773

Recibido: Octubre, 2005. Aprobado: Agosto, 2006.

Publicado como ARTCULO en Agrociencia 40: 773-782. 2006.
RESUMEN
Seestudi la patognesis deColletotrichumfragariae Brooks en
frutos inmaduros dechirimoya (Annona cherimola Mill.) porque
se desconocen las fases de este proceso, as como los cambios
anatmicos inducidos por el hongo en estecultivo. Los conidios
germinaron sobre la epidermis y tricomas, y formaron un
apresorio globoso o clavado 6-9 h despus dela inoculacin. El
hongo indujo la produccin de polifenoles en las clulas
epidermales y del parnquima, pero la infeccin no sedetuvo.
La penetracin fue directa a travs de clulas epidermales y
tricomas 24 h despus de la inoculacin. La colonizacin del
hongo en el mesocarpio fueinter eintracelular. Despus sepro-
dujo colapso y necrosis celular. El hongo form conidiforos
cortos libres o agrupados en acrvulos subcuticulares y
subepidermales en el tejido daado. C. fragariae complet el
ciclo dela enfermedad en 72 h en frutos inmaduros y sanos de
chirimoya.
Palabras clave: Annona cherimola, cambios estructurales, ciclo de
la enfermedad, polifenoles.
INTRODUCCIN
P
or sus excelentes caractersticas organolpticas,
la chirimoya (Annona cherimola Mill.) se con-
sidera la mejor fruta dentro de las anonceas
(Nakasone y Paull, 1998). Su cultivo en Mxico es
incipiente, se desarrolla en traspatio o como parte de
un sistema agrcola de subsistencia. Dentro de las
limitantes fitosanitarias de la produccin de chirimo-
ya, destacan las enfermedades causadas por hongos,
particularmente de los gneros Colletotrichum, Elsinoe,
Fusarium, Olpitrichum, Phoma, Phomopsis,
Phyllosticta, Rhizopus y Stigmella, los cuales reducen
la calidad comercial del producto (Andrs y Rebollar,
1996; Nava-Daz et al., 2000; Villanueva-Arce et al.,
2003). De estos gneros, se considera a Colletotrichum
como el patgeno ms importante (Andrs y Rebollar,
1996) por causar prdidas considerables si no se
PATOGNESIS DE LA ANTRACNOSIS (Colletotrichum fragariae)
EN FRUTOS DE CHIRIMOYA
PATHOGENESIS OF ANTHRACNOSE (Colletotrichum fragariae) IN CHERIMOYA FRUITS
Ramn Villanueva-Arce
1
, Elizabeth Crdenas-Soriano
2
, Ana M. Hernndez-Anguiano
2
, Antonio Mora-Aguilera
2
, Daniel Tliz-Ortz
2
1
UPIBI-IPN. Avenida Acueducto s/n. 07340. Ticomn, Mxico, Distrito Federal. (rarce@ipn.mx).
2
Campus Montecillo. Colegio de Postgraduados. 56230. Montecillo, Estado de Mxico.
ABSTRACT
Thepathogenesis of Colletotrichumfragariae Brooks on immature
cherimoya fruits (Annona cherimola Mill.) was studied because
thestages of this process, as well as theanatomical changes induced
by thefungus on this crop areunknown. Conidia germinated on
theepidermis and trichomes, and formed a globoseor clavate
appressorium 6-9 h after inoculation. The fungus induced the
production of polyphenols in epidermal cells and in thoseof the
parenchyma, but the infection did not cease. Penetration was
direct through epidermal cellsand trichomes24 h after inoculation.
Colonization of thefungus in themesocarp was inter- and intra-
cellular. Later cells collapsed and necrosis set in. The fungus
formed short conidiophores, freeor grouped in subcuticleand
subepidermal acervuli in the damaged tissue. C. fragariae
completed its diseasecyclein 72 h in healthy, immaturecherimoya
fruits.
Key words: Annona cherimola, structural changes, disease cycle,
polyphenols.
INTRODUCTION
B
ecause of its excellent organoleptic
characteristics, cherimoya (Annona cherimola
Mill.) is considered the best of annonas
(Nakasone and Paull, 1998). Its cultivation in Mxico
is incipient, grown in home gardens or as part of a
subsistence agricultural system. Among the major
limiting phytosanitary factors for cherimoya
production there are diseases caused by fungi,
particularly of the genera Colletotrichum, Elsinoe,
Fusarium, Olpitrichum, Phoma, Phomopsis,
Phyllosticta, Rhizopus, and Stigmella, which reduce
the commercial quality of the product (Andrs and
Rebollar, 1996; Nava-Daz et al., 2000; Villanueva-
Arce et al., 2003). Of these genera, Colletotrichum
is considered the most important pathogen (Andrs
and Rebollar, 1996), causing considerable losses if
it is not controlled in time. The disease caused by
Colletotrichumgloeosporioides (Penz.) Penz & Sacc
in cherimoya leaves is known as black spot (Nava-
Daz et al., 2000).
AGROCIENCIA, NOVIEMBRE-DICIEMBRE 2006
774 VOLUMEN 40, NMERO 6
controla oportunamente. La enfermedad causada por
Colletotrichumgloeosporioides (Penz.) Penz & Sacc
en hojas de chirimoya se conoce como mancha negra
(Nava-Daz et al., 2000).
C. gloeosporioides es el agente causal de la
antracnosis en papaya (Carica papaya L.), mango
(Mangifera indica L.), aguacate (Persea americana
Miller), chirimoya y otras especies de anonceas (An-
drs y Rebollar, 1996; Dickman, 1998; Ploetz, 1998;
Prusky, 1998). Puede encontrarse ms de una especie
atacando frutos u otros rganos de un mismo hospedante
(Freeman, 2000). Por ejemplo, en fresa, un complejo
de especies patognicas que incluyen a C. acutatum,
C. gloeosporioides y C. fragariae, inducen la pudricin
de frutos o partes vegetativas (Howard, 1972; Smith y
Black, 1987, 1990; Gunnell y Gubler, 1992). En
anonceas, y en especial en frutos de chirimoya, se
han identificado otras especies adems de C. gloeospo-
rioides (Farr et al., 1989; Villanueva-Arce et al., 2005).
En peciolos de fresa inoculados con C. fragariae,
la germinacin de conidios y formacin de apresorios
ocurri entre 6-16 h y 12-24 h despus de la inocula-
cin (Milholland, 1982; Curry et al., 2002). La pene-
tracin del hongo fue directa a travs de las clulas
epidermales y las basales de los tricomas. La coloniza-
cin del tejido fue inter e intracelular con una corta
fase biotrfica previa a la fase necrotrfica. Los cam-
bios estructurales se relacionaron con necrosis y colap-
so celular, desorganizacin del xilema y crecimiento
anormal de los elementos del haz vascular, as como la
formacin de tlides dentro de los vasos del xilema.
Despus de 72 h aparecieron sntomas de antracnosis y
se formaron acrvulos, los cuales irrumpieron por la
cutcula y liberaron conidios secundarios.
En frutos de chirimoya se desconocen las fases del
proceso de patognesis inducido por C. fragariae. Por
tanto, el objetivo del presente estudio fue estudiar los
cambios anatmicos inducidos por el hongo dentro de
los tejidos y las fases del desarrollo del hongo para
proponer el ciclo de la enfermedad de la antracnosis en
frutos de chirimoya.
MATERIALES Y MTODOS
Material vegetal
En julio de 2003 se recolectaron 25 frutos de chirimoya Concha
Lisa de cinco meses de edad (aproximadamente 750 horas-calor) en
el municipio de Coatepec Harinas, al sur del Estado de Mxico (18
57 54 N, 99 46 38 O; 2200 mde altitud; temperatura media
anual 14.9 C; 1057 mm precipitacin pluvial; clima templado
subhmedo). Los frutos se trasladaron al Laboratorio de Histopatologa
Vegetal del Colegio de Postgraduados en cajas de cartn a 20 C
para ser procesados.
C. gloeosporioides is the causal agent of anthracnose
in papaya (Carica papaya L.), mango (Mangifera indica
L.), avocado (Persea americana Miller), cherimoya
and other species of annonas (Andrs and Rebollar,
1996; Dickman, 1998; Ploetz, 1998; Prusky, 1998).
More than one species can be found attacking fruits or
other organs of a single host (Freeman, 2000). For
example, in strawberries a complex of pathogenic
species that includes C. acutatum, C. gloeosporioides
and C. fragariae, induce rot in fruits or vegetative
parts (Howard, 1972; Smith and Black, 1987, 1990;
Gunnell and Gubler, 1992). In annonas, and especially
in cherimoya fruit, other species besides C.
gloeosporioides have been identified (Farr et al., 1989;
Villanueva-Arce et al., 2005).
In strawberry petioles inoculated with C. fragariae,
conidial germination and appressorium formation took
place between 6-16 h and 12-24 h after inoculation
(Milholland, 1982; Curry et al., 2002). Penetration of
the fungus was directly through the epidermal and
trichome base cells. Colonization of tissue was inter-
and intra-cellular with a short biotrophic phase before
the necrotrophic phase. Structural changes were related
to necrosis and cell collapse, disorganization of the
xylem and abnormal growth of elements of the vascular
bundle, as well as formation of tilides within the vessels
of the xylem. After 72 h anthracnose symptoms
appeared and acervuli formed; these broke through the
cuticle and released secondary conidia.
In cherimoya fruit, the phases of the pathogenesis
process induced by C. fragariae are unknown.
Therefore, the objective of this study was to study the
anatomical changes induced by the fungus within tissues
and the phases of fungus development in order to
propose the disease cycle of anthracnose in cherimoya
fruit.
MATERIALS AND METHODS
Plant material
In J uly 2003, 25 five-month-old (approximately 750 hours heat)
Concha Lisa cherimoya fruits were collected in the municipality of
Coatepec Harinas, south of the State of Mxico (18 57 54 N, 99
46 38 W; 2200 maltitude; mean annual temperature 14.9 C;
1057 mm precipitation; subhumid temperate climate). The fruits
were transferred to the Histopathology Laboratory of the Colegio de
Postgraduados in cardboard boxes at 20 C for processing.
Innoculum obtention
M-35 monoconidial of C. fragariae, characterized by Villanueva-
Arce et al. (2005), obtained fromcherimoya fruits with anthracnose
and suspended in glycerol (20%) at -84 C, was reactivated in PDA
775 VILLANUEVA-ARCE et al.
PATOGNESIS DE LA ANTRACNOSIS (Colletotrichumfragariae) EN FRUTOS DE CHIRIMOYA
Obtencin del inculo
El monoconidial M-35 de C. fragariae, caracterizado por
Villanueva-Arce et al. (2005), obtenido de frutos de chirimoya con
antracnosis y mantenido en glicerol (20%) a 84 C, se reactiv en
PDA durante 5 d (271 C, luz blanca fluorescente continua). De
este cultivo se obtuvo una suspensin de conidios en agua destilada
estril.
Inoculacin defrutos
En los frutos de chirimoya lavados y desinfestados con hipoclorito
de sodio a 1.5% durante 5 min y lavados con agua destilada estril,
se aplic, con una micropipeta en un punto especficamente delimi-
tado de su superficie, aproximadamente 110
5
conidios contenidos
en 50 L de agua destilada estril. En los testigos se aplic slo
agua destilada estril. Los frutos inoculados se colocaron en charo-
las de plstico, sobre toallas de papel hmedas, se cubrieron con
bolsas de polietileno y se incubaron a temperatura de laboratorio
(252 C) y humedad relativa de 95-100%. Se utilizaron cuatro
frutos por tratamiento.
En septiembre 2003, en ocho frutos inmaduros de 7 meses de
edad (1100 horas-calor) en campo, se aplic de igual forma 110
5
conidios. Se cubrieron con bolsas de polietileno y se incubaron en
las condiciones ambientales de la regin (15-28 C, 95-100% HR).
En cuatro frutos testigo se aplic slo agua destilada estril.
Preparacin demuestras para microscopa deluz
De cada fruto se tom un trozo de tejido (1 cm
3
), en el rea
inoculada, a las 6, 9, 12, 24, 48 y 72 h despus de la inoculacin y
se fij con una solucin FAA (etanol:cido actico glacial:
formaldehdo: agua; 50:5:10:35) por 72 h. Estas muestras se lava-
ron tres veces cada 15 min, se deshidrataron e infiltraron en un
procesador de tejidos automtico Tissue-TekII (Mod. 4640-B, Sakura
Finetechnical Co., LTD. Tokio, J apan). Para la deshidratacin se
utilizaron soluciones de etanol a 50, 70, 96 y 100%, y etanol abso-
luto-xileno (1:1), con 3 h en cada una. La infiltracin e inclusin se
realizaron en paraplast (SIGMA Chemical Co., USA). Se hicieron
cortes (10 mgrosor) con un microtomo rotatorio (Mod. Spencer
820, American Optical Company); los cortes se tieron con la tincin
diferencial safranina 0-verde rpido (Curtis, 1986). Las preparacio-
nes se observaron en un microscopio de luz Ultraphot II Carl Zeiss.
Preparacin demuestras para microscopia
electrnica debarrido
De cada fruto se tom un trozo de tejido (1 cm
3
) del rea inocu-
lada a las 6, 9, 12, 24, 48 y 72 h despus de la inoculacin y se fij
con FAA por 72 h. Las muestras se lavaron tres veces con agua y
una vez con amortiguador de fosfatos 0.2 M pH 7.2. Despus, se
hicieron porciones ms pequeas del material (555 mm) que se
deshidrataron con soluciones graduales de alcohol (30, 40, 50, 60,
70, 80, 90 y 100%; las ltimas tres soluciones dos veces), durante
for 5 d (271 C, continuous white fluorescent light). Fromthis
culture, a suspension of conidia was obtained in sterile distilled
water.
Inoculation of fruits
Cherimoya fruits were washed and disinfected with 1.5% sodium
hypochlorite for 5 min and washed with sterile distilled water.
Approximately 110
5
conidia contained in 50 L of sterile distilled
water were applied to the fruits with a micropipette on a specifically
delimited point on their surface. Only sterile distilled water was
applied in the controls. Inoculated fruits were placed on plastic trays
on moist paper towels, covered with polyethylene bags and incubated
at laboratory temperature (252 C) and relative humidity (95-
100%). Four fruits per treatment were used.
In September 2003, in a like manner, 110
5
conidia were applied
on eight 7-month-old (1100 hours heat) immature fruits in the field.
These were covered with polyethylene bags and incubated under the
environmental conditions of the region (15-28 C, 95-100% RH).
On four control fruits, only sterile distilled water was applied.
Samplepreparation for light microscopy
Fromeach fruit, a piece of tissue (1 cm
3
) was taken fromthe
inoculated area 6, 9, 12, 24, 48, and 72 h after inoculation and
placed in a FAA fixing solution (ethanol:glacial acetic
acid:formaldehyde:water in a 50:5:10:35 proportion) for 72 h. The
portions of tissue were washed three times with water in intervals of
15 min, dehydrated and infiltrated in an automatic tissue processor,
Tissue-Tek II
TM
(Mod. 4640-B, Sakura Finetechnical Co,. LTD,
Tokyo, J apan). For dehydration, tissue was kept in ethanol solutions
at 50, 70, 96 and 100% and absolute ethanol-xylene (1:1) for 3 h
each. Infiltration and inclusion were performed in paraplast (SIGMA
Chemical Co., USA). Sections (10 mthick) were cut with a rotary
microtome (Mod. Spencer 820, American Optical Company); the
sections were dyed with differential fast saffranine 0-green (Curtis,
1986). The preparations were observed in an Ultraphot II Carl Zeiss
TM
light microscope.
Samples preparation for electronic scanning microscopy
Fromeach fruit, a 1 cm
3
piece of tissue was taken fromthe
inoculated area 6, 9, 12, 24, 48, and 72 h after inoculation and fixed
with FAA for 72 h. The samples were washed three times with
water and once with phosphate buffer, 0.2 M pH 7.2. Later, smaller
portions of the material were cut (555 mm) and dehydrated
with gradually higher solutions of alcohol (30, 40, 50, 60, 70, 80,
90 and 100%; the last three solutions were used twice), for 15 min
each. Samples were dried to critical point with CO
2
in a Samdri-
780A
TM
drier (TOUSIMIS Research corporation, Rockville, USA),
placed on a slide and covered with gold in a metal ionizer J FC-
110
TM
(J EOL LTD, Tokyo, J apan). The samples were observed
using a scanning electron microscope J SM-35C
TM
J EOL LTD,
Tokio, J apan).
AGROCIENCIA, NOVIEMBRE-DICIEMBRE 2006
776 VOLUMEN 40, NMERO 6
15 min en cada una. Las muestras se secaron a punto crtico con
CO
2
en una secadora Samdri-780A (TOUSIMIS Research
Corporation, Rockville, USA); se colocaron en un portamuestras y
se recubrieron con oro en una ionizadora de metales J FC-1100
(J EOL LTD, Tokio, J apan). Las muestras se observaron en un mi-
croscopio electrnico de barrido J SM-35C(J EOL LTD, Tokio,
J apan).
RESULTADOS Y DISCUSIN
Anatoma del fruto de chirimoya sano
El fruto sano de chirimoya Concha Lisa tuvo una
cutcula delgada y epidermis unicelular con tricomas
simples con ms de una clula (Figura 1). En el
mesocarpio, haba, adems de clulas de parnquima,
grupos de esclereidas, haces vasculares distribuidos irre-
gularmente, canales resinferos y clulas tanferas in-
dividuales o en grupos. Los polifenoles en las clulas
tanferas tenan colores rojos y guindas por la tincin
empleada (safranina-verde rpido). Las clulas con
polifenoles rojos fueron ms abundantes que las que
tuvieron polifenoles guindas, stos ltimos fueron gr-
nulos finos mientras que los rojos exhibieron formas
redondas semejantes a gotas de lpidos. En la porcin
ms interna del mesocarpio no se encontraron grupos
de esclereidas, las clulas taniferas fueron escasas y
los canales resinferos ms evidentes. El mesocarpio
estuvo delimitado por una epidermis interna. Tales ca-
ractersticas coincidieron parcialmente con la descrip-
cin de Roth (1975); las diferencias pudieron deberse
a las variedades estudiadas.
Figura 1. Fotomicrografas al microscopio de luz de cortes transversales que muestran la anatoma de la epidermis y parte del
parenquima deun fruto dechirimoya (Annona cherimola Mill.) sano. Tricomas (tr), epidermis (ep), esclereidas (sc), tejido
vascular (tv), clulas epidermales (ce).
Figure1. Light microscopephotomicrographs of cross sections that show theanatomy of theepidermis and part of theparenchyma of
healthy cherimoya fruit (Annona cherimola Mill.). Trichomes (tr), epidermis (ep), sclereids (sc), vascular tissue(tv), epidermal
cells (ce).
clornquima
sc
tv
tr
ep
10x
A
clornquima
ce
tr
40x
B
RESULTS AND DISCUSSION
Anatomy of healthy cherimoya fruit
Healthy Concha Lisa cherimoya fruit had a thin
cuticle and single-cell epidermis with simple trichomes
of more than one cell (Figure 1). In the mesocarp,
besides parenchyma cells, there were groups of
sclereids, irregularly distributed vascular bundles,
resiniferous channels, and tanniferous cells, single or
in groups. The polyphenols in the tanniferous cells
were red and dark red due to the dye used (saffranine-
fast green). The cells with red polyphenols were more
abundant than those with dark red polyphenols; these
were fine granules, while the red polyphenols exhibited
round forms similar to drops of lipids. In the innermost
portion of the mesocarp, groups of sclereids were not
found, tanniferous cells were scarce, and resiniferous
channels were more evident. The mesocarp was
delimited by an internal epidermis. These characteristics
coincide partially with the description by Roth (1975);
the differences could be due to the varieties under study.
Histopathology and development of the disease in
cherimoya fruits
Pre-infection
Pathogenesis began with the germination of the
conidia 6 to 9 h after inoculation, coinciding with the
studies of Milholland (1982) and Curry et al. (2002) in
strawberry plants. The conidia germinated on the fruit
777 VILLANUEVA-ARCE et al.
PATOGNESIS DE LA ANTRACNOSIS (Colletotrichumfragariae) EN FRUTOS DE CHIRIMOYA
Histopatologa y desarrollo de la enfermedad
en frutos de chirimoya
Preinfeccin
La patognesis se inici con la germinacin de los
conidios 6 a 9 h despus de la inoculacin y concord
con los estudios de Milholland (1982) y Curry et al.
(2002) en plantas de fresa. Los conidios germinaron
sobre la superficie del fruto y tricomas (Figura 2A-D).
Segn Sela-Buurlage et al. (1991), la adhesin de los
conidios en la superficie del hospedante podra estar
directamente relacionada con la naturaleza de las ceras
epicuticulares, ya que en el aguacate estas ceras indu-
cen la germinacin de los conidios (Krishna et al.,
1993; Prusky y Keen, 1993); en chirimoya, tales ceras
podran tambin haber favorecido la germinacin. El
proceso de germinacin involucr la formacin del tubo
germinativo, emitido en uno o ambos extremos del
conidio. En los extremos de los tubos germinativos se
form un apresorio globoso o clavado 12-24 h des-
pus de la inoculacin, que se tornaron caf a las 24 h
Figura 2. Fotomicrografas al microscopio electrnico debarrido dela superficiedefrutos dechirimoya (Annona cherimola Mill.)
inoculados con Colletotrichumfragariae. A, conidio (co) germinando sobrela superficiedel fruto; B, en el tricoma (tr); C y
D, conidio germinado y apresorio (ap) sobrela superficiedel fruto y tricomas; hifas (hi) y tubo germinativo (tg).
Figure2. Electronic scanning microscope photomicrographs of cherimoya fruit (Annona cherimola Mill.) surface inoculated with
Colletotrichumfragariae. A, condium (co) germinating on the fruit surface; B, in trichome (tr); C and D, germinated
conidium and appressorium (ap) on thefruit surfaceand trichomes; Hyphae(hi) and germ tube(tg).
15 KV X1000 0501
tr
tg
co
A
10.0U
tr
tg
co
B
tg
ap
co
C
ap
tr
D
15 KV X2000 0503 10.0U
1
5

K
V



X
2
0
0
0


0
8
0
9
1
0
.
0
U
15 KV X1300 0104 10.0U
C.P.
C
.
P
.
C.P.
surface and trichomes (Figure 2A-D). According to
Sela-Buurlage et al. (1991), the adhesion of the conidia
on the surface of the host could be directly related to
the nature of the epicuticular waxes, since in avocado
these waxes induce germination of conidia (Krishna et
al., 1993; Prusky and Keen, 1993); in cherimoya, they
could also have favored germination. The process of
germination involved formation of the germ tube,
emitted on one or both ends of the conidium. On the
tips of the germ tubes a globose or clavate appressorium
formed 12-24 h after inoculation; this turned brown at
24 h (Figure 2C), similar to that reported by Milholland
(1982) and Curry et al. (2002). Frequently the
production of brown or chestnut polyphenols was
observed in the epidermal cells that were in contact
with the conidia. These results contrast with those
reported by Mould et al. (1991a), who found that in
the pathosystem C. trifolii-alfalfa (Medicago sativa)
the reactions of the host cells begin only after contact
with the penetration point, and this was observed
because the infected cells were dyed intensely by the
polyphenols.
AGROCIENCIA, NOVIEMBRE-DICIEMBRE 2006
778 VOLUMEN 40, NMERO 6
(Figura 2C), similar a lo reportado por Milholland
(1982) y Curry et al. (2002). Frecuentemente se ob-
serv la produccin de polifenoles caf castao en
las clulas epidermales que tuvieron contacto con los
conidios. Estos resultados contrastaron con los repor-
tados por Mould et al. (1991a) quienes encontraron
que en el patosistema C. trifolii-alfalfa (Medicago
sativa), las reacciones de las clulas del hospedante se
iniciaron slo despus del contacto con la punta de
penetracin y se observ porque las clulas infectadas
se tieron intensamente por la presencia de los
polifenoles.
Infeccin
La penetracin del hongo fue directa a travs de
una punta de penetracin desarrollada en la base del
apresorio, como se ha reportado para varias especies
de Colletotrichum (Mould et al., 1991b; Mims y
Vaillancourt, 2002; Curry et al., 2002). La penetra-
cin tuvo lugar en las clulas epidermales y en las
clulas de los tricomas (Figura 3A-B) 24-48 h despus
de la inoculacin. Esto demuestra que la susceptibili-
dad de variedades de chirimoya al hongo puede estar
asociada con elevadas cantidades de tricomas, ya que
estas estructuras no evitaron que el hongo penetrara al
fruto. Este resultado puede ser til en programas de
mejoramiento gentico para desarrollar variedades re-
sistentes a la enfermedad.
Colonizacin
No se observ fase de latencia del hongo en la
etapa de apresorio, tal como ocurre cuando C. gloeos-
porioides infecta al aguacate u otros frutos tropica-
les o subtropicales (Binyamini y Schiffmann, 1971;
Dodd et al., 1997). Los frutos inmaduros, de siete
meses de edad, inoculados en campo con conidios
de C. fragariae desarrollaron sntomas de antracnosis
9-13 d despus. Esto demuestra que este hongo, en
chirimoya, puede infectar frutos inmaduros en
precosecha, lo cual contrasta con lo reportado para
los patosistemas C. gloeosporiodes-aguacate, mango
o papaya donde el hongo infecta slo en postcosecha
(Dickman, 1998; Ploetz, 1998; Prusky, 1998).
La colonizacin del hongo fue inter e intracelular y
avanz de las clulas epidermales y tricomas al
mesocarpio. En los tricomas se observ como la hifa
del hongo creci hacia la clula basal y de sta al
parnquima (Figura 3B).
En las clulas epidermales no slo se indujo la pro-
duccin de polifenoles al contacto de los conidios con
la cutcula sobre la pared de las clulas, sino tambin
cuando la hifa del hongo penetr al citoplasma. No
Infection
Penetration of the fungus was directly through a
penetration point developed at the base of the
appressorium, as has been reported for several species
of Colletotrichum(Mould et al., 1991b; Mims and
Vaillancourt, 2002; Curry et al., 2002). Penetration
took place in the epidermal cells and in the cells of the
trichomes (Figure 3A-B) 24-48 h after inoculation. This
demonstrates that susceptibility of cherimoya varieties
to the fungus may be associated with large quantities
of trichomes, since these structures did not prevent the
fungus from penetrating the fruit. This result could be
useful in genetic improvement programs for the
development of disease-resistant varieties.
Colonization
No latent phase of the fungus was observed in the
appressorium stage, as occurs when C. gloeosporioides
infects avocado or other tropical or subtropical fruits
(Binyamini and Schiffmann-Nadel, 1971; Dodd et al.,
1997). Seven-month-old immature fruits inoculated with
C. fragariae conidia developed symptoms of
anthracnose 9-13 d later. This demonstrates that this
fungus in cherimoya can infect immature fruits before
harvest, contrasting with reports that the fungus infects
only in postharvest in the pathosystems C.
gloeosporioides-avocado, mango, or papaya (Dickman,
1998; Ploetz, 1998; Prusky, 1998).
Colonization was inter- and intra-cellular, and
progressed from the epidermal and trichome cells to
the mesocarp. In the trichomes, it was observed that
the fungus hyphae grew toward the base cell and from
there to the parenchyma (Figure 3B).
In epidermal cells, not only was the production of
polyphenols induced when the conidia made contact
with the cuticle on the cell walls, but also when the
fungus hyphae penetrated the cytoplasm. In spite of
the induction of brown or chestnut polyphenols in the
infected cells, the progress of the fungus advanced to
the mesocarp parenchyma cells (Figure 3C). After 48-
72 h, the cells that were higher toward the surface
began collapsing. After this, the first macroscopic
symptoms were observed: brown or dark brown necrotic
lesions with bordered edges, sunken, typical of
anthracnose, and in these lesions conidia and short free
conidiophores were produced (Figure 4A), constituting
a source of secondary inoculum.
As the tissues were colonized by the fungus, red
and dark red polyphenols were metabolized, leaving
the former disposed in the cell walls, the reason that
alterations were more evident in the damaged area. In
contrast, the dark red polyphenols turned brown and
779 VILLANUEVA-ARCE et al.
PATOGNESIS DE LA ANTRACNOSIS (Colletotrichumfragariae) EN FRUTOS DE CHIRIMOYA
obstante la induccin de polifenoles caf o castao en
las clulas infectadas, el avance del hongo continu a
las clulas del parnquima del mesocarpio (Figura 3C).
A las 48-72 h las clulas dispuestas ms hacia la super-
ficie del fruto colapsaron. Despus, se observaron los
primeros sntomas macroscpicos: lesiones necrticas
caf o caf oscuro, con bordes delimitados, hundidas,
tpicas de antracnosis y en ellas se produjeron conidios
y conidiforos cortos libres (Figura 4A) que constitu-
yeron una fuente de inculo secundario.
Conforme los tejidos fueron colonizados por el hon-
go, los polifenoles rojos y guindas se metabolizaron
quedando los primeros dispuestos en las paredes celu-
lares, por lo que las alteraciones fueron ms evidentes
en la zona daada; en contraste, los guindas se torna-
ron caf y permanecieron en el citoplasma. Es proba-
ble que la alteracin metablica de los compuestos
fenlicos ocasione la muerte celular, y sta, junto con
los polifenoles, sean los responsables del color y del
lmite de la lesin. En infecciones ms desarrolladas
que alcanzaron la epidermis interna del mesocarpio, se
indujo hiperplasia celular con la diferenciacin de abun-
dantes esclereidas y clulas tanferas. En este estado
Figura 3. Fotomicrografas al microscopio deluz quemuestran la penetracin (A, B) y colonizacin (C, D) por Colletotrichumfragariae
a travs declulas epidermales (ce) y tricomas (tr). Colonizacin inter eintracelular (C) y colapso celular (D) delas primeras
capas declulas. Apresorio (ap), hifa (hi), conidios (co), epidermis (ep).
Figure3. Light microscopephotomicrographs that show penetration (A, B) and colonization (C, D) by Colletotrichumfragariae through
epidermal cells (ce) and trichomes (tr). Inter- and intra-cellular colonization (C) and cell collapse(D) of thefirst layers of
cells. Appressorium (ap), hypha (hi), conidia (co), epidermis (ep).
hi
ce
ap
100x
A
hi
tr
40x
B
C
D
100x
hi
co
tr
ep
Clulas del parnquima
stayed in the cytoplasm. It is likely that the metabolic
alteration of phenolic compounds cause cells to die, and
this, together with the polyphenols, is responsible for
the color and the border of the lesion. In more developed
infections that reached the internal epidermis of the
mesocarp, cell hyperplasia was induced with the
differentiation of abundant sclereids and tanniferous cells.
In this stage of development of the lesion (72 h), it was
observed that the fungus established on the fruit surface
produced a mycelial stroma, from which the acervulus
developed. The acervuli were subcuticular and
subepidermal, and their eruption was carried out by setae
with 4-9 septa which produced a large amount of conidia
once emerged (Figure 4B), but the largest production of
conidia occurred in the short acervular conidiophores
(Figure 5) imbibed in orange gelatinous masses. In this
manner the disease cycle caused by C. fragariae in
immature fruits completed in 72 h (Figure 6).
The production of secondary inoculum in cherimoya
coincided with that reported by Milholland (1982) and
Curry et al. (2992) for C. fragariae in strawberries,
which is apparently much faster than C. gloeosporioides
in papaya (Chau and Alvarez, 1983). The excessive
AGROCIENCIA, NOVIEMBRE-DICIEMBRE 2006
780 VOLUMEN 40, NMERO 6
de desarrollo de la lesin (72 h) se observ que el
hongo establecido en la superficie del fruto produjo un
estroma micelial, del cual se desarroll el acrvulo.
Los acrvulos fueron subcuticulares y subepidermales
y su erupcin se llev a cabo por setas de 4-9 septos
que produjeron una gran cantidad de conidios una vez
emergidas (Figura 4B), pero la mayor produccin de
conidios ocurri en conidiforos cortos acervulares
(Figura 5) embebidos en masas gelatinosas anaranja-
das. As, el ciclo de la enfermedad por C. fragarie en
frutos inmaduros se complet en 72 h (Figura 6).
La produccin de inculo secundario en chirimoya
coincidi con lo reportado por Milholland (1982) y
Curry et al. (2002) para C. fragariae en fresa, que
aparentemente es mucho ms rpida que la de C. gloeos-
porioides en papaya (Chau y Alvarez, 1983). La pro-
duccin excesiva de nueva fuente de inculo por C.
fragariae en frutos de chirimoya puede aumentar
production of the new source of inoculum by C.
fragariae in cherimoya fruits can increase the progress
of the disease significantly in the field when conditions
of humidity and temperature are favorable for the
infection. The above could explain why C. fragariae is
more virulent than C. gloeosporioides (Mass and
Howard, 1985; Smith and Black, 1990).
CONCLUSIONS
The pathogenesis of Colletotrichumfragariae in five-
month-old picked immature cherimoya fruits was
characterized by the well-defined phases of pre-
infection, infection and colonization. The main structural
changes induced by the fungus were accumulation of
phenolic compounds in the cell walls and cytoplasm,
collapse of the epidermal and parenchyma cells, and
necrosis. The disease cycle was completed in 72 h.
Figura 4. Fotomicrografas al microscopio electrnico debarrido quemuestran la produccin deconidios (co) en conidioforos libres (A)
y setas fertiles (B) deColletotrichumfragariae en frutos dechirimoya (Annona cherimola Mill.). Tricoma (tr), conidiforo
libre(cd), setas (st), estoma (s).
Figure4. Electronic scanning microscopephotomicrographs that show conidium (co) production in freecondiophores (A) and fertile
setae(st) (B) of (Colletotrichumfragariae in cherimoya fruit (Annona cherimola Mill.). Trichome (tr), freeconidiophore(cd),
setae(sf), stoma (s).
C
.
P
.
15 KV X1200 0103 10.0U
C.P.
tr
B
1
5

K
V


X
6
6
0


0
6
0
8
C.P.
st
s
1
0
.
0
U
co
A
co
cd
Figura 5. Fotomicrografas al microscopio electrnico debarrido quemuestran la produccin deconidios (co) en setas frtiles (st) (A)
y conidiforos cortos acervulares (cd) tricomas (tr) (B), deColletotrichumfragariae en frutos dechirimoya (Annona cherimola
Mill.).
Figure5. Electronic scanning microscope photomicrographs that show conidium production (co) in fertile setae (st) (A) and short
acervular conidiophores (cd) and trichomes (tr) (B) of Colletotrichumfragariae in cherimoya fruits (Annona cherimola Mill.).
C.P.
B
co
cd
acrvulos
15 KV X1500 0804 10.0U
C.P.
acrvulos
V X400 0805 10.0U
A
tr
st
st
781 VILLANUEVA-ARCE et al.
PATOGNESIS DE LA ANTRACNOSIS (Colletotrichumfragariae) EN FRUTOS DE CHIRIMOYA
significativamente el progreso de la enfermedad en cam-
po cuando las condiciones de humedad y temperatura
son favorables para la infeccin. Lo anterior podra
explicar por qu C. fragariae es ms virulento que C.
gloeosporioides (Mass y Howard, 1985; Smith y Black,
1990).
Figura 6. Ciclo dela enfermedad dela antracnosis en frutos dechirimoya (Annona cherimola Mill.) por Colletotrichumfragariae.
Fuenteprincipal deinculo (a). Produccin deconidios en setas (b) y acrvulos (c). Adhesin deconidios en frutos sanos (d),
en clulas epidermales y tricomas (e). Germinacin deconidios 6-9 h (f), formacin deapresorios y punta depenetracin 12-
24 h (g). Penetracin directa por las clulas epidermales y tricomas (g). Colonizacin inter eintracelular delas clulas
epidermales y basales detricomas al mesocarpio 24 h (h). Aparicin delesiones 24-48 h (i-j). Formacin de acrvulos 48-
72 h y produccin denueva fuentedeinculo 72 h (b-c).
Figure6. Diseasecycleof anthracnosein cherimoya fruits (Annona cherimola Mill.) by Colletotrichumfragariae. Principal sourceof
inoculum (a). Production of conidia in setae(b) and acervuli (c). Adhesion of conidia on healthy fruits (d), in epidermal cells
and trichomes (e). Germination of conidia 6-9 h (f), formation of appressoria and penetration point 12-24 h (g). Direct
penetration through epidermal cells and trichomes (g). Inter- and intra-cellular colonization of theepidermal cells and basal
cells of trichomes to mesocarp 24 h (h). Appearance of lesions 24-48 h (i-j). Formation of acervuli 48-72 h (b-c), and
production of new sourceof inoculum 72 h (b-c).
a
b
c
d
Conidios
C
o
n
i
d
i
o
Tricoma
Arribo del
conidio
Fruto sano
Fruto momificado
Epidermis
Produccin deconidios en
setas frtiles y conidiforos
cortos acervulares 72 h
Emergencia de setas y
produccin de conidios
48-72 h
Lesin macroscpica avanzada
(necrsis y colapso)
Lesin microscpica
24-48 h
Formacin de apresorios
y penetracin 12-24 h
Germinacin de
conidios 6-9 h
Colonizacin 24 h
Parnquima
e
f
g
h
i
j
Immature fruits on the plant or picked are susceptible
to anthracnose caused by C. fragariae.
End of the English version

AGROCIENCIA, NOVIEMBRE-DICIEMBRE 2006

782 VOLUMEN 40, NMERO 6
CONCLUSIONES
La patognesis de Colletotrichumfragariae en fru-
tos inmaduros de chirimoya cortados a los cinco me-
ses, se caracteriz por presentar las fases de
preinfeccin, infeccin y colonizacin bien definidas.
Los principales cambios estructurales inducidos por el
hongo fueron: acumulacin de compuestos fenlicos
en las paredes celulares y citoplasma, colapso de las
clulas epidermales y del parnquima, y necrosis. El
ciclo de la enfermedad se complet en 72 h. Frutos
inmaduros en la planta o cortados son susceptibles a la
antracnosis por C. fragarie.
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