411
Plrysis\ttir.1a.ndLIolemLurPlant Patltologt(1992) 40, +li-421
l n f e c t i o no f v a s c u l atri s s u e sb y t h e a u t o e c i o u rsu s t s
Puccinia punctiformis and Puccinia lagenophorae: a
c y t o l o g i c asl t u d y
I
Z. A. Bera*
4:
I
and D. M. Ldsnr,
Unioersit2
of $hefield,Shefuld5102TtY, U.K.
oJAnimalandPLantSciences,
Departmetzt
I
I
t--
:l -
futa11992)
(Accepted
for publication
The distribution of two autoecious rust fungi within host leaves has been investigated with
particular reference to the vascular system. Vascular infection by the thistlc rust Puccinia
punctiformis, was low at the spermogonial stage but increased during development of the
were accompanied by dense fungal growth in leaf
uredinoid aecia. Aecial stagesofP. Iagenophorae
velns of Seneciowtlgaris. In both cases,host cells were penetrated by unspecialized, filarnentous,
fungal structures.No vaschiar infection by the dikaryon was found in uredinial-telial phasesofP.
punctiJormison thistle leaves, although specialized D-haustoria were present with high frequency
in mesophyll cells. These observations are consistent with the hypothesis that access to host
translocates, afforded by vascular penetration, may compensate for the probably lower efficiency
of the unspeciaiized filamentous haustoria found in spermogonial and aecial stages.
INTRODUCTION
l
I
The invasion of host vascular tissue by rust fungi has been relatively infrequently
recorded 14,5, 10,12,13,15,16,19,21,22,24,2n. During the spermogonial-aecialstages
poarum,monokaryotic intercellular and intracellular hyphae
of the life-cy cle of Puceinia
in
can be readily observed the vascular tissLieof Tussilagofarfara,inthe phloem region,
xylem parenchyma and bundle sheath. However, in the dikaroytic, uredinial-telial
stagesof the same pathogen on the alternate host Poapratensis,only the mesophyll ancl
bundle sheath cells contain haustoria [a]. The question of whether similar differences
occur also in autoeciousrusts, infecting only a single host species,was investigatedin
the following study.
The taxonomically related, autoeciousrusts,Pucciniapunctiformis(Str.) Rohl and P.
L. and
lagenophorae
Cooke were selected,since their respectivehosts Cirsiumaruense
Senecio
uulgarisL., are in the same family as T.farfara, the host of the monokaryon of
P. paarum.The occurence of infection in the leaf vascular system was investigated
throughout the macrocyclic life-cycle o{ P. puncti"formisar,d in the aecial stage of P.
lagenophorae.
P. punctiformrsproducesspermogonia on systemicallyinfected shootsin iate spring,
followed by uredinoid aecia 123), also termed primary uredinia [8]. The spores in
these sori are morphologically identical with urediniospores but the associated
*Present address: Botany Department, Mansoura University, Mansoura, Egypt.
0885-57
65/92 /060411+ I I $0B.00/0
@ 1992 Academic PressLimited
Z. A. Bakaand D. M. Losel
412
Frc. 1. Filamentous haustorium in xylem parenchymaolCirsiun anazsrieaf, during spermogonial
stage of Pucci.niapunctiformis. The host cell cytoplasm contains mitochondria and profiles of
endoplasmic reticulum (arrows), closely associated with extrahaustorial rnembrane and
plasmalemma. Larger arrows indicate secondary wall in adjacent tracheary element. BS, bundie
sheath; C, chloroplast; cc, companion cell; g, Golgi body; h, haustorium, I, intercellular hypha;
L, lipid; M, mitochondrion; MC, mesophyll ce11;N,host nucleus; n, fuqgal nucleus; Ph, phloem;
pp, phloem parenchyma; se, sieve element; sw, lignified secondary wall; wi, wall ingrowth; X,
xylem element; XP, xylem parenchyma.
mycelium bears unspecialized,filamentous intracellular structures l4' of the type
normally found in monokaryotic phasesof the rust life cycle [/7]. Infection of heaithy
thistles by primary urediniospores initiates a dikaryotic mycelium with typical Dnative to
haustoria and bearing secondary uredinia and telia ln. P. lagenophorae,
in the
mainly
investigated
1961
was
since
found
in
Europe
only
and
Australia
124,
in
in
telia
which,
and
aeciospores
later
among
the
develop
aecial phase. Teliospores
the
pith
aavtty.
within
even
develop
communication),
some cases(N. Paul, personal
M A T E R I A L SA N D M E T H O D S
Primary.and secondary stages of P. punctiformis inlection of Cirsium aruense,were
collectedlocally or obtained from systemicallyinfected plants maintained in a growth
room (16 h day, B0-100 pmoles m-2 sec-l, 20+2 "C) . Secondary infections were also
obtained in growth room conditions by inoculating young plants with primary
(: u."iorpores),followingtheprocedureofMcGeeetal.ll9l.Secondary
urediniospores
uredinia emerged about l5 days after inoculation. The common annual weed Senecio
was collectediocally, mainiy in iate
uulgarislbearing aecial infections of P. lagenophorae,
summer and autumn.
V a s c u l airn f e c t i o nb y r u s tf u n g i
413
At snccessivestagesof development of rust infections, portions of leaves were fixed
in 2'5o/o (v/v) glutaraldehydein lo/o cacodylatebuffer at pH 7 for 24h at room
temperature, washedin buffer, then post fixed with I o/oOsOn (v/v) in the same br-rffer
[25], cleirydrateclin a graded ethanol series and embedded in Araldite. Semi-thin
(0.5-l pm) sections,stainedwith toluidine blue, were examinedwith a liglit microscope.
Ultrathin sectionswere cut on a Reichert Ultramicrotome, using a diamond knife,
stained with uranyl acetate[/4] then lead citrate 120)and examined with a Phillips 200
elecron microscope.For each stageof infection, EM observationswere basedon three
plants from each of which nirle blocks were prepared, three from each of three leaves.
From each b1ock,serial sectionson three grids were examined.
RESULTS
P uccinia p uncti-formis
In infected leaves of C. aruensebearing spermogonia, monokaryotic filamentous
haustoria were common in bundle sheath cells (Fig, l) but sparselydistribr"rtedwithin
the vascular tissue.During the subsequentdevelopment of aecia, bundle sheath cells
often became denselyinfectedand intercellular hyphae grew within the vascular region
(Fig. 2). Filamentous haustoria were freqlient in phloem parenchyma (Figs. 3 & 4),
Frcs. 2-5. Sections of leaf veins of C. araenseduring aecial stage of P. punctifornis.
Frc. 2. Light micrograph of semi-thin section of small vascular bundle showing numerous
sectionsoffungal structuresin bundle sheath cells. Intercellular hyphae are present in vascular
tissueas well as in mesophvll. Abbreviations as for Fie. 1.
Z . A . B a k aa n d D . M . L o s e l
$:
5Fm
Fic. 3. Phloem region oflarger vascular bundle showing the infection ofphloem parenchyma
cells. Note intercellular hyphae. Abbreviations as for Fig. l.
Frc. 4. Haustoria and intercellular hypha in phloem parenchyma. Compare the staining ofhost
wall material and extra-haustorial matrix (arrow). Abbreviations as for Fie. i.
Vascularinfectionby rust fungi
415
F r c . 5 . S e c t i o no f f u n g a i c e l l e m b e d d e di n b a n d o f l i g n i f i e ds e c o n d a r yt h i c k e n i n go f x y l e m v e s s e l
(arrow). Note healthy appeararlceoffungal nucleus and cytoplasm. Abbreviations as for Fig. l.
xylem parenchyma and even xylem elements where, in some instances, they were
embedded in bands of lignified wall thickening (Fig. 5).
The cytoplasm of infected celis of the vascular region was of relatively healthy
appearance,with abundant profiles of endoplasmicreticulum and mitochondria. The
hypha-like intracellular strllctures,like thoseprevior-rslydescribedin the mesophyll,l7),
were not observed to exit from the host cells. These structllres grew mainly
longitudinally in vascularelements,commonly presentingcircular profiies in transverse
sectionsof leaf veins. Later in aecial development, morphologically similar haustoria
were seenin vascular tissue.These intracellular structures,like those previously noted
in the mesophyll [6, 7], lacked the narrow neck and neck-band typical of the
specializedD-haustoria [17] of uredinial-telial phasesof rlrst life cycles.As in mesophyll
cellsof theseand other [/, 2, 17,2q rust species,material resemblingthe host wall in
its staining reactionstended to accumulate around older filamentous haustoria, in the
extra-haustorial matrix between the invaginated regions of host plasmalemma and
fungal walls (Fig.4). This layer was absentin young haustoria,as in Fig. 1.
examination
In secondary uredinial and telial stagesof infection by P. punctiforruzs,
of many tissue blocks from different plants failed to reveal any penetration of the
vascular system by the fungus, although the bundle sheath cells were infected by
clavate, D-haustoria of the type familiar in dikaryotic infections by rust fungi.
Puccinialagenophorae
was consistently
In leavesof Senecio
uulgaris,the development of aecia of P. lagenophlrae,
associatedwith vascular infection which light microscopy showeclto be more densely
concentrated in this region than in the mesophyll (Fig. 6). Filamentous haustoria were
Z . A . B a k aa n d D . M . L o s e l
416
Frcs. 6-10. Sections ofieafveins of Seneciouulgaris duy'rrg aeciai stage of Puccinialagenophorae.
Flc. 6. Light micrograph oflongitudinal section, showing inter- and intra-celluiar iofection of
bundle sheath, phloem and xylem. Compare the density of infection of the vascular strand and
adjacent mesophyll. Abbreviations as for Fig. I
found in all types of cel1sof both phioem and xylem (Figs 7-10). The cytoplasm of
infected phloem parenchyma, transfercellsand xylem parenchyma, remained relatively
electron-dense,even after infection, with a well-developed membrane system and
abundant mitochondria, giving the impression of a metaboiically active condition
(Figs 7-9).
host wall-like
In some older infections of transfer cells of the phloem of Senecio,
material was occasionally found to be deposited in the extrahaustorial matrix
surrounding fiiamentoushaustoria in a manner similar to the wali ingrowths projecting
where xylem vesselshad
from the rest of the host cell wall (Fig. B). As in P. punctiformis,
been penetrated, fungal cells were sometimesembeddedwithin the lignified bands of
secondary wall thickening (Fig. 10).
DISCUSSION
The above observations appear to be the first ultrastructural investigation of the
relationship of autoeciousrustswith host vascular tissues.The intensive invasion of the
contasts
vascular system of Seneciouulgaris by the aecial phase of P. lagenophorae
and
punctiformis
in
C.
aruense
P.
infection
by
limited
vascular
more
with
the
strikingly
poarum
T.
in
leaves
of
of
P
,
the
monokaryon
of
the
distribution
closelyresembles
farfara
14).
auenaell2f and P' poarum[4]' the
In the caseof the heteroeciousrusts, P. coronata
Rhamnuscatharticaand T.farfara,
systems
of
infect
the
vascular
monokaryons of which
the
dikaryon from vascular tissues
respectively,it could be argued that the exclusionof
might be determined by anatomical
of the alternate hosts,A. satiuaand Poapratensis,
or physiologicalcharacteristicsof thesegrasses,such as the lignified bundle sheath.The
presentstudy has shown this pattern persistingeven where there is no differencein host
V a s c u l a irn f e c t i o nb y r u s tf u n g i
417
Frc. 7. Transversesectionofsmall vascular bundle, showing lungal structures adjacent to and
within cells of bundle sheath, phioem and xylem (arrowhead). Note binucleate cells of
intercellular hyphae (arrow). Abbreviations as for Fig. 1.
speciesduring the life-cycle.The dikaryon initiated by the uredinoid aeciosporesof P.
punctiformis
appearsto beincapdble of penetrating the vascularsystemof C. aruense.The
lacks uredinia and spermogonia,and
situation is still unclear in P. lagenophorae,lvhich
where the limited amount of telial materiai examined proved too brittle to provide good
sections.
The only evidenceso far for the occurrenceof D-haustoria in host vascular tissueis
from a study of wheat varietiesinfected by P. graminis,where vascular penetration was
recorded in the most susceptibleof a range of varieties examined [5].
During the spermogonial and aecial stages of infection by P. punctiformisand P.
lagenophorae,
the fungal structures within iiving cells of the vascular system, like those
in the mesophyll [6, 7], correspondto descriptionsof P-haustoria [12], or M-haustoria
[17]. Since,like thoseof P. poarum14,2fJ,they have not been observedto exitfrom a
418
Z . A . B a k aa n d D . M . L o s e l
#**w$
rr;liii
J[ul
*',{
.,i
:,'Ifr'l
t, i:a
::,
2Fm
Frc. B. Longitudinal section through heavily-infected transfer cells in phloem. Note long,
intracellular, fungal structures with moderately electron-dense extracellular matrlr which, in the
one on the right, resembieswali in-growths of transfer cell. The upper cell contains large numbers
of mitochondria and deposits of electron-dense granules. The haustoria in the lower celI are
probably younger, having no wall-like material visible in matrix. Abbreviations as for Fig. 1.
host cell, they fit Bushnell's definition of a haustorium [7], rather than intracellular
hypha, as employed by Gold et al. llll. These intracellular structures like those of the
monokaryons of P. poarumL3,2q, P. coronataU2), P. menthae
llQ and other rursts[17],
lack the strongly-constrictedneck with osmiophilic neck-band, characteristic of the
specialized D-haustoria of uredinial-telial dikaryons 17,17f. They share common
featuresof a wider point of entry into the host cell and similar cytochemical reactions
of the extra-haustorialmatrix 16,7, lfl, elegantly demonstratedby Woods and Gay in
monokaryotic infections of P. poarum pey -Ihe distinction between intracellular
hyphae, as found in P. reconditalll) and P. menthae
[16], and filamentous haustoria is
likely to be functionally lesssi.gnificantthan their differencesfrom D-haustoria.
The encasementof the fungal wall in older regionsof filamentous haustoria by host
wall-like material has been studied cytochemically in P. paarum1261,P. punctiformisand
P. lagenophorae
16,n and other rust fungi 116,Ln. A more specializedreaction of this
type has been noted here in some transfer cellsof leaf veins of S. uulgaris,where transfer
celilike projections of wall material, have occasionally been observed around
419
Vascularinfectionby rust fungi
,
, .$iL
:i:,.
il:r
9
*.
'r;,lt;'.,'l
rlfrm
Frc. 9. Transverse sections of two filamentous haustoria in xylem parenchyma ce1l. The host
cytoplasm is of healthy appearance, unvacuolated, containing mitochondria and numerous
profiles of endoplasmic reticulum (arrows). The extra-haustorial matrix surrounding the
haustorium on the left is moderately electron-dense but that on the right (arrow) shows no
deposition of wall-like material. Abbreviations as for FiE. l.
question ariseswhether the proiiferation
intracellular structLrresof P. lagenophtrae.Tlle
of the enclosingextrahaustorialmembrane, associatedwith such wall deposition,may
facilitate movement of solutesfrom host celi to hypha, by a mechanism comparable to
the loading of conducting eiementsadjacent to transfer cells.
Such deposition of wall material, like the embedding of fungal cells within bands of
Iignified secondary wall thick€ning, in xylem elements of C. aruenseand S. uttlgaris,
supports the cytochemical evidence 16,16,2eJ that the invaginated region of
plasmalemma, surrounding the fungal wall of older regions of filamentous haustoria,
carries out similar wall-synthesizins reactions to those of the uninvaginated region
lining the host cell wall. The hyphae found within non-living xylem elementsmay have
developed initially as haustoria in young, undifferentiated cells. Following the
maturation and death of the host cell, however, they become essentiallyapoplastic,
lack the extrahaustorialmatrix offunctional haustoria, and frequentiy appear necrotic.
As suggestedin the caseof P. poarum,the ability of the spermogonial-aecialthallus
of these rust fungi to gain accessto host nutrients of the vascular system may
compensatefor the unspeciaiizednature of the associatedfilamentous haustoria 14,267,
MPP 40
424
Z . A . B a k aa n d D . M . L d s e l
. .Ira.. 10. Tracheary elements of xylem with interceliular hypha and intracellular fungal
.'strlrcture6, partiatly embedded in secondary wall thickening. Note breakdown of primary wali
(aiiqws). Abbreviations as for Fig. 1.
The lack of this characteristic in the uredinial-telial dikaryon points to fundamentaL
physiological and morphogeneticdifferencesin genomeexpressionwhich merit further
investigation.
J
Z. A. Baka wishes to thank the Egyptian Educational Bureau for financial support
during the period of this work and the Committee of Vice-Chancellors and Principals
for an Overseas Research Students Scholarship. The assistanceof Dr N. Paul in
and of Mrs E. E. M. McGee in the
providing teliospore stages of P.' lagenophorae
maintenance of rust infections is gratefully acknowledged.
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Zakaria Baka