Ul as uc u e o he ea ly s ages o
Colle o ichum acu a um in ec ion o s awbe y
issues
F ancisco T. A oyo, Ja ie Mo eno, G ego io Ga cía-He dugo,
Be a De los San os, Ca men Ba au, Ma ía Po as, Césa Blanco,
and Fe nando Rome o
Abs ac : The ea ly s ages o he in ec ion o a ached lea es and pe ioles o s awbe y (F aga ia ×ananassa Duch.
‘Cama osa’) by Colle o ichum acu a um Simmonds we e s udied using scanning and ansmission elec on mic oscopy.
P e-pene a ion e en s o hese issues we e simila , bu he p oduc ion o seconda y conidia (mic ocyclic conidia ion)
was de ec ed only on lea es. A he ul as uc u al le el, di e en s ages o ma u a ion o app esso ia we e obse ed
and desc ibed. In young app esso ia, he cell wall was composed o wo laye s and he plasma memb ane displayed a
wa y appea ance. In he ollowing s age, he app esso ium de eloped a hi d elec on- anspa en laye be ween he
cell wall and he plasma memb ane. This new elec on- anspa en ma e ial was especially isible in he egion o he
app esso ium nea he cu icle. The plasma memb ane o his app esso ium showed a smoo h appea ance. A e wa ds, a
pene a ion peg eme ged h ough he po e pene a ing he cu icle and eached he epide mal wall whe e i enla ged o
o m an in amu al in ec ion esicle. Bo h s uc u es o in ec ion, he pene a ion peg and he in amu al in ec ion esi-
cle, p oduced du ing he ea ly phases o in ec ion o s awbe y issues by C. acu a um, ha e no been p e iously e-
po ed and con i m ha i s in asion s a egy is ha o a subcu icula in amu al pa hogen. Once he in ec ion was well
es ablished, abundan subcu icula and in amu al hyphae we e p oduced on pe ioles, causing se e e deg ada ion o he
hos cell walls. Occasionally, he cu icle appea ed dis up ed in hose egions whe e he hos walls we e e y deg aded
and dila ed. Di e ences be ween coloniza ion o pe ioles and lea es we e obse ed.
Key wo ds: Colle o ichum acu a um,F aga ia ×ananassa, in ec ion esicle, in asion s a egy, pene a ion peg.
Résumé : À l’aide de la mic oscopie élec onique pa balayage e pa ansmission, les au eu s on é udié les p emie s
s ades de l’in ec ion des euilles a achées e des pé ioles du aisie (F aga ia ×ananassa Duch. ‘Cama osa’), pa le
Colle o ichum acu a um Simmonds. Les é ènemen s de p é-péné a ion de ces issus son similai es, mais la p oduc-
ion de conidies secondai es (conidia ion mic ocyclique) ne s’obse e que su les euilles. Les au eu s on obse é e
déc i en les ul as uc u es, aux di é en s s ades de ma u a ion des app esso iums. Chez les jeunes app esso iums, la
pa oi cellulai e es composée de deux couches e la memb ane plasmique mon e une appa ence ondulée. Au s ade sui-
an , l’app esso ium dé eloppe une oisième couche anspa en e aux élec ons, en e la pa oi cellulai e e la plasma-
lemme. Ce nou eau ma é iel anspa en aux élec ons es pa iculiè emen isible dans la égion de l’app esso ium
oisin de la cu icule. La plasmalemme de ce app esso ium es d’appa ence lisse. Pa ap ès, l’hyphe de péné a ion
éme ge à a e s le po e e pénè e la cu icule pou a eind e la pa oi épide mique, où il s’éla gi pou o me une ési-
cule d’in ec ion in amu ale. Les deux s uc u es d’in ec ion, hyphe de péné a ion e ésicule d’in ec ion in amu ale,
p odui es au cou s des p emiè es phases d’in ec ion des issus du aisie pa le C. acu a um, n’on jamais é é appo -
ées jusqu’ici e con i men sa s a égie d’in asion pa pa hogénèse subcu iculai e in a mu ale. Une ois l’in ec ion bien
é ablie, on obse e la o ma ion de nomb eux hyphes subcu iculai es e in amu aux su les pé ioles, en aînan la dé-
g ada ion des pa ois des cellules de l’hô e. Occasionnellemen , la cu icule appa aî écla ée dans les égions où les pa-
ois de l’hô e son aimen dég adées e dila ées. On obse e des di é ences en e la colonisa ion des pé ioles e des
euilles.
Mo s clés : Colle o ichum acu a um,F aga ia ×ananassa, ésicule d’in ec ion, s a égie d’in asion, hyphe de péné a-
ion.
[T adui pa la Rédac ion] A oyo e al. 500
Can. J. Bo . 83: 491–500 (2005) doi: 10.1139/B05-022 © 2005 NRC Canada
491
Recei ed 21 July 2004. Published on he NRC Resea ch P ess Web si e a h p://canjbo .n c.ca on 21 May 2005.
F.T. A oyo,1B. De los San os, C. Ba au, M. Po as, C. Blanco, and F. Rome o. Cen o de in es igación y o mación ag a ia
Las To es-Tomejil, Apdo. de Co eos O icial, 41200 Alcalá del Río (Se illa), Spain.
J. Mo eno and G. Ga cía-He dugo. Depa amen o de Biología Celula , Uni e sidad de Se illa, Se illa, Spain.
1Co esponding au ho (e-mail: [email p o ec ed]).
In oduc ion
S awbe y (F aga ia ×ananassa Duch.) an h acnose is
caused by species o he gene a Colle o ichum and
Gloeospo ium. In he Uni ed S a es his disease is mainly
caused by C. aga iae B ooks, C. gloeospo ioides (Penz.)
Penz. & Sacc., and C. acu a um Simmonds (Howa d e al.
1992). In Eu ope, only C. gloeospo ioides and C. acu a um
ha e been iden i ied as pa hogens causing an h acnose in
s awbe y (De los San os and Rome o 1999; Denoyes and
Baud y 1995).
Spain is he majo p oduce o s awbe y ui in he Eu-
opean Union. In his coun y mo e han 90% o he na ional
p oduc ion is loca ed in he p o ince o Huel a (sou hwes -
e n Spain) (Hancock 1999). An h acnose, caused by
C. acu a um, is one o he mos impo an diseases a ec ing
s awbe y p oduc ion in his egion, causing signi ican e-
duc ions in yield (De los San os 1998).
Hos coloniza ion and pa hogenesis a e well cha ac e ised
o se e al species o Colle o ichum (Dickman 2000;
O’Connell e al. 1985). The ini ial s ages o hos in ec ion
by Colle o ichum spp. include conidial adhesion o he hos
su ace, ge mina ion o conidia, p oduc ion o ge m ubes
ha di e en ia e o o m melanized app esso ia, and pene-
a ion o he hos cu icle ia app esso ia (Dickman 2000).
Acco ding o Bailey e al. (1992), Colle o ichum spp. use
p ima ily wo in ec ion s a egies: in acellula hemibio-
ophic in asion and subcu icula in amu al in asion. In he
i s s a egy, he pa hogens exhibi a wo-phase in ec ion
p ocess: an ini ial symp omless o bio ophic phase, wi h
hyphae g owing wi hin he cell lumen by means o a globose
in ec ion esicle wi hou pe u bing he hos plasma mem-
b ane, ollowed by a nec o ophic phase. Colle o ichum
lindemu hianum in ec ing bean (Phaseolus ulga is)o cow-
pea (Vigna unguicula a) (Bailey e al. 1990; Me ce e al.
1975; O’Connell e al. 1985; Skipp and De e all 1972),
C. i olii in ec ing al al a (Medicago sa i a) (Mould e al.
1991a, 1991b), and C. gloeospo ioides in ec ing Mal a spp.
(Mo in e al. 1996) a e examples o species exhibi ing his
s a egy. The subcu icula in amu al in asion s a egy is
cha ac e ised by he g ow h o he pa hogen benea h he
cu icle and wi hin he pe iclinal walls o epide mal cells
(Bailey e al. 1990). Colle o ichum capsici (P ing e al.
1995), C. unca um (Manandha e al. 1985), C. ci cinans,
and C. phomoides (Bailey e al. 1992) ha e been epo ed o
use his s a egy.
Colle o ichum acu a um is a pa hogen a ec ing impo an
c ops wo ld-wide; howe e , ew s udies ha e in es iga ed
he ul as uc u e in s awbe y sys ems (Cu y e al. 2002).
Leand o e al. (2001) epo ed he ge mina ion and he o -
ma ion o seconda y conidia o his pa hogen on he su ace
o de ached s awbe y lea es. Ho owi z e al. (2002) s udied
he de elopmen o C. acu a um on s awbe y and o he
plan species by using ansgenic isola es. Cu y e al. (2002)
s udied he his opa hology o C. acu a um and C. aga iae
in s awbe y s olons and pe ioles and desc ibed he p ocess
o in ec ion, showing, a he ul as uc u al le el, s ages o
in asion o hese pa hogens wi h hyphae unde hos cu icle
and in an iclinal hos wall. Howe e , in ec ion s uc u es
du ing he pene a ion phase, such as a pene a ion peg o in-
ec ion esicle, o C. acu a um ha e no been desc ibed a
he ul as uc u al le el. The e a e s ill some aspec s o he
in ec ion p ocess o C. acu a um on s awbe y ha emain
poo ly unde s ood, especially hose ela ed o he ea lies
s ages o pene a ion.
The pu pose o his s udy was o cha ac e ise he in ec ion
s uc u es o C. acu a um in ol ed du ing he pene a ion
phase and he ea ly s ages o in ec ion in wo s awbe y is-
sues, pe ioles and lea es, ha exp ess di e en suscep ibili y
o his pa hogen and o cla i y he in asion s a egy used by
C. acu a um wi h espec o s a egies p e iously desc ibed
by o he au ho s.
Ma e ials and me hods
Plan ma e ial
S awbe y plan s (F aga ia ×ananassa ‘Cama osa’) ha
a e highly suscep ible o C. acu a um (De los San os 1998)
we e ob ained by mic op opaga ion echniques. Plan s we e
plan ed in 10.5 cm × 13.5 cm plas ic po s con aining s e il-
ized pea (Klansmann-Deilmann, Gees e, Ge many) and
g own o 75 d be o e inocula ion wi h C. acu a um in a
g eenhouse main ained a 25±5°Cday:15±5°Cnigh .
Inocula ion
Colle o ichum acu a um isola e CECT-20240 ob ained
om a na u ally in ec ed s awbe y (‘Chandle ’) c own was
used in his s udy. The ungus was g own on po a o dex ose
aga (Di co Labo a o ies, De oi , Michigan) o 7da 25°C
unde con inuous luo escen ligh (Os am L 18 W/21-840
Hellweiss Lumilux Cool Whi e, 75 µE·m–2·s–1) (De los
San os 1998, De los San os and Rome o 1999).
Conidial suspensions we e p epa ed by looding he cul-
u e pla es wi h 4–5 mL o s e ile dis illed wa e , sc aping
he colony su ace wi h a scalpel, and il e ing he suspen-
sion h ough s e ile cheeseclo h. The concen a ion was ad-
jus ed o1×10
6conidia·mL–1 using a haemocy ome e .
A ached pe ioles and bo h abaxial and adaxial su aces o
s awbe y lea es we e inocula ed by applying 50-µL d op-
le s o conidial suspension (A oyo e al. 2001). Inocula ed
plan s we e enclosed in plas ic bags o 48 h o main ain
high ela i e humidi y and we e incuba ed in a g ow h cham-
be a 25 °C, wi h a 16-h pho ope iod benea h luo escen
ligh (Syl ania Luxline Plus F58W/840 Cool Whi e Deluxe,
100.5 µE·m–2·s–1). Con ol plan s we e ea ed wi h 50 µLo
s e ile dis illed wa e and incuba ed as desc ibed abo e.
Scanning elec on mic oscopy
To s udy conidial de elopmen o C. acu a um on s aw-
be y issues, o p e-pene a ion e en s, h ee pe ioles and
six lea es we e sampled a 4, 8, 12, 24, 48, and 72 h a e in-
ocula ion. Pe iole segmen s 1 cm long and lea disks 1 cm2
om benea h inocula ion d ople s we e emo ed and ixed
o e nigh a 4 °C in 4% ( / ) glu a aldehyde in 0.1 mol·L–1
phospha e bu e (pH 7.2), insed in bu e , and dehyd a ed
h ough g aded ace one o 100%. Segmen s and disks we e
d ied in a c i ical poin d ying appa a us (model No. CDP-
030, BAL-TEC Company, Balze s, Leich ens ein) and
coa ed wi h gold. Obse a ions and image collec ion we e
made using a Philips XL-20 scanning elec on mic oscope
(SEM) ope a ing a 10 kV.
© 2005 NRC Canada
492 Can. J. Bo . Vol. 83, 2005
T ansmission elec on mic oscopy
To s udy he ea ly s ages o C. acu a um in ec ion on
s awbe y issues, h ee pe ioles and six lea es we e sam-
pled a 24, 36, and 48 h a e inocula ion. S ips o issue ap-
p oxima ely 1 mm hick and 1–2 mm long we e emo ed
om benea h he inocula ion d ople s on pe ioles and lea es
and we e ixed in 4% ( / ) glu a aldehyde in 0.1 mol·L–1
cacodyla e bu e (pH 7.2) o 3ha 4°C.A e insing in
he same bu e , he issues we e pos - ixed in 1% (w/ )
osmium e oxide o 2ha 4°C, hen dehyd a ed in
a g aded ace one se ies, and embedded in EMBED-812
(Polysciences, Wa ing on, Penn.) acco ding o he manu ac-
u e ’s ins uc ions.
Slides wi h semi hin sec ions (0.5 µm) we e placed on a
ho pla e a 50 °C, s ained o 1 min wi h 0.1% aqueous
oluidine blue O, and examined using a ligh mic oscope
(Lei z A is oplan). Ul a hin 60–80 nm sec ions we e made
wi h a Reiche -Jung Ul acu E ul amic o ome and a dia-
mond kni e, and collec ed on 300-mesh coppe g ids
(Dashek and May ield 2000). G ids we e s ained wi h 7%
aqueous u anyl ace a e and lead ci a e. Sec ions we e ob-
se ed and images collec ed using a Philips CM-10 ans-
mission elec on mic oscope (TEM).
Resul s
Scanning elec on mic oscopy
Mos conidia began o ge mina e on pe iole and olia su -
aces wi hin 4 h a e inocula ion by o ming a ge m ube
om ei he end o he conidium, and occasionally om bo h
ends. La e al ge mina ion was a ely obse ed. Ge mina ed
conidia ypically de eloped one o wo ans e se sep a. By
8 h a e inocula ion, globose and subglobose app esso ia
we e de ec ed as swellings o he ge m ube ips. On lea es,
especially on he adaxial su ace, app esso ia o igina ed
om sho ge m ubes (sessile app esso ia) (Fig. 1). How-
e e , app esso ia obse ed on pe ioles,a 24 h a e inocula-
ion, usually de eloped om elonga ed ge m ubes, which
o en eached a leng h se e al imes he conidium size. SEM
e ealed a sep um be ween he ge m ube and app esso ium,
nea he base o he app esso ium (Fig. 2). App esso ia we e
de ec ed p ima ily nea o he junc ion o he epide mal cells
and, a ely, on s oma a.
Mic ocyclic conidia ion also was obse ed on ei he
adaxial o abaxial su aces o inocula ed lea es, p oducing
abundan seconda y conidia om conidial and hyphal
phialides (Figs. 3–5). Howe e , hese conidiogenic s uc u es
© 2005 NRC Canada
A oyo e al. 493
Figs. 1–5. Conidial de elopmen o Colle o ichum acu a um on pe ioles and lea es o s awbe y (SEM). Fig. 1. Sessile app esso ium
(A) and conidium (CO) wi h wo ans e se sep a (a ows) on he adaxial lea su ace a 8 h pos -inocula ion (hpi). Scale ba = 5 µm.
Fig. 2. App esso ia (A) on pe iole a 24 hpi, wi h basal sep a (a ows) o igina ing om long ge m ubes. Scale ba = 5 µm. Fig. 3.
Seconda y conidium on he adaxial lea su ace a 48 hpi, o igina ing om hyphal phialide (a ow). Scale ba = 5 µm. Fig. 4. Second-
a y conidia on he adaxial lea su ace a 72 hpi, o igina ing om hyphal phialide (a owhead) and conidial phialide (a ows). Scale
ba =10µm. Fig. 5. Seconda y conidium (a ow) and app esso ium (A) o igina ed om a sho ge m ube on abaxial lea su ace a
72 hpi. Scale ba = 10 µm.
we e no de ec ed on pe ioles. A 72 h a e inocula ion,
anas omosed ungal masses we e de ec ed on inocula ed is-
sues, especially on lea es, o ming ball-shaped accumula-
ions nea ly 100 µm in size (da a no shown).
T ansmission elec on mic oscopy
App esso ia o C. acu a um we e p ima ily loca ed nea
he junc ion o he epide mal cells. A 24 h a e inocula ion,
app esso ia wi h di e en ma u a ion s ages and he ea lies
s ages o pene a ion we e obse ed on pe ioles (Figs. 6–10).
Young (imma u e) app esso ia, which had no ye de eloped
he app esso ial po e in he basal egion, we e obse ed. The
cell wall o hese app esso ia was composed o wo mo pho-
logically dis inc laye s: an ou e elec on-dense laye and an
inne elec on- anspa en laye . In addi ion, a mucilaginous
ma ix, which had a ib illa aspec , co e ed he cell wall. In
hese app esso ia he plasma memb ane had a wa y appea -
ance and mi ochond ia we e loca ed in he cellula pe iphe y
(Fig. 6). In a mo e ad anced app esso ium s age, he wid h
o he cell wall appea ed qui e educed in he egions close
o he cu icle, and bo h inne and ou e laye s we e indis in-
guishable, displaying a mode a ely dense aspec (Fig. 7). In
u he s ages o app esso ia ma u a ion, he cell wall close
o he cu icle was comple ely dissol ed o o m a po e. In
his egion, he o ma ion o a new elec on- anspa en laye
be ween he cell wall and plasma memb ane was obse ed
connec ing di ec ly wi h he cu icle (Figs. 7, 8). The plasma
memb ane o hese app esso ia had a smoo h aspec excep
in he po e egion, whe e i displayed a wa y appea ance
(Fig. 9). A he e ophagosome, wi h a myelin appea ance, was
obse ed a ound he po e egion. The nuclei o hese
app esso ia showed decondensed ch oma in and i s en elope
was agmen ed (Figs. 7–9).
Subsequen ly, he app esso ium ini ia ed he o ma ion o
a V-shaped pene a ion peg, which pushed he hos cu icle
(Fig. 10). The cell wall o he pene a ion peg was indis inc
om he elec on- anspa en laye desc ibed abo e
(Fig. 11). Some app esso ium cy oplasmic o ganelles mi-
g a ed h ough he pene a ion peg (Fig. 11). The peg was
abou 400 nm wide whe e i passed h ough he cu icle.
When i eached he uppe epide mal wall a small in ec ion
esicle was o med (Fig. 12). This in ec ion esicle, which
de eloped in amu ally, had an elec on-dense con en and
an elec on- anspa en wall simila o he pene a ion peg
(Fig. 13). Su ounding he in ec ion esicle, elec on-dense
deposi s in he hos wall we e obse ed (Figs. 12, 13).
Du ing he pene a ion phase, no mo phological signs o
he deg ada ion o cu icula componen s we e obse ed, bu
cu icle s uc u e emained in ac in hose egions close o he
in ec ion po e (Figs. 12, 13).
La e on du ing he pene a ion phase, he cu icle loca ed
be ween well-de eloped in ec ion esicles and collapsed
app esso ia showed da ks bands co esponding o olds and
dis up ions (Fig. 14).
The pa hogen de eloped abundan hyphae om he in ec-
ion esicle in he subcu icula and in amu al spaces. Hos
cell walls we e highly deg aded in he in ec ed egions and
his deg ada ion could be occasionally seen a ound he in a-
sion hyphae (Fig. 15). The highly deg aded hos wall
showed an aspec o dila a ion and i could ha e caused he
olding and he dis o ion o he cu icle (Fig. 16). In ac ,
whe e he epide mal wall and he cu icle we e sepa a ed he
cu icle was o en dis up ed. In hose egions o hos issue
whe e he cu icle had la ge dis up ions seconda y in ec ions
by hyphae we e acili a ed (Fig. 17). Di ec pene a ion om
ge mina ed conidia h ough small dis up ions was also ob-
se ed (Fig. 18).
A 24 h a e inocula ion, in e - and in a-cellula hyphae
we e de ec ed in pe ioles using ligh mic oscopy and TEM.
These hyphae we e loca ed in he second and hi d laye o
he co ex, causing a high deg ee o diso ganiza ion in hos
cells and des oying o e 10–20 hos cells in his egion
(Figs. 19, 20). Concu en ly, mac oscopic symp oms ap-
pea ed on he pe iole-inocula ed egion as b ownish spo s o
up o 1 mm in size. Fo y-eigh hou s a e inocula ion, in e -
and in a-cellula hyphae ex ended h oughou he co ex,
inc easing he nec osis o he pe iole issue. A he mac o-
scopic le el, lesions ha we e lineal-shaped, 2–8 mm long,
no sunken, b own and g eenish-b own o o ange-b own
we e obse ed.
The pene a ion p ocess o C. acu a um in lea issues was
simila o ha in pe ioles; howe e , he pa hogen was e-
s ic ed o he subcu icula laye and did no p og ess in o
he lea mesophyll spaces. Fo y-eigh hou s a e inocula-
ion, in inocula ed lea issues, in a- and in e -cellula
hyphae we e de ec ed in he abaxial epide mis, bu only in-
amu al subcu icula hyphae we e obse ed on he adaxial
lea side. Mac oscopic symp oms, such as b ownish spo s
(1 mm in diame e ), we e only obse ed on he abaxial lea
su ace.
Discussion
This is he i s epo , a he ul as uc u al le el, o he
ea lies s ages o pene a ion o C. acu a um in s awbe y
issues by he o ma ion o a pene a ion peg ia an app es-
© 2005 NRC Canada
494 Can. J. Bo . Vol. 83, 2005
Figs. 6–10. App esso ia o Colle o ichum acu a um in di e en ma u a ion s ages on s awbe y pe ioles a 24 hpi (TEM). Fig. 6.
Young app esso ium on cu icle (C) wi h a wa y plasma memb ane (a owhead), app esso ial wall wi h an elec on- anspa en inne
laye (open a ow) and elec on-dense ou e laye (solid a ow), su ounded by a mucilaginous coa ing (MU). Scale ba = 1 µm. Fig. 7.
Po e o ma ion in he basal po ion o app esso ium (open a ow) whe e a new elec on- anspa en ma e ial (solid a ow) is o med be-
ween app esso ial wall and plasma memb ane (a owhead). The agmen ed nuclea en elope (whi e a ows) and nucleus (N) a e also
shown. Scale ba = 0.5 µm. Fig. 8. App eso ium wi h o med po e, dissol ed wall (open a ow) in po e egion, smoo h plasma mem-
b ane (a owhead), and elec on- anspa en ma e ial in po e (solid a ow). The agmen ed nuclea en elope (whi e a ows) and he nu-
cleus (N) a e also shown. Scale ba = 1 µm. Fig. 9. Enla gemen o Fig. 8. The app esso ium plasma memb ane appea s smoo h excep
o he po e egion whe e i is wa y (a owhead). Scale ba = 0.5 µm. Fig. 10. App esso ium wi h in ec ion po e and ini ia ion o he
pene a ion peg (PP) o ma ion. Scale ba = 0.5 µm. AW, app esso ial wall; C, cu icle; HE, he e ophagosome; HW, hos cell wall; M,
mi ochond ia.
© 2005 NRC Canada
A oyo e al. 495

© 2005 NRC Canada
496 Can. J. Bo . Vol. 83, 2005
Figs. 11–13. Fo ma ion and de elopmen o pene a ion peg by Colle o ichum acu a um in s awbe y pe ioles a 24 hpi (TEM).
Fig. 11. App esso ium o ganelles (a owhead) in pene a ion peg (PP) and peg wall (a ow). Scale ba = 0.5 µm. Fig. 12. Pene a ion
peg (PP), small in amu al in ec ion esicle (IV), and elec on-dense deposi s (open a ows) in hos cell wall (HW) su ounding in ec-
ion esicle (IV). Scale ba = 0.5 µm. Fig. 13. In amu al de elopmen o in ec ion esicle (IV), peg wall (solid a ows), elec on-dense
deposi s in hos cell wall (open a ow), mi ochond ia (M) nea po e, and wa y plasma memb ane (a owhead). Scale ba = 0.5 µm.
AW, app esso ial wall; C, cu icle; HE, he e ophagosome; HW, hos wall.
© 2005 NRC Canada
A oyo e al. 497
Figs. 14–17. Ad anced s ages o in ec ion in pe ioles by Colle o ichum acu a um a 24 hpi. Fig. 14. Cu icle (C) be ween collapsed
app esso ium (A) and in amu al in ec ion esicle (IV) showing da k bands ha co espond o olds (a owhead) and dis up ion (a -
ow). Deg ada ion o he hos cell wall (HW) is isible (*). Scale ba = 0.5 µm. Fig. 15. Deg ada ion (*) o hos cell wall (HW) in e-
gion close o in asion hypha (H). Scale ba = 0.5 µm. Fig. 16. Cu icle (C) is olded and dis o ed in he p esence o a subcu icula
hypha (H). Subjacen epide mal cell (EC) wi h no isible signs o al e a ion is also shown. Scale ba = 1 µm. Fig. 17. Seconda y in-
ec ions owing o massi e in asion o hyphae (H) in a egion whe e cu icle (C) is dis o ed and b oken (a ows). High deg ees o deg-
ada ion o he hos cell wall (*) a e also shown. Scale ba = 1 µm. Fig. 18. Di ec pene a ion om a ge mina ed conidium (CO) o
Colle o ichum acu a um in s awbe y issues a 24 hpi (TEM). Pene a ion is h ough a small cu icle (C) dis up ion (a ow) ha p o-
duces an in asion hypha (H) in o hos cell wall (HW). Scale ba = 1 µm.
so ium and a subcu icula and in amu al in ec ion esicle.
The pene a ion peg has been obse ed and desc ibed a he
ul as uc u al le el in o he Colle o ichum spp. ha in ec
o he hos s (Coa es e al. 1993; La unde-Dada e al. 1996;
Me ce e al. 1975; Mould e al. 1991b; O’Connell e al.
1985; Poli is and Wheele 1973; Wha on e al. 2001; Xuei
e al. 1988). Cu y e al. (2002) men ioned, bu did no de-
sc ibe, he o ma ion o he pene a ion peg om app esso ia
o C. aga iae in ec ing s awbe y issues, a he ligh mi-
c oscope le el bu no he ul as uc u al le el. This is also
he i s epo showing he subcu icula and in amu al in-
ec ion esicle om C. acu a um du ing he ea ly in ec ion
p ocess o s awbe y issues and demons a es ha i be-
ha es as a subcu icula and in amu al pa hogen acco ding
o in ec ion s a egies p e iously desc ibed by Bailey e al.
(1992).
P e-pene a ion e en s om ge mina ion o app esso ium
o ma ion we e simila o hose desc ibed o o he species
o Colle o ichum (Dickman 2000). Mic ocyclic conidia ion
o conidiogenesis was obse ed on bo h adaxial and abaxial
su aces o a ached s awbe y lea es. This p ocess has
al eady been desc ibed in C. capsici on cowpea lea es
(La unde-Dada e al. 1999) and in C. acu a um on de ached
(Leand o e al. 2001) and a ached (Ho owi z e al. (2002)
s awbe y lea es, which may ha e implica ions on disease
epidemics. Howe e , on s awbe y pe ioles, conidiogenic
s uc u es we e no ound. In p e ious wo k, s awbe y
lea es we e less suscep ible o C. acu a um in ec ion han
pe ioles (A oyo e al. 2001; De los San os 1998). Leand o
e al. (2001) sugges ed ha he p oduc ion o seconda y
conidia may inc ease inoculum when suscep ible issue is
no a ailable. This is co ela ed wi h he obse a ion o mo e
se e e mac oscopic symp oms on pe ioles han on olia
issues, ha may be explained by he limi ed pene a ion
obse ed on lea es.
The pene a ion phase o C. acu a um on s awbe y is-
sues was no a synch onous p ocess, as p e iously desc ibed
o o he Colle o ichum spp. on o he hos s (O’Connell e
al. 1985; Makowski and Mo ensen 1998). Ou wo k shows
a de ailed desc ip ion, a he ul as uc u al le el, o di e en
app esso ia ma u a ion s ages o C. acu a um, be o e, du -
ing, and a e hei di e en ia ion o melaniza ion, which
occu s a app oxima ely he same ime ha app esso ial po e
o ma ion begins (Leand o e al. 2001). Du ing he pene a-
ion phase, he obse a ion o a clean dis up ion o he cu i-
cle, which does no display signs o deg ada ion, suppo s
© 2005 NRC Canada
498 Can. J. Bo . Vol. 83, 2005
Figs. 19–20. Coloniza ion o s awbe y pe ioles issues by Colle o ichum acu a um. Fig. 19. Ligh mic og aph o he in asion o
C. acu a um in pe ioles issues a 24 hpi. Seconda y in ec ions h ough dis up ed cu icle egions (open a ow) and discolo a ion o he
hos cell wall a e isible in deg ada ed egions (*). Subcu icula , in amu al, in e cellula , and in acellula hyphae (solid a ows) in he
second and hi d laye o co ex a e shown. Scale ba = 20 µm. Fig. 20. TEM mic og aph o C. acu a um in pe iole co ex cells a
24 hpi. In amu al and in acellula hyphae (H) wi h deg aded hos cell wall (*) and hyphae in he hos cell (a ow) a e shown. Scale
ba =2µm.
he hypo hesis ha pene a ion could be based on mechani-
cal o ces (Me ce e al. 1975; O’Connell e al. 1985). Ne -
e heless, o he ac o s such as he ac ion o ex acellula
enzymes du ing he pene a ion p ocess can no be excluded
(Manandha e al. 1985; O’Connell e al. 1985). The exis-
ence o app esso ia whose plasma memb anes show a
smoo h appea ance a he pene a ion ime, which could be
ela ed o an inc eased u go p essu e, also suppo s his
hypo hesis. Howa d e al. (1991) epo ed ha high u go
p essu e in he app esso ium is essen ial o mechanical pen-
e a ion. Ho owi z e al. (2002) obse ed collapsed ap-
p esso ia a e pene a ion, which hey ela ed o losses o
u go p essu e in hese s uc u es.
Whe eas o he species o Colle o ichum p oduce a pene-
a ion peg and de elop an in acellula in ec ion esicle
(Bailey e al. 1990; La unde-Dada e al. 1996; O’Connell e
al. 1985; Wha on e al. 2001), C. acu a um p oduces a pen-
e a ion peg ha de elops a subcu icula and in amu al in-
ec ion esicle. These esul s demons a e ha his pa hogen
uses a subcu icula in amu al in asion s a egy, which is e-
la ed o species wi h a wide hos ange ha a e conside ed
gene alis in ade s (Bailey e al. 1992). Howe e , Cu y e
al. (2002) le some unce ainly abou calling his ungus
hemibio ophs because o he b e i y o he bio ophic phase
in C. acu a um. In ou obse a ions, he bio ophic phase on
pe ioles was e y b ie , occu ing du ing he pe iod be ween
12 and 24 h a e inocula ion, simila o ha desc ibed by
Cu y e al. (2002).
A 24 h a e inocula ion, hos cell nec osis is less se e e
in olia isues han in pe ioles, so he bio ophic phase may
be p olonged in he olia issue and he pa hogen may
emain wi hin he plan cell wall longe wi hou causing
mac oscopically isible lesions. The e o e, his symp omless
s age in he lea issues would acili a e he su i al o
C. acu a um and ep esen a sou ce o inoculum o u he
in ec ions. The p esence o subcu icula , in amu al, and
in a- o in e -cellula hyphae in lea issues disag ees wi h
esul s o Leand o e al. (2001), who did no de ec hos pen-
e a ion o s awbe y lea es by C. acu a um. Howe e , ou
obse a ions ag ee wi h hose o Ho owi z e al. (2002), who
de ec ed he ungus inside s awbe y lea es.
The obse a ion o elec on-dense deposi s o unknown
na u e a ound he in ec ion esicle sugges s a de ence eac-
ion o he plan o he p esence o C. acu a um. In o he
hos -pa hogen in e ac ions in ol ing Colle o ichum spp., an
elec on-dense ma e ial called papilla has been ound in he
plan cell wall. This is a nonspeci ic esponse o he hos in
eac ion o he p esence o he pa hogen (Bailey e al. 1990;
Me ce e al. 1975; Mould e al. 1991b; O’Connell e al.
1985). In mos ins ances, hese elec on-dense deposi s a e
no capable o s opping he in asion o he pa hogen (Mould
e al. 1991b).
Du ing he hos issue in asion p ocess he hos walls ap-
pea ed highly deg aded, p esumably because o pa hogen
enzymes (Fe nando e al. 2001). This deg ada ion and dis-
o ganisa ion o he hos walls caused dila a ion and occa-
sionally he dis up ion o he cu icle. This phenomenon has
al eady been obse ed in al al a in ec ed by C. i olii (Po o
e al. 1988).
This ul as uc u al s udy o he in ec ion p ocess in pe i-
oles and lea es o s awbe y by C. acu a um suppo s a di -
e en esponse o bo h issues o his pa hogen, which had
al eady been obse ed a he mac oscopic le el. I is un-
known which mechanisms could be used by he olia issue
o limi he in asion o C. acu a um and keep i in a
symp omless s age.
Acknowledgmen s
We g a e ully acknowledge he collabo a ion o Asunción
Sánchez Con e as (CIFA Las To es-Tomejil), Remedios
Ga cía Na a o (Depa amen o Biología Celula de la
Uni e sidad de Se illa), and José Ma ía Sanab ia (Se icio
de Mic oscopía Elec ónica de la Uni e sidad de Se illa) o
hei in aluable echnical suppo .
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