P. hordei r e s u l t from reduced appressorium f o r mation (10). With both PR and non-host r e a c t i o n s , the stoma pene-
54
tration, SSV and infection hyphae formation were not affected
(Tables1, 3 ) .With cowpearust,inducedby Uromyces phaseoli var.
vignae, reduced stoma penetration and SSV formation in somenonhost species was reported (4,5 ) .Rustpathogens ofgrasses,however, formed readily appressoria andSSVsingraminaceous non-hosts
(14, 21).PR and non-host reactions toleafrustswere associated
with ahigh degree of early abortion with little or no cellnecrosis (Tables2,4)andareduced sizeoftheestablished colonies
at the time of sampling (Table 4 ) .Inthenon-hostreactionthese
effects were more extreme than with PR.Notonlywere theproportions of early abortion higher innon-hostreactions,butthedeposition of callose containingmaterial atthecontactpointswith
HMCs of P. recondita
secalis suggested amore extreme reaction to
infection by anon-pathogen. A substantial colony abortion immediately after the formation of the firstHMCs was mentioned also
for other non-host combinations (5,9, 21).Deposition ofcallose
aspart of anon-hostreactiondoesnotseemtobecommon (5).The
resemblance between PR and non-host reactions was furthermoreexpressedbythe factthattheearlyabortionof P.hordei coloniesin
both non-host wheat and partially resistant barleyVadawasassociatedwithafailureintheformationofhaustoria(12).
The results suggested thatbarley is an intermediate form of
host and non-host to P.recondita
tritici:
fiveoftheninebarley
genotypes gave a symptomless reactiontothe fungusand innoneof
thegenotypesthesporulationwas abundant. P. recondita
tritici
was
reported to attack Hordeum spp. occasionally (1),which leavesit
undecided whether barley is to be considered ahost ornon-host.
The established colonies of P. recondita
tritici
inbarleywereassociated with chlorosis and necrosis ofplant tissue (Table4 ) .
Such ahypersensitive type of reaction inhosts inoculatedwitha
'wrong' forma specialis
isconventionally foundwithrustsofGramineae (2,7). Established colonies of P.hordei inpartially resistant genotypes ofbarley are (bydefinition)ofafullysusceptibleinfectiontype (Table4 ) .
The level of PR of the barley genotypes was notobviouslyrelatedwithdifferences innatureofnon-hostreactionto
P.recondita
secalis,
but the results suggested a relationship between PR and
thenature ofreactionto P.recondita
triciti.
Thebarley genotypes
55
i n w h i c h P. recondita
i n f e c t i b l e t o P. hordei
tritici
produced u r e d i o s p o r e s were a l l
highly
i n t h i s s t u d y , e x c e p t f o r L98, b u t t h i s g e -
n o t y p e h a d a l o w l e v e l o f PR i n p r e v i o u s s t u d i e s ( 1 8 , 2 0 ) .
s u g g e s t s t h a t a l l e l e s f o r l o w PR t o P. hordei
f e c t i v i t y o f t h e r e a c t i o n t o P. recondita
This
also reduce the
t r i t i c i . There i s ,
efhow-
e v e r , evidence t h a t slow r u s t i n g genes or genes for h o r i z o n t a l
re-
sistance are pathogen species-specific
more
( 3 , 17, 2 2 ) . T h e r e f o r e ,
b a r l e y g e n o t y p e s h a v e t o b e t e s t e d w i t h m o r e P. recondita
tritici
i s o l a t e s t o e s t a b l i s h w h e t h e r t h e r e l a t i o n b e t w e e n l e v e l o f PR t o
P. hordei
a n d l e v e l o f n o n - h o s t r e a c t i o n t o P. recondita
s u g g e s t e d by t h e p r e s e n t s t u d y i s r e a l o r m e r e l y due t o
tritici
co-
incidence .
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
A n i k s t e r , Y. , and I . Wahl. 1979. Coevolution of the r u s t fungi on Gramineae
and L i l i a c e a e and t h e i r h o s t s . Ann. Rev. Phytopathol. 17: 367-403.
Eshed, N . , and A. Dinoor. 1980. Genetics of p a t h o g e n i c i t y in Puccinia
coronata: pathogenic s p e c i a l i z a t i o n a t the host genus l e v e l . Phytopathology
70: 1042-1046.
G a v i n l e r t v a t a n a , S . , and R.D. Wilcoxson. 1978. I n h e r i t a n c e of slow r u s t i n g
of s p r i n g wheat by Puccinia r e c o n d i t a f . s p . t r i t i c i and host p a r a s i t e r e l a t i o n s h i p s . Trans. Br. Mycol. Soc. 71: 413-418.
Heath, M.C. 1974. Light and e l e c t r o n microscope s t u d i e s of the i n t e r a c t i o n s
of host and non-host p l a n t s with cowpea r u s t - Uromyces p h a s e o l i v a r .
v i g n a e . P h y s i o l . P l a n t P a t h o l . 4: 403-414.
Heath, M.C. 1977. A comparative study of non-host i n t e r a c t i o n s with r u s t
fungi. P h y s i o l . P l a n t P a t h o l . 10: 73-88.
Johnson, D.A., and R.D. Wilcoxson. 1979. I n h e r i t a n c e of slow r u s t i n g of b a r ley i n f e c t e d with Puccinia hordei and s e l e c t i o n of l a t e n t period and number
of u r e d i a . Phytopathology 69: 145-151.
Johnson, T . , and K.W. Buchannon. 1954. The r e a c t i o n of b a r l e y v a r i e t i e s t o
rye stem r u s t , Puccinia graminis v a r . s e c a l i s • C a n . J . A g r i c . S e i . 34: 473-482.
McNeal, F . H . , C F . Konzak, E.P. Smith, W.S. T a t e , and T.S. R u s s e l l . 1971. A
uniform system for recording and p r o c e s s i n g c e r e a l r e s e a r c h d a t a . USDA,
Agric. Res. S e r v . , Washington, D.C. ARS 34-121.
Mendgen, K. 1978. Attachment of bean r u s t c e l l wall m a t e r i a l t o host and
non-host p l a n t t i s s u e . Arch. Microbiol. 119: 113-117.
Niks, R.E. 1981. Appressorium formation of Puccinia hordei on p a r t i a l l y r e s i s t a n t b a r l e y and two non-host s p e c i e s . Neth. J . P l a n t P a t h o l . 87: 201-207.
Niks, R.E. 1982. Early a b o r t i o n of c o l o n i e s of l e a f r u s t , Puccinia h o r d e i ,
in p a r t i a l l y r e s i s t a n t b a r l e y s e e d l i n g s . Can.J.Bot. 60: 714-723.
Niks, R.E. 1982. Haustorium formation of Puccinia hordei in leaves of hypers e n s i t i v e , p a r t i a l l y r e s i s t a n t and non-host p l a n t genotypes. Phytopathology
72: (This i s s u e ) .
Niks, R . E . , and H.J. Kuiper. 1982. Histology of the r e l a t i o n between minor
and major genes for r e s i s t a n c e of b a r l e y to leaf r u s t . Phytopathology
72: (This i s s u e ) .
56
14. Ogle,H.J., and J.F. Brown. 1971. Quantitative studies of thepost-penetration phase of infection by Puccinia graminis tritici. Ann. Appl. Biol.
67: 309-319.
15. Parlevliet, J.E. 1978.Race-specific aspects ofpolygenic resistance ofbarley to leaf rust,Puccinia hordei. Neth.J.PlantPathol.84:121-126.
16. Parlevliet, J.E. 1979. Components of resistance that reduce the rate of
epidemic development.Ann.Rev. Phytopathol. 17:203-222.
17. Parlevliet, J.E. 1981.Disease resistance inplants and its consequences for
breeding. Proc. Plant Breeding Symp. II (Ed. by K.J. Frey), Iowa State
Univ.,Ames:309-364.
18. Parlevliet, J.E., and H.J. Kuiper. 1977. Partial resistance of barley to
leaf rust, Puccinia hordei. IV.Effect of cultivar and development stage on
infection frequency. Euphytica 26:249-255.
19. Parlevliet, J.E., W.H. Lindhout,A. van Ommeren, and H.J. Kuiper. 1980.
Level of partial resistance to leaf rust,Puccinia hordei,inWest-European
barley and how to select for it.Euphytica 29: 1-8.
20. Parlevliet, J.E., and A. van Ommeren. 1975.Partial resistance ofbarley to
leaf rust, Puccinia hordei. II. Relationship between field trials, micro
plot tests and latentperiod. Euphytica 24:293-303.
21. Tani, T., H. Yamamoto, Y. Ohasa, and Y.Yamashita. 1978. Non-host response
of oat leaves against rust infection. Ann. Phytopathol. Soc. Jpn
44: 325-333.
22. Van der Plank,J.E. 1978.Genetic and molecular basis ofplantpathogenesis.
Springer-Verlag, Berlin,Heidelberg,New York. 167pp.
57
5. Haustorium formation of Puccinia hordei
in leaves of hypersensitive, partially
resistant and non-host plant genotypes
by
R.E.Niks
Institute of Plant Breeding, Agricultural University, Lawickse
Allee 166, 6709 DB Wageningen, The Netherlands.Presentaddress:
ICARDA,P.O.B. 5466,Aleppo,Syria.
IwishtothankT.O.Al-Khesraji andD.M. Lösel,DepartmentofBotany, University of Sheffield, England, for their helpful suggestions. The critical remarks and stimulating interest of
J.E.Parlevliet, M.S.Ramanna, J. Sneep and J.C. Zadoks,AgriculturalUniversity ofWageningen,TheNetherlands,aregratefullyacknowledged.
Accepted 21May1982
ABSTRACT
Inbarley genotypes with early acting hypersensitivity orwith
partial resistance and innon-host species likewheat,alargeproportion of the colonies of Puccinia hordei aborted aftertheformation of the first infection hyphae and a few haustorial mother
cells.(The early abortion without collapse ofplantcellswasassociated with a"failure in the formation of haustoria. This was>
particularly evident inpartially resistantbarley.(Early abortion
with host cell necrosis occurred after the formationofthefirst
haustorium. This type ofabortionwasimportant inbarleywiththe
early acting Pa3 gene forhypersensitivity. Innon-hostwheatmost
of the infection units aborted before the formation of thefirst
haustorium.
58
INTRODUCTION
There arethreetypesofresistance associatedwithearlycessationofcolony growthoftheleafrustpathogenofbarley, Puccinia
hordei Otth.This phenomenon has been called 'earlyabortion' (6)
and ischaracterizedby arrested growth justafterthe formationof
the firstinfectionhyphae andafewhaustorialmothercells (HMC).
Itwas found in seedlings of partially resistantbarley genotypes
(6), inbarley genotypes carrying an early actinggene forhypersensitivity such as Pa3 (8),andinnon-host species suchaswheat
(7). Since a clearing and staining technique was used in those
studies which did not permit observations of haustoria, research
was initiated to determine whether early abortionoccurredbefore
orafterthe formationofthe firsthaustoria.
MATERIALSANDMETHODS
For each type of resistance (6-8)one representative genotype
wasused:wheat (Triticum
aestivum L.)cultivarDuri (non-host [7])
and barley (Hordeum vulgare L.)genotypesVada (highlevelofpartial resistance [6]),P3-Ze-2 (carrying the Pa3 allele forhypersensitivity inabackgroundwithamoderate levelofpartialresistance [8])and L94 (no detectable host resistance, control). The
plants were grown in a 37x39cm flat.The primary leaves were
inoculatedwithurediospores of Puccinia hordei, isolate 121A.This
isolate is avirulent to Pa3 and causes no symptoms inwheatcultivarDuri. Sevenofthe12primary leavespergenotypeweredensely
inoculatedwithabrush.These leaveswereused forthe observations
on the haustorium formation.Theremaining leavesreceived aninoculum doseofabout100sporespercm2 using asettling tower.These
leaveswere investigated afterpreparation forfluorescencemicroscopy (6,8), to assess the degree ofearly abortion andthedegree
of host cell necrosis associated with the early abortion. The
moderate inoculum density was necessary to preventintertwinement
of the growing colonies.The plants were transferred to agreenhouse compartment where during the firstnight the relativehumiditywaskeptatsaturation.
59
Sections of upper epidermis and mesophyll ofthedenselyinoculated leaves were sampled 20,35 and 45hours post inoculation
(h.p.i.). They were hand sectioned inaplaneparallel totheleaf
surface. The sections offourtosixleavespergenotypewereprocessed together.Theywere fixedinacetic acid:ethanol (1:3,v/v)
for 12h, boiled in 0.03% aniline blue in lactophenol: ethanol
(1:2,v/v)forca15minandcleared inanearly saturated solution
of chloral hydrate (5:2,w/v) for ca 45min at roomtemperature.
The sections were mounted in glycerol and the observations were
madebymeansofaphase contrastmicroscope (x1200).The stomatal
cavities were screened forthepresenceof P. hordei colonies.Per
colony, two infection hyphae developed antipodally forming two
poles whichwere oriented parallel to the long axis oftheleaf.
Thenumberofhaustoriawasdeterminedperpole.Colonies developed
from doubly penetrated stomata were discarded. Observations were
made on 80colonies per genotype and persamplingtime,ifavailable.
The five or six leaves that had been inoculated lessdensely,
weresampledca90h.p.i.Fiftypenetrated infectionunitsperleaf
segmentwere screened fordevelopmentalstage.
Theexperimentwasrepeated once.
RESULTSANDDISCUSSION
To determine whether or not early abortion occurs before the
formation of the first haustoria, the proportion of colonies
failing toproduce haustoriahadtobecomparedwiththe frequency
of early abortion. In all four genotypes, some colonies did not
formhaustoria (Table 1). At20h.p.i.thisproportiontended tobe
higher than at 35 h.p.i., indicating that apart ofthecolonies
produced their first haustorium between 20 and 35 h.p.i.At 45
h.p.i. counting ofhaustoriawasdifficultinL94duetothelarge
amount ofmycelium andinP3-Ze-2 andDuribecauseofanexcessive
uptakeofstainbycollapsingmesophyllcells.
No detectable
host resistance.
At35h.p.i.lessthan10%ofthe
colonies had not formed at least onehaustorium inL94 (Table1 ) .
Thisproportionwasinagreementwiththelowdegreeofearlyabortionintheleavesthatwere investigated using fluorescencemicroscopy (Table 1). The colonies without haustoria had the typical
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61
HMC
D/
HMC
f
F,
&fy %&°^
Fig. 1. Reactions to haustorium formation by Puccinia hordei; A, young haustorium and B, mature haustorium in barley cultivar L94 (no detectable resistance); C, deeply stained collapsing mesophyll cell of barley cultivar P3-Ze-2
(hypersensitivity) containing ahaustorium;D,E, failed haustorium formation in
barley cultivar Vada (partial resistance) and F, in L94 (no detectable resistance). The cell wall shows a slight thickening (arrow); G, lobed hyphal tip
(arrow) in Vada; H, partially encased, and I, completely encased haustorium in
wheat cultivar Duri (non-host). Indicated are: H, haustorium; HMC, haustorial
mother cell;E,encasement.All figures x1085.
64
REFERENCES
1. Heath,M.C. 1971.Haustorial sheath formation incowpea leaves immune to rust
infection. Phytopathology 61:383-388.
2. Heath, M.C. 1974. Light and electron microscope studies of the interactions
of host and non-host plants with cowpea rust-Uromycesphaseoli var.vignae.
Physiol. Plant Pathol.4:403-414.
3. Heath, M.C. 1977. A comparative study of non-host interactions with rust
fungi. Physiol.PlantPathol. 10:73-88.
4. Mendgen, K. 1978.Der Infectionsverlauf vonUromycesphaseoli bei anfälligen
und resistenten Bohnensorten. Phytopathol.Z. 93:295-313.
5. Mendgen,K. 1978.Attachment ofbean rust cellwall material tohost andnonhostplant tissue.Arch.Microbiol. 119:113-117.
6. Niks, R.E. 1982. Early abortion of colonies of leaf rust, Puccinia hordei,
inpartially resistant barley seedlings.Can.J.Bot. 60:714-723.
7. Niks, R.E. 1982. Comparative histology of partial resistance and non-host
reaction to leaf rusts in barley and wheat seedlings. Phytopathology 72:
(This issue).
8. Niks, R.E., and H.J. Kuiper. 1982. Histology of the relation between minor
and major genes for resistance of barley to leaf rust.Phytopathology 72:
(This issue).
9. Rohringer, R., W.K. Kim, and D.J. Samborski. 1979. A histological study of
interactions between avirulent races of stem rust and wheat containing resistance genes Sr5,Sr6,Sr8,or Sr22. Can.J.Bot.57:324-331.
10. Tani, T., H. Yamamoto, G.Kadota and N.Naito. 1976. Development of rust
fungi in oat leaves treated with blasticidin S, a protein synthesis inhibitor.Techn.Bull. Fac.Agr.Kagawa Univ. 27:95-103.
65
General discussion
Theobjective oftheinvestigationspresented inthisthesiswas,
to deepen the insight in the natureofpartialresistance (PR)in
barley to leaf rust (Puccinia
hordei Otth). This resistance is
thoughttobehorizontal sensuVanderPlank (18,19), andtheunderlying principles may also apply for horizontal resistance (HR)in
otherhost/pathogenrelationships.
Intheexperiments,thehistologyofPRhasbeeninvestigated in
relationtootherdefencemechanisms ofplantstorust fungi.Three
importantbarrierswerediscernable,acting inthreedistinctphases
ofthe infectionprocess (Fig.1 ) .
First,theplantmay lackthestimuli thatarerequired todirect
thegermtubesofarustfungustowardsthestomata.Thegermtubes
grow randomly, and seldom find stomata to form appressoria over.
This barrier was demonstrated inthe firstpaper (13)withlettuce
and P. hordei. Itmaybethemostcommondefencemechanism of(nonhost)plants to rustfungi.Failing appressorium formationdidnot
appear to be the cause of low infectibility due to PR inbarley
seedlings.
Second, an important barrier occurs at the interphase ofthe
haustorial mother cell (HMC)and the firstmesophyllcellwallto
be penetrated. Failing formation of the firsthaustoria leads to
early abortionofcolonies:thecolonies arearrested aftertheformation of a few HMCs, without provoking host cell necrosis. It
couldbedemonstrated thatPRofbarley seedlingsto P. hordei rests
on such ahampered haustorium formation bytherustcolonies (secondand fifthpaper [14,16]). Inpartially resistant adultplants,
early abortionofcolonies isalsoanimportant feature (unpublished
results).Thisbarrier isalsopertinent forthe infectionunitsof
rustthatsucceed inpenetrating stomataofnon-hostspecies (fourth
and fifthpaper [15,16]).
Third, abarrier mayoccurduringthecolonizationphase,after
the formation of at least one haustorium.Abortionofcoloniesin
this phase (late abortion) is important withthesimply inherited
66
NH
Phases of the
infection
process
NH
PR
HyR
No
PR
Incompl.
resist. +
NH
incompl.
HyR
Barrier
Appressorium
formation
First haustorium
development
II
Colonization
III
Reproduction
Fig. 1: Diagrammatic representation of the barriers a pathogen may encounter
while invading a plant. The widths of the arrows are proportional to the fractions of the colonies encountering the respectivebarriers.
Abbreviations: HyR, hypersensitive resistance; NH, non-host reaction; PR, partial resistance.
The following host/pathogen systems are given asexamples:
1.Lettuce/P. hordei(13).
2.Wheat cv.Duri/P. hordei(16).
3.Barley cv.Vada/P. hordei (14,16).
4. Barley cv.Rika x (Rika xBaladi)/P. hordei, 121A(17).
5. Barley cv.L94/P. hordei (14,16).
6. Barley P7-Va/P.hordei, 121A(17).
7.Barley cv.L92/P. recondita tritici(15).
hypersensitive resistance (third and fifthpaper [16,17]),butit
occurs also with colonies in non-hosts that succeed to pass the
second barrier. Late abortion innon-hostcombinationsmay (wheat/
P.hordei)
or may not (barley/P. recondita
secalis)
be associated
with hypersensitivity. In partially resistant barley seedlings
abortion of established P. hordei colonies isrelatively rare,and
usually not associated with host cell necrosis. In adultplants,
67
late abortion probably is amore important component of PR (unpublished results).
PR, assumed to representHR, andhypersensitive resistance,assumedtorepresentvertical resistance (VR)(18,19)inthebarley/
P. hordei relation,appeartobeessentially differentdefence systems.Theycanbedistinguished histologically,HRbeing 'pre-haustorial incompatibility',VRbeing 'post-haustorial incompatibility'
in the terminology ofHeath (8).There isneitherareasontoconsider adivision ofbothtypesofresistance artificial (2)norto
consider them representing the same kind ofresistance (12).Also
HRdoesnotseemtobean 'artifact'sensuEllingboe(5).
The data presented here suggest that treatises inwhichHRand
VR are compared (1,2, 4, 10,12)areoflittlerelevance ingaining abetter understanding ofthemechanisms causingPR inbarley
to P. hordei. PR appears to be more akin to non-hostreactionat
barrier II.There is noessential differencebetweenthehistology
of fungal development inapartially resistanthostwithincompletely acting hypersensitive resistance and that in an incomplete
non-host (Fig.1, arrow 6and 7). Itisalsoconceivable thatwhen
enough alleles forPRarecompiled intoonebarleygenotype,abarleyvariety is obtained that is a 'non-host'to P. hordei because
ofacompleteblockingofcolonies atbarrierII.
TreatisesthatrelateHRtonon-hostreactionshavenotyetcome
to the authors notice.Inarecentpaper,however,Parlevliet (11)
stated that "The former (ie,stable pathogen-specific resistance
genes,REN)consistsofresistance genes,thatareassumedtooperate within the basic resistance-pathogenicity system and confer
partialresistance.Thelatter (ie,veryunstablepathogen-specific
resistance,REN)genesarethoughttoactwithin anincompatibility
system giving low ITreactions, superimposed uponthisbasic system...". The concept ofabasicresistance-pathogenicity system on
which VR is supposed to be superimposed has been deltwith by
several authors (3,6, 9 ) .Day (3)connects theconcepttonon-host
reaction, but none of the authors relate the system to HR, as
Parlevliet (11)does. IfPRofbarley to P. hordei actswithinthe
basic system indeed (and the histological observations indicate
so), thestatementofGabrielet al. (7)thattherearenoexamples
ofnaturally occuringvariability ingenesbelongingtothissystem
68
does not hold t r u e . Genotypic d i f f e r e n c e s with r e s p e c t to the b a s i c
system are d e f i n i t e l y p r e s e n t in b a r l e y as well as i n P. hordei
(second paper [ 1 4 ] ) .
The r e s u l t s presented in t h i s t h e s i s are mainly of importance as
a c o n t r i b u t i o n to the understanding of the nature of HR. There a r e ,
however, i m p l i c a t i o n s for r e s i s t a n c e breeding. The discovery t h a t
HR and VR in the b a r l e y / P . hordei r e l a t i o n are h i s t o l o g i c a l l y d i s cernable, suggests t h a t a screening of breeding m a t e r i a l for HR by
fluorescence microscopy may be worthwhile. The methods are too l a borious t o j u s t i f y l a r g e s c a l e screening, but easy enough to be
used in p r o j e c t s t h a t are aimed t o compile a l l e l e s for HR i n t o imp o r t a n t p a r e n t s , t o be used i n a c r o s s i n g program.
REFERENCES
1.
Abdalla, M.M.F. & J.G.Th. Hermsen, 1971. The concept of breeding for uniform
and d i f f e r e n t i a l r e s i s t a n c e and t h e i r i n t e g r a t i o n . Euphytica 20: 351-361.
2. C l i f f o r d , B.C., 1975. Stable r e s i s t a n c e t o c e r e a l d i s e a s e : problems and p r o g r e s s . Rep. Welsh P I . Breed. Stn. for 1974: 107-113.
3 . Day, P . R . , 1976. Gene functions in h o s t - p a r a s i t e systems. I n : S p e c i f i c i t y in
p l a n t d i s e a s e s . Ed. by R.K.S. Wood & A. G r a n i t i . Plenum P r e s s , New York and
London: 65-73.
4. Eenink, A.H., 1976. Genetics of h o s t - p a r a s i t e r e l a t i o n s h i p s and uniform r e s i s t a n c e . Neth. J . P l a n t P a t h o l . 82: 133-145.
5 . E l l i n g b o e , A.H. 1975. H o r i z o n t a l r e s i s t a n c e : an a r t i f a c t of experimental
procedure? Aust. P l a n t P a t h o l . Soc. Newsl. 4: 44-46.
6. E l l i n g b o e , A.H., 1976. Genetics of h o s t - p a r a s i t e i n t e r a c t i o n s . I n : P h y s i o l o g i c a l p l a n t pathology, encyclopedia of p l a n t physiology. New s e r i e s . Vol.
4. Ed. by R. Heitefuss & P.H. Williams. Springer Verlag, New York: 761-778.
7. G a b r i e l , D.W., A.H. Ellingboe &E.C. Rossman, 1979. Mutations a f f e c t i n g v i r ulence in P h y l l o s t i c t a maydis. Can. J . Bot. 57: 2639-2643.
8. Heath, M.C., 1974. Light and e l e c t r o n microscope s t u d i e s of the i n t e r a c t i o n s
of host and non-host p l a n t s with cowpea rust-Uromyces p h a s e o l i v a r . v i g n a e .
P h y s i o l . P l a n t P a t h o l . 4: 403-414.
9. Loegering, W.Q., 1978. Current concepts i n i n t e r o r g a n i s m a l g e n e t i c s . Ann.
Rev. Phytopathol. 16: 309-320.
10. Nelson, R.R., 1978. Genetics of h o r i z o n t a l r e s i s t a n c e to p l a n t d i s e a s e s .
Ann. Rev. Phytopathol. 16: 359-378.
11. P a r l e v l i e t , J . E . , 1981. Race-non-specific d i s e a s e r e s i s t a n c e . In: S t r a t e g i e s
for the c o n t r o l of c e r e a l d i s e a s e . Ed. by J . F . Jenkyn and R.T. Plumb.
Blackwell S c i e n t . Publ. Ltd. Oxford, Edinburgh: 47-54.
12. P a r l e v l i e t , J . E . & J . C . Zadoks, 1977. The i n t e g r a t e d concept of d i s e a s e r e s i s t a n c e ; a new view i n c l u d i n g h o r i z o n t a l and v e r t i c a l r e s i s t a n c e in
p l a n t s . Euphytica 26: 5 - 2 1 .
69
13. Niks,R.E.,1981.Appressorium formation ofPuccinia hordei onpartially resistantbarley and two non-host species.Neth. J. Plant Pathol.87:201-207.
14. Niks, R.E., 1982.Early abortion of colonies of leaf rust,Puccinia hordei,
inpartially resistant barley seedlings.Can.J. Bot.60:714-723.
15. Niks, R.E., 1982. Comparative histology of partial resistance and non-host
reaction to leaf rusts in barley and wheat seedlings. Phytopathology
72:000-000.
16. Niks,R.E., 1982. Haustorium formation of Puccinia hordei in leaves of hypersensitive, partially resistant and non-host plant genotypes. Phytopathology 72:000-000.
17. Niks, R.E.&H.J. Kuiper, 1982.Histology of the relation between minor and
major genes for resistance of barley to leaf rust. Phytopathology
72:000-000.
18. Van der Plank, J.E., 1963. Plant diseases: epidemics and control. Academic
Press,NewYork and London. 349pp.
19. Van derPlank,J.E., 1968.Disease resistance inplants.Academic Press,New
York and London. 206pp.
70
Samenvatting
Indegerst (Hordeum vulgare)
- dwergroest {Puccinia hordei) relatie zijn twee typenvan resistentie te onderscheiden, namelijk
overgevoeligheidsresistentie en partiële resistentie. Deze typen
kunnen beschouwd worden alsvoorbeeldenvanVanderPlank'sverticaleenhorizontale resistentie respectievelijk. Laatstgenoemd type
is vermoedelijk beduidend duurzamer dan de overgevoeligheidsresistentie,maar over hetmechanisme dataanpartiële resistentie
tengrondslag ligtisnogmaarzeerweinigbekend.
Partiële resistentie (PR)wordtgekenmerktdooreenverminderde
epidemie opbouw in hetveld ondanks eenvatbaarreactietype tussen
waardenpathogeen.Uitmacroscopischonderzoek isgeblekendateen
langeontwikkelingsduurvandeschimmel,eenlage infectiedichtheid
en een lage sporenproduktie per urediosorus debelangrijkstecomponentenvanPRzijn.
Onderzoeknaardehistologische achtergrondvandelageinfectiedichtheid inpartieel resistente genotypenvormthetonderwerpvan
de eerste twee artikelen van ditproefschrift. Indelaatstedrie
artikelen wordt door middel van histologisch onderzoek PR vergeleken met overgevoeligheidsresistentie en met de niet-waard reactie.
Het eerste artikel betreft een onderzoek naar de appressorium
vorming door P. hordei opzaailingenvandezeervatbaregerstlijn
L94 envan 11gerstlijnendieeenmatigtothoogniveauvanPRbezaten.De11weinigvatbaregerstlijnenuitdezestudieblekenniet
reproduceerbaar teverschillen van L94 in hetaantal doorappressoria bezette huidmondjes per vierkante centimeter bladoppervlak.
Ditwijsteropdatinfectiedichtheidsverschillen tengevolgevanPR
veroorzaakt worden door mechanismen diewerkennadeappressoriumvorming.Hetbleek dat zelfs op de niet-waardsoort tarwe,waarop
P. hordei geen symptomen veroorzaakt, niet minder appressoria
werden gevormd dan op L94. Gerst entarwehebbeneen soortgelijke
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epidermisstructuurwelkewaarschijnlijkdestimuliverschaftdiede
schimmel instaatstellendehuidmondjes tevindenener appressoria
optevormen.Opeenslagenotypetradgeenappressoriumvormingop.
Op deze niet-waardsoort, meteenepidermisstructuur diesterkverschiltvan die van Gramineae, ontbreken waarschijnlijk destimuli
diedeschimmel instaatstellenhuidmondjes tevinden.
Ineen onderzoek beschreven in het tweede artikelwerd gebruik
gemaakt van fluorescentie microscopie om de ontwikkeling van
P.hordei in zaailingen vanpartieel resistentie gerstlijnentebestuderen. Hetbleek datdeinfectiedichtheidsverschillentussende
partieel resistente genotypen en de zeer vatbare L94berusten op
verschillen in frequentie van "vroegeabortie"van P. hordei kolonies.Dezevormvanabortie tradopwanneerééntotzeshaustoriummoedercellenwaren gevormd. Vroege abortie inpartieel resistente
lijnengingslechtszeldensamenmetnecrosevanmesophylcellenin
dewaard. Er werd aangetoond datookverschillen in aggressiviteit
tussendwergroestisolatenhoofdzakelijkberustenopverschillen in
frequentievanvroege abortie.Het feitdateenhoge frequentievan
vroege abortie werd gevonden inverscheidene nietverwantegerstgenotypen wijst erop dat hetmechanisme dat aan PR tengrondslag
ligtdeeluitmaaktvaneenalgemeen ingerstvoorkomend systeem.
HeteffectvangenenvoorPRwerdvervolgensbestudeerdbijaanenafwezigheidvangenenvoorovergevoeligheid (derde artikel).Het
bleek dat devroege abortie ten gevolgevanPRnietalleenoptrad
bijafwezigheidvaneenovergevoeligheidsreactie maarookbijovergevoeligheid ten gevolge van het resistentiegen Pa7.OokhetvertragendeeffectvanPRgenenopdeschimmelgroeibleefmerkbaarbij
aanwezigheid van Pa7.Wanneereenovergevoeligheidsreactie tengevolgevanhetPa3genoptrad,warendeeffectenvanPRgenenminder
duidelijk waarneembaar. De reactie van Pa3 isveel eerder inhet
infectieproces waarneembaar dan dievanPa7.Pa3veroorzaaktevenalsPRgenenabortievaneenbelangrijk deelvandekolonies direct
na devorming van één tot zeshaustoriummoedercellen.Eenbelangrijkverschil tussendevroege abortiedoorPRgenenenhetPa3gen
is dat de abortie inhetlaatstegevalgeassocieerd ismetnecrose
vanwaardcellen.
Hetonderzoekbeschreven inhetvierde artikeltoonde aandater
een grote overeenkomst bestaat in histologie vandePRreactieen
de niet-waard reactie bij dwergroest enbruine roest in gersten
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tarwe. Beide reacties werden gekarakteriseerd door een hoge frequentievanvroegeabortiemetweinigofgeennecrosevanmesophylcellen.Bij P. hordei optarween P. recondita
secalis opgerstbestonddeniet-waard reactieuiteenvroege abortievanvrijwel alle
kolonies. P. recondita
tritici
was,ondankseenhoge frequentievan
vroege abortie, in staat tot reproductie te komen op viergerstlijnen met een zeer laag niveau vanPRtegen P. hordei, maarniet
op vier lijnen met een hoog niveauvanPR.Mogelijk gaateenlaag
niveauvanPRtegen P. hordei samenmeteenmindereffectievenietwaard reactietegen P. recondita
tritici.
Onderzoek naar de haustoriumvorming door P. hordei invatbare,
partieel resistente en overgevoelige gerst zaailingen endenietwaardsoorttarwe (vijfdeartikel)bevestigde deovereenkomst tussen
dePRenniet-waard reacties.Bijbeide afweersystemenbleekvroege
abortie geassocieerd te zijn met eenmislukkenvandehaustoriumvorming zonder necrose van demesophylcel.De vroege abortie met
necrose ten gevolge van het Pa3 overgevoeligheidsgen bleek op te
tredenkortnadevormingvanheteerstehaustorium.
Uit de studies kangeconcludeerdwordendatPRenovergevoeligheidsresistentie essentieel verschillende afweersystemen zijn.PR
is, evenalsdeniet-waard reactie,voornamelijk een"pre-haustorium
incompatibiliteit", overgevoeligheidsresistentie een"post-haustoriu
incompatibiliteit". Men neemt wel aan dat overgevoeligheidsgenen
werkenbinneneensysteemdatgesuperponeerd isopeenbasisresistentie-pathogeniteitssysteem. Deniet-waard reactiezouberustenop
eenniet-herkenningsreactie inditbasis systeem.Deresultatenvan
het onderzoek waarop ditproefschrift isgebaseerdwijzeneropdat
PRgenenbehorentothetbasisresistentie-pathogeniteitssysteem.