J Phytopathol 158:270277 (2010) doi: 10.1111/j.1439-0434.2009.01608 .

x
2009 Blackwell Verlag GmbH

Universidade Federal de Lavras (UFLA), Lavras, Brazil

Gejala dan kejadian Prepenetration yang berhubungan dengan infeksi pada buncis oleh Anamorph
danTeleomorph dari Glomerella cingulata f.sp. phaseoli
Francine H. Ishikawa1, Quelen L. Barcelos1, Eduardo Alves2, Osnil A. Camargo Jr1 and Elaine A. de
Souza1
Authors addresses: 1Laboratorio de Resistencia de Plantas a Doencas, Departamento de Biologia,
Universidade Federal de
Lavras, 37200-000 Lavras, Minas Gerais, Brazil; 2Laboratorio de Microscopia Eletronica e Analise
Ultraestrutural,
Departamento de Fitopatologia, Universidade Federal de Lavras, 37200-000 Lavras, Minas Gerais,
Brazil (correspondence to
E. A. de Souza. E-mail: easouza@ufla.br or F. H. Ishikawa. E-mail: francinehi@hotmail.com)
Received February 26, 2009; accepted May 15, 2009

Keywords: anthracnose, appressoria, ascospores, Colletotrichum lindemuthianum, germination,

Phaseolus vulgaris, sexual
reproduction

Abstract
Glomerella cingulata f.sp. phaseoli dan Colletotrichum lindemuthianum adalah teleomorph dan
anamorph, dari pathogen yg menyebabkan anthracnose pada buncis. Mekanismenya berhubungan
dengan reproduksi seksual dari tanaman pathogen, seperti infeksi dan gejala produksi. Dalam penelitian
ini, tanaman buncis dinokulasi dengan ascospores dan conidia, kejadian tersebut menempati tempat
selama 120 h yang diperiksa dengan menggunakan mikrskop cahaya dan scanning dengan mikroskop
elektron. Gejala yang diperlihatkan oleh tanaman yang diinfeksi dengan ascospores lebih kecil daripada
yang diinokulasi dengan conidia. Microscop memperlihatkan bahwa sebagian besar dari ascospores
memproduksi germ tubes dan appressoria pada tahap awal (24 jam setelah inokulasi). Mulai 48 jam
kedepan, formasi dari hyphae dan produksi dari germ tubes and appressoria sangat tinggi. Berbeda
dengan infeksi dari perkembangan konidia sangat rendah pada 24 dan 48 jam, banyak conidia yang
tidak bergerminasi, hanya sedikit conidia yang mengembangkan germ tubes dan appressoria. Ascospore
germination and appressorium formation were similar on both resistant and susceptible cultivars. Hence,
the symptoms and the temporal sequence of events associated with the infection of bean plants by the
two fungal forms differed, although the structures produced were similar. Ini merupakan laporan
pertama yang membadingkan symptoms and kejadian prepenetrasi antara anamorph dan teleomorph of
G. cingulata f.sp. phaseoli dalam buncis.

ntroduction
Glomerella cingulata SymptomsandPrepenetration Events 271

Information concerning the prepenetration events and symptoms resulting from infection by
theteleomorphof Glomerella cingulata f.sp. phaseoli is scarce. It has been reported that teleomorph G.
cingulata f.sp. phaseoli (Kimati and Gali, 1970) produces milder symptoms compared with those
induced by anamorph Colletotrichum lindemuthianum (Sacc. and Magnus) ( Camargo et al., 2007). The
causal agent of anthracnose in common bean (Phaseolus vulgaris L.) is normally found in
natureinthemitospore-producingform(Sutton,1992).
Colletotrichum lindemuthianum adalah contoh klasik dari jamur hemibiotrophic because the
pathogen forms an intracellular infection vesicle followed by primary hyphae a few days after
penetration of the cuticle and cell wall of the host plant. The primary hyphae subsequently produce
ramifications of secondary hyphae, causing cell death, and from this point onwards fungal growth is
necrotrophic (OConnell et al., 2000). The symptoms induced following infection by this plant pathogen
include the emergence of dark-brown necrotic lesions in the main veins of the lower surface of the leaf
blade, and superficial or internal elongated lesions in the hypocotyls, which may cause strangulation
and, ultimately, plant death (Kimati et al., 1997).
The occurrence of a sexual phase of the fungus was first reported almost a century ago by Shear and
Wood (1913), who named this form G. lindemuthiana. More recently, however, Kimati and Galli (1970)
proposed the name G. cingulata f.sp. phaseoli. During the past 20 years, a number of genetic studies
using morphological and molecular markers have demonstrated the formation of recombinant forms
during the sexual phase (Batista and Chaves, 1982; Bryson, 1990 ; Mendes-Costa, 1996; Rodriguez-
Guerra et al., 2005 ; Camargo et al., 2007; Luna-Martinez et al., 2007) , results which may explain the
remarkable variability that has been observed amongst different populations of the fungus (Rodriguez-
Guerra et al., 2005; Camargo et al., 2007). The meiospore producing form of the ascomycete has been
observed in field-collected isolates maintained under laboratory conditions in the absence of artificial
inducers (Camargo et al., 2007; Damasceno e Silva et al., 2007; Souza et al., 2007).
Afewyearsago,the initial infection events (formation of appressoria and fungal penetration) of many
plant pathogens, including C. lindemuthianum, were extensively revised
(Hardham,2000;TuckerandTalbot,2001; Perfect et al., 2001; Veneault-Fourrey et al., 2005), but nothing
related to the infection by meiosporic producing form was described. We here describe the symptoms
exhibited by bean plants infected by theteleomorp hand anamorph of G. cingulata f.sp. phaseoli. The
events preceding fungal penetration into the host cells have been analyzed using light microscopy and
scanning electron microscopy (SEM) for the first time and compared with the mitosporic form.

Materials and Methods

Origin and maintenance of fungal isolates
The isolates (Table 1) of G. cingulata f.sp. phaseoli employed were from the fungal collection of the
Plant Resistance Laboratory of the Biology Department of the Universidade Federal de Lavras (Lavras,
MG, Brazil). All isolates were obtained from anthracnose lesions on leaves and pods on common bean
cultivars in the field. Teleomorphs were obtained from the field and isolates were obtained without any
induction (specific medium or nutrient).The isolates were maintained in M3 culture medium (Junqueira
et al., 1984), and subsequently inoculated into sterilized bean pods, partially submerged in 2% agar
contained in glass tubes, and incubated at 22 2C for 1520 days in darkness for the production of
spores. The same conditions were used for both the anamorph and teleomorph.

Slide culture
All the strains (Table 1) were evaluated in microscope slide wells filled with distilled water. Spores
were harvested from a 15- to 20-day-old culture and washed twice in water to remove all mucilage.
Spore concentration was adjusted to 5 105 cells ml for all strains and spores were incubated at 22
2C. Germination was monitored on a phase contrast microscope ( Leica DMLS, Wetzlar, Germany) at
272 Ishikawa etal.

6, 12 and 24 h. The statistical significance of differences between mean values was assessed using an
anova and Scott Knott range test.

Inoculation for microscopy studies

Seeds of the cultivars were cultivated in polystyrene trays containing substrate Plantmax(
Eucatex,Paulina, Sao Paulo, Brazil). After 15 days, the primary leaves were removed and placed in
Petridishes lined with filter paper moistened with sterile water in orderto maintain humidity. Spores
were harvested from a 15- to 20dayold culture and washed twice in water to remove all mucilage. Drops
of spore suspensions (1.2 106 spores ml) were place don leaves,which were then incubated under
humid condition sas described above,forperiods of24,48,72,96and120 h.

Light microscopy
Ascospores of UFLAG03, and conidia of LV116
(non-pathogenic control) and LV117 (pathogenic control) were tested (Table 1). Spores were harvested
as described in the previous section. A susceptible and a resistant cultivar, Perola and G2333,
respectively, were used in this experiment. Inoculated leaves were cleared using a modification of the
method of Ryan and Clare (1974). Pieces of leaf (approximately 1 cm2) were placed on Petri dishes
containing filter paper humidified in acetic acid and ethanol (1 : 1 v v) solution for 48 h. The leaves
were then transferred to filter paper humidified in sterilized water for 30 min. The cleared leaves were
finally mounted with a drop of solution 67 mm K2HPO4 (pH 9.0) containing 0.1 % aniline blue and 50%
glycerol, and were observed with a phase contrast microscope (Leica DMLS). The
Table1
Strains of Glomerella cingulata f.sp. phaseoli and Colletotrichum lindemuthianuminvestigated
Isolate Racea Phase Origin b
UFLAG01 0 Teleomorph Guarapuava, PR
UFLAG02 0 Teleomorph Lavras,MG
UFLAG03 0 Teleomorph Ribeira
oVermelho,MG
UFLAG04 0 Teleomorph RibeiraoVermelho,MG
UFLAG05 0 Teleomorph RibeiraoVermelho,MG
UFLAG06 0 Teleomorph RibeiraoVermelho,MG
UFLAG07 0 Teleomorph Lavras,MG
UFLAG08 0 Teleomorph Lavras,MG
LV51 73 Anamorph Lavras,MG
LV77 81 Anamorph Lavras,MG
LV115 65 Anamorph PatosdeMinas,MG
LV116 Non-pathogenic PatosdeMinas,MG
Anamorph
LV117 65 Anamorph Lavras,MG
a

Race 0 symptom observed on Perola (employed as a positive control);non-pathogenicnone of

cultivars were susceptible. b Isolates obtained from anthracnose lesions on leaves and pods on common
bean cultivars in the field. PR, Parana; MG, Minas gerais
(BrazilianStates).
Glomerella cingulata SymptomsandPrepenetration Events 273

statistical significance of differences between mean values was assessed using an anova and Scott Knott
range test.

Analysis of events preceding host cell penetration and SEM

Ascospores of UFLAG01 and conidia of LV117 strains were inoculated onto the susceptible cultivar
(Perola) at the same concentration (1.2 106 spores ml). Following each time period (24, 48, 72, 96
and 120 h), leaf samples were prepared for SEM analysis by fixing for 48 h in modified Karnovsky
solution [2.5% glutaraldehyde and 2.5% formaldehyde in
0.05 m sodium cacodylate buffer (pH 7.2) containing 0.001 m calcium chloride], washing with 0.05 m
sodium cacodylate buffer (pH 7.2) and fixing for 1 h with 1% osmium tetroxide in sodium cacodylate
buffer at room temperature. Following fixation, leaf samples were dehydrated in water : acetone step
gradient with acetone concentrations of 25, 50, 75, 90 and 100%. Exposure to each step was for 10 min,
and the 100% step was repeated three times. Acetone was removed using a Bal-Tec (Balzers,
Liechtenstein) model CPD 030 critical point drier. Samples were fixed to aluminium stubs with double-
sided adhesive carbon tape, and coated under vacuum with a thin film of metallic gold using a Bal-Tec
model SCD 050 evaporator. A Nano Technology Systems (Carl Zeiss, Oberkochen, Germany) model
Evo 40 VP SEM was used with an accelerating voltage of 20 kV and a working distance of 9 mm to
obtain digital images at varying magnifications. The images were processed using corel draw 9
photopaint software (Corel Corporation, Ottawa, Canada).

Pathogenicity tests
Sixteen seeds each of 12 differential bean cultivars (Centro Internacional de Agricultura Tropical, 1990)
togetherwithcv.Perola,which issusceptible to various C. lindemuthianum races and served as a positive
control, were cultivated in polystyrene trays (128 wells each) containing the commercial substrate
Plantmax. After the emergence of primary leaves, seedlings were sprayed with 200 ml of the suspension
of fungal spores (1.2 106 spores ml) that were harvested from 15- to 20-day-old culture and the trays
were placed in a humid incubator for 72 h at a temperature of 20 2C with a photoperiod of 12 h dark
12 h light. Seedlings were then transferred to a green house for 10days, after which the symptoms
exhibited by the infected plants were evaluated on a scale from 1 to 9, in which scores 1 to 3 represented
resistant plants and scores4to9 represented susceptible plants(Schoonhoven and Pastor-Corrales, 1987).
The Habgood (1970) binary system was employed for the purpose of race identification.

Re-isolation of Glomerella cingulata f.sp. phaseoli

Leaf segments with symptoms of anthracnose were treated with 70% ethanol for 2 min, followed by 1%
sodium hypochlorite solution for 2 min, and then thoroughly rinsed with sterile distilled water. The
sterilized leaves were incubated on M3 culture medium at 22 2C to allow fungal growth.
Results
Ascospores germinate faster than conidia in vitro
The germination rates of ascospores were faster than conidia in slide culture conditions (Table 2). This
assay allows the evaluation of a large number of strains, whereas quantification performed on beans
leaves has been very difficult. The statistical analyses performed showed that all factors were
significantly different: Strain (P < 0.0001), incubation time (P < 0.0001) and the interaction strain time
(P < 0.001). Different groups were formed according the percentage of germination. In general, one
group (A) was formed by C. lindemuthianum strains and the other groups (B, C and D) by G. cingulata
f sp. phaseoli strains (Table 2). It was possible to observe statistical difference between the teleomorph
strains at 6 and 12 h incubation time. At 6 h, two groups were formed (A and B) and the strain UFLAG07
showed low germination rate and was grouped together with anamorph strains. However, at 12 h
incubation time, teleomorph strains formed three different groups (B, C and D). UFLAG06 showed the
274 Ishikawa etal.

higher percentage of germination at 12 h (37.89%). All G. cingulata f sp. phaseoli strains (teleomorph)
were classified in the same group (B) at 24 h.

Ascospores and conidia germinate and form appressorium on the leaf surface
Twenty-four hours after inoculation, most of ascospores of strain UFLAG03 had germinated on the
leaves of resistant (75.66%) and susceptible (76.33%) cultivars (Table 3). The UFLAG03 germination
percentages were significantly different (P < 0.0001) for the pathogenic (LV117) and non-pathogenic (
LV 116) controls. After 48 h following inoculation, almost 100% of ascospores were germinated,
whereas few conidia had germinated in the pathogenic control. It was not possible to quantify the non-
pathogenic control at 48 h because few conidia were observed and the conidia seemed to disintegrate.
The statistical analyses performed using UFLAG03 and LV117 showed that all factors were
significantly different: Strain
Table2
%Sporegermination
Isolates 6h 12h 24 h
a
UFLAG01(ascospores) 10.336.40 B 9.165.35 B 100.00 B
UFLAG02(ascospores) 6.575.89B 11.007.02B 100.00 B
UFLAG03(ascospores) 6.891.89B 9.944.63 B 100.00 B
UFLAG04(ascospores) 10.334.91B 22.3116.01C 100.00 B
UFLAG05(ascospores) 7.784.29B 14.786.91B 100.00 B
UFLAG06(ascospores) 16.229.63B 37.8922.22D 100.00 B
UFLAG07(ascospores) 4.671.66A 11.452.54B 100.00 B
UFLAG08(ascospores) 15.0017.03B 19.6725.54C 100.00 B
LV51(conidia) 1.440.50A 2.000.33 A 2.890.19
A
LV77(conidia) 0.110.19A 0.440.19 A 2.330.33
A
LV115(conidia) 0A 1.111.92 A 2.442.22
A
LV116(conidia) 0A 1.00A 3.671.88
A
LV117(conidia) 0A 1.160.23 A 1.330.46
A
Percentageofspore germination from differentstrains of
Glomerella cingulata f.sp. phaseoli and Colletotrichum lindemuthiamum at 6 ,12and24h
incubationtimein water(slide culture)

Meanvalueswiththesameletterarenot significantly differentaccordingtoaScottKnottstest5%.

a
Meanandstandarddeviationforthreereplicates(300sporesobserved).
Glomerella cingulata SymptomsandPrepenetration Events 275

(resistant )
a a
Germinatedspores(%) Germinatedsporesformingappressoria(%)

Perola G2333 Perola G2333

Fungalstrain 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h

UFLAG03 76.3317.47 97.662.51 75.666.43 98.331.52 63.712.74 96.331.15 65.78.37 97.332.08

Table3
Spore germination and appressorium formation of Glomerella cingulata f.sp. phaseoli strain
(UFLAG03) and Colletotrichum lindemuthianum strains (LV116 non-pathogenic control; LV117
pathogenic control) on two Phaseolus vulgaris cultivar, Perola (susceptible) and G2333
LV116 26.6611.54 NA 42.663.51 NA 25.669.81 NA 303.46 NA

LV117 9.338.32 4.65.68 21.73 1.331.15 9.338.32 4.65.68 21.73 1.331.15

300 sporesobserved.
120 (a) (b)
UFLAG03 G2333
UFLAG03 Perola
100 LV117 G2333
LV117 Perola

80
10 m

60 (c)

40

20
20 m 10 m

0
24 h 48 h Fig. 2 Germ tubes and appressoria of Glomerella cingulata f.sp.
a
Meanandstandard deviationforthreereplicates.
Incubation time

Fig.1 UFLAG03 and LV117 spore germination percentage on resistant (G2333) and susceptible
(Perola) cultivars, 24 and 48h after inoculation

(P < 0.0001), cultivar (P < 0.04), time (P < 0.001) and the interactions strain cultivar (P < 0.05) and
strain time (P < 0.001). UFLAG03 exhibited a similar percentage of germination as the resistant and
susceptible cultivars (Fig. 1). The same was not observed to LV117, which exhibited higher percentage
of germination on the susceptible cultivar.
All the strains evaluated developed appressoria (Table 3). The formation of two appressoria at the
extremitie soft he ascospores attached to the sporeorto the end of germ tubes was typically observed
(Figs 2a and 3a). After 72 h, it was not possible to quantify the percentage of spore germination and
appressorium formation due to the intense production of mycelia. It was possible, however, to quantify
the conidial germination of LV117 on the resistant cultivar (1.8% germination). The differences between
the susceptible and resistant cultivar were clear. Disease symptoms became visible in the susceptible
cultivar once the primary hyphae developed inside host cells (96 h after inoculation)when infected by
conidia.Structures such as infection vesicles and primary hyphae were not visualized when ascospores
were applie dusing this approach.
Observation by SEM of leaves inoculated with ascospores of G. cingulata f.sp. phaseoli showed that
276 Ishikawa etal.

phaseoli (UFLAG03) in bean leaf epidermal cells stained with aniline blue, 24h after inoculation. (a)
Formation of two appressoria (arrow) at the extremities of the ascospore ( As). (b) Spherical appressoria
emitted by conidia,(c) appressoria emitted by ascospore fungal growth was more vigorous at 24 and 48
h (Table 4) than that observed after conidial inoculation. These observations confirmed the light
microscopy results. Moreover, during this period, the production of mycelia, the emergence of
ascospores germ tubes and the formation of appressoria could be detected (Fig. 3ac). All of these
structures were present 72 h after inoculation (Fig. 3d), while after 96 and 120 h only mycelia could be
observed. Appressoria were clearly formed from the sexual spore of the fungus. The ascospores
exhibited an allantoidal shape and were bigger than conidia, as described previously by Roca et al.
(2003a).

Table4
Characteristics of Glomerella cingulata f.sp. phaseoli and Colletotrichum lindemuthinum
strainsunderleavessurfaceusingSEM

AppresoriumdifferGermination entiation
a a a
rate(%) rate(%) CAT (% )

Isolate 24 h 48 h 24 h 48 h 48 h

LV117(conidia) 1 34 0 5 5
UFLAG01(ascospores) 59 NA b 34 NA b 0
a
100 sporesamples.
b It wasnotpossibletoquantify,hyphaedeveloped.
(a) (b)

20 m 20 m

(c) (d)

20 m 10 m

20 m 10 m

Fig.3 Scanning electron micrographs of the surface of bean leaves (cultivar Perola) after inoculation
with Glomerella cingulata f.sp. phaseoli (UFLAG01): (a) 24h after inoculation showing ascospores (As)
producing germ tubes (white arrows) and appressoria (a), and the development of hyphae (H); (b) and
(c) 48h after inoculation showing ascospores (As) with germ tubes (white arrows), hyphae (H) and
appressoria (a) [in (c) details are amplified in the upper right insert];(d)72 h after inoculation showing
ascospores with appressoria ( detailsareamplifiedintheupperrightinsert ) Analysis of leaf material
collected 24 and 48 h after inoculation with the conidia revealed that the fungus had grown rather slowly
and that numerous non-germinated conidia together with a few conidia germ tubes and appressoria were
presented (Fig. 4a,b). After 48 and 72 h, it was possible to observe conidia anastomosis tubes (CAT)
Glomerella cingulata SymptomsandPrepenetration Events 277

fusion (Fig. 4c), and germ tube and appressoria emerging from conidia (Fig. 4d). After 96 and 120 h,
there was a large amount of mycelia present but no conidia.

Teleomorph Glomerella cingulata causes symptoms in bean cultivar

The commercial cv. Perola (employed as a positive control) was susceptible to both fungal forms, albeit
with different symptoms. However, none of the 12 differential cultivars employed in the study were
susceptible to the UFLAG01 teleomorph of G. cingulata f.sp. phaseoli applied. The LV117 isolate used
as pathogenic control in microscopy analyses was classified asrace65. None of cultivars were
susceptible to the non-pathogenic strain (LV116); for this reason, it was chosen for non-pathogenic
control. Plants inoculated with ascospores showed milder symptoms (score = 4.5) than those normally
associated with conidial inoculation. Lesions occurred in the vicinity of the primary vein of the leaf
blade, while those on the hypocotyls were barely visible (Fig. 5 ae ).
In order to confirm that the symptoms observed on cv. Perola were the result of infection by G.
cingulata f.sp. phaseoli, diseased leaves were surface-sterilized and re-cultured on M3 medium. The
formation of
(a) (b)

20 m 20 m

(c) (d)

20 m 10 m

Fig.4 Scanning electron micrographs of the surface of bean leaves (cultivarPerola)after inoculation
with Colletotrichum lindemuthianum (LV117): (a) 24h after inoculation showing conidia forming germ
tubes (white arrows) and appressoria (a); (b) 24h after inoculation showing various non-germinating
conidia, conidia (c) forming germ tubes (white arrow) and appressoria (a); (c) 72h after inoculation
showing conidia forming chain anastomosis (white arrow); and ( d ) 72h after inoculation showing
conidia with germ tube (white arrow) and appressoria (a)
278 Ishikawa etal.

(a)

(b) (c) (d) (e)

Fig.5 Symptoms of anthracnose 10 days after inoculation (a) Bean plants inoculated with ascospores
Glomerella cingulata f.sp. phaseoli (UFLAG01) on the left and conidia (LV117) on the right; (b) and
(c) leaf and hypocotyls symptoms inoculated with teleomorph; ( d ) and(e) leaf and hypocotyls
symptoms inoculated with anamorph perithecia (Fig. 6b) could be observed in growing cultures 1520
days after re-isolation, together with an orange coloured sporulation mass typical of the asexual phase
of the fungus (Fig. 6c). When slides from

(a) (b)

(c) (d)

Fig.6 (a) Colonies of the UFLAG01 Glomerella cingulata f.sp. phaseoli isolate; (b) G. cingulata f.sp.
phaseoli re-isolated from bean leaves and cultured on M3 medium; (c) G. cingulata f.sp. phaseoli
colonies producing an orange sporulation mass typical of the asexual phase (Colletotrichum
lindemuthianum); (d) C. lindemuthianum colonies re-isolated from bean leaves and cultured on M3
medium: The black spots constitute perithecia the sporulation mass were prepared, ascospores and
conidia were clearly visible. Fungal colonies originating from diseased leaves that had been inoculated
with C. lindemuthianum also produced perithecia (Fig. 6d), thus demonstrating that inoculation of plants
with either of the fungal forms (sexual or asexual) gave rise to both types of colonies.
Glomerella cingulata SymptomsandPrepenetration Events 279

Discussion
We have demonstrated that the growth and development of ascospores of G. cingulata f.sp. phaseoli in
vitro and on common bean leaves is more rapid than that of conidia(Tables2and3).Coloniesof G.
cingulata f.sp. phaseoli and C. lindemuthianum re-isolated from diseased bean leaves produced
perithecia, ascospores and conidia, indicating that these pathogens had undergone a complete life cycle.
Although the isolates were dissimilar with respect to the symptoms and prepenetration events, both
forms produced, at different times, germ tubes, appressoria and hyphae on the surface of the leaves.
The emergence of germ tubes and the growth of mycelia occurred at a very early stage of infection
(24 to 48 h) with the sexual form of the fungus, while with the asexual form, most conidia were either
in a non germinated state or just beginning to germinate during the same periods. Moreover, the
percentages of ascospore germination on the resistant and susceptible cultivar were similar which was
not observed to pathogenic anamorph control (LV117). The pathogenic control exhibited a higher
percentage of conidial germination on the susceptible than the resistant cultivar. These findings are in
agreement with those reported by Jerba et al. (2005), in which the number of appressoria formed during
the initial stages of infection was higher in susceptible cultivars compared with their resistant
counterparts. Although these authors reported the occurrence of conidia during the preinfection stage,
there was no mention of the presence of ascospores.
In general, the isolates were divided into groups according to the reproduction phase. One group ( A
) was formed by C. lindemuthianum strains ( anamorph ) that germinate slowly and the other groups (B,
C and D) by G. cingulata f sp. phaseoli strains ( teleomorph ) that germinate faster (Table 2).
In Colletotrichum species, the formation of appressoria is important for the penetration of the plant
pathogen into the plant cell and subsequent development of the disease. However, there are examples,
such as the invasion of mulberry by C. gloesporioeides (Kumar et al., 2001), in which the fungus does
not produce appressoria or other infection structures. Normally, such structures are formed either inside
or over the stomata prior to the penetration of leaf cells, but in the case of C. lindemuthianum,
penetration occurs directly through the leaf cuticle as a consequence of the pressure exerted by the
appressoria. In respect of teleomorph G. cingulata f.sp. phaseoli, the mechanisms of penetration into
cells of common bean plants have not been described. In this context, the present study revealed the
formation of germ tubes and appressoria from ascospores (Fig. 3ad), although penetration through
stomata could not be verified. Mature appressoria were also characterized by the presence of melanin
pigments in their walls such as the anamorph (Fig. 2ac). However, structures such as infection vesicles
and primary hyphae were not observed when ascospores were applied using this approach, thus other
methodologies such as transmission electronic microscopy now need to be tested.
Structures such as conidia, germ tubes, appressoria, infection vesicles and primary hyphae play
important roles in the interaction between the plant pathogen and the host cells, serving in various
processes such as adhesion, cell signalling, and maintenance of fungal viability and recognition
mechanisms (Perfect et al., 2001). Thus, the absence or malfunction of such structures may lessen the
manifestation of the disease. According to Veneault-Fourrey et al. (2005), failures in the development
or maturation of appressoria may produce isolates that are non-pathogenic towards bean plants. As the
number of appressoria formed during the initial stages of infection is higher in susceptible cultivars
compared with their resistant counterparts (Jerba et al., 2005), the choice of a positive control (cv.
Perola) which was susceptible to both sexual and asexual isolates was very appropriate because the
appressoria could be clearly seen in the SEM samples studied, although teleomorph development was
similar on both, resistant and susceptible cultivars.
In confirmation of the findings of Camargo et al. (2007), the symptoms of anthracnose in bean plants
inoculated with ascospores were mild compared with those presented by plants inoculated with conidia.
The small number of reports concerning G. cingulata f.sp. phaseoli infection may be explained by the
less aggressive nature of the sexual form of the fungus. It is possible that the occurrence of G. cingulata
f.sp. phaseoli in the field could be more common than has been previously believed, and this might
280 Ishikawa etal.

explain the exceptional genetic variability of this pathogen (Mahuku and Riascos, 2004; Damasceno e
Silva et al., 2007; Ishikawa et al., 2008).
According to Brygoo et al. (1998), the organization and genetic diversity of C. lindemuthianum
resembles those of sexually reproduced fungal populations rather than of populations undergoing
asexual reproduction. Although such evidence constitutes a strong indication that sexual reproduction
may occur naturally in the field, there is a great deal of uncertainty concerning this aspect. In the absence
of a sexual phase, the genetic variability of C. lindemuthianum could be rationalized by the existence of
a parasexual cycle, chromosome polymorphism and CATs (OSullivan et al., 1998; Roca et al., 2003a,b,
2004; Ishikawa et al., 2008). As CAT fusion was also observed in the present study (Fig. 4c) such an
event might play a role in the magnification of the genetic variability of this pathogen (Roca et al.,
2004).
These results based on events preceding host cell penetration show that it is possible that the
differences regarding the symptoms produced by anamorph and teleomorph of G. cingulata f.sp.
phaseoli may result from events occurring after penetration. Therefore, more studies are required in
order to identify the genetic and physiological alterations responsible for the milder symptoms produced
by ascospores of G. cingulata f.sp. phaseoli in the common bean. This is the fist report comparing
symptoms and prepenetration events between anamorph and teleomorph of G. cingulata f.sp. phaseoli
in common bean.

Acknowledgements
The authors thank Dr Gabriela Roca and Dr Nick Read (University of Edinburgh) for helpful advice and
critical reading of the manuscript. This work was supported by grants from CNPQ and
FAPEMIG(Brazil).

References
Batista UG, Chaves GM. (1982) Patogenicidade de culturas monoascosporicas de cruzamento entre
racas de Colletotrichum lindemuthianum (Sacc.andMagn.)Scrib. Fitopatol Bras 7:285293.
Brygoo Y, Caffier V, Carlier J etal. (1998) Reproduction and population structure in phytopathogenic
fungi. In: Bridge P, Couteaudier Y, Clarkson J. (eds) Molecular Variability of Fungal Pathogens.
Wallingford, Great Britain, CAB International, pp 133148.
Bryson RJ. (1990) Sexual hybridization and the genetics of pathogenic specificity in Colletotrichum
lindemuthianum. Birmingham, Great Britain,UniversityofBirmingham, PhDThesis.
Camargo OA Jr, Souza EA, Mendes-Costa MC, Santos JB, Soares MA. (2007) Identification of
Glomerella cingulata f.sp. phaseoli recombinantsbyRAPDmarkers. Genet Mol Res 6:607615.
CIAT.(1990) Informe Anual 1988. Programa de Frijol Cali.Colombia,
CIAT,DocumentodeTrabajo72,pp128129.
Damasceno e Silva KJ, Souza EA, Ishikawa FH. (2007) Characterization of Colletotrichum
lindemuthianum isolates from the state of MinasGerais,Brazil. J Phytopathol 155:241247.
Habgood H. (1970) Designation of physiological races of plant pathogens. Nature 227:12681269.
Hardham AJ. (2000) Cell biology of fungal infection of plants. In: Howard RJ, Gow NAR. (eds) The
Mycota: Biology of the Fungal Cell. Vol.VIII.Berlin,Springer, pp91124.
Ishikawa FH, Souza EA, Davide LMC. (2008) Genetic variability within isolates of Colletotrichum
lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil. Biologia 63: 156161.
JerbaVF,RodellaRA,FurtadoEL.(2005)Relacaoentreaestrutura foliar do feijoeiro e a pre-infeccao
por Glomerella cingulata f.sp. phaseoli. Pesqui Agropecu Bras 40:217223.
Junqueira NTV, Chaves GM, Zambolin L, Romero RS, Gasparoto L. (1984) Isolamento, cultivo e
esporulacao de Microcylus ulei, agente etiologico do mal das folhas da seringueira. Rev Ceres
31:322331.
Glomerella cingulata SymptomsandPrepenetration Events 281

KimatiH,GalliF.(1970) Glomerella cingulata ( Stonem.)Spauld.etv. Schrenk. f.sp. phaseoli n.f., fase

ascogena do agente causal da antracnose do feijoeiro. An Esc Super Agric Luiz de Queiroz 27:411
437.
Kimati H, Amorim L, Bergamin Filho A, Camargo LEA,
Rezende JAM. (1997) Manual de fitopatologia: doenc as das plantas cultivadas. Vol. 2, 3rd edn.
Sao Paulo, Brazil, Editora Agronomica Ceres.
Kumar V, Gupta VP, Babu AM, Mishra RK, Thiagarajan V, Datta RK. (2001) Surface ultrastructural
studies on penetration and infection process of Colletotrichum gloesporioides on mulberry leaf
causingBlackspotdisease. J Phytopathol 149:629633.
Luna-MartinezF,Rodrguez-GuerraR,Victoria-CamposM,SimpsonJ. (2007) Development of a
molecular genetic linkage map for Colletotrichum lindemuthianum and segregation analysis of two
avirulencegenes. Curr Genet 51:109121.
Mahuku GS, Riascos JJ. (2004) Virulence and molecular diversity within Colletotrichum
lindemuthianum isolates from Andean and Mesoamerican bean varieties and regions. Eur J Plant
Pathol 110:253263.
Mendes-Costa MC. (1996) Genetics of Glomerella cingulata f.sp. phaseoli I. Sexual compatibility.
Caxambu, Minas Gerais, Brazil. Rev Bras Genet, Ribeirao Preto, Suplemento do Congresso
NacionaldeGenetica42 19:350.
OConnell RJ, Perfect SE, Hughes HB, Carzaniga R, Bailey JA, Green JR. (2000) Dissecting the cell
biology of Colletotrichum infection processes. In: Prusky D, Freeman S, Dickman M. ( eds )
Colletotrichum: Host Specificity, Pathology, and Host-Pathogen
Interaction.StPaul,MN,USA,APSPress,pp5777.
OSullivan D, Tosi P, Creusot F, Cooke M, Phan TH, Dron M, Langin T. (1998) Variation in genome
organization of the plant pathogenic fungus Colletotrichum lindemuthianum. Curr Genet 33:291298.
Perfect SA, Green JR, OConnell RJ. (2001) Surface characteristics of necrotrophic secondary hyphae
produced by the bean anthracnose fungus, Colletotrichum lindemuthianum. Eur J Plant Pathol
107:813819.
Roca MG, Davide LC, Mendes-Costa MC. (2003a) Citogenetica de Colletotrichum lindemuthianum
(Glomerella cingulata f.sp. phaseoli). Fitopatol Bras 28:367373.
Roca MG, Davide LC, Mendes-Costa MC, Wheals A. (2003b)
Conidial anastomosis tubes in Colletotrichum. Fungal Genet Biol 40:138145.
RocaMG,DavideLC,DavideLMC,Mendes-CostaMC,SchwanR, Wheals AE. (2004) Conidial
anastomosis fusion between Colletotrichum spp. Mycol Res 108:13201326.
Rodriguez-Guerra R, Ramrez-Rueda MT, Cabral-Enciso M, Garca-Serrano M, Lira-Maldonado Z,
Guevara-Gonzalez RG, Gonzalez-Chavira M, Simpson J. (2005) Heterothallic mating observed
between Mexican isolates of Glomerella lindemuthiana. Mycologia 97:793803.
RyanCC,ClareBG.(1974)Coatingofleafsurfaceswhiteagaroseto reactionfungalinoculuminsitustaining.
Stain Technol 49:1518.
Schoonhoven AV, Pastor-Corrales MA. (1987) Standard System for the Evaluation of Bean
Germoplasma. Cali, Colombia, Centro Internacionalde AgriculturaTropical.
Shear CL, Wood AK. (1913) Studies of Fungus Parasites Belonging to the Genus Glomerella.
Washington DC, USDA Bureau of Plant Industry,BulletinNo252.
Souza BO, Souza EA, Mendes-Costa MC. (2007) Determination of variability in isolates of
Colletotrichum lindemuthianum based on morphological and cultural markers. Ciencia Agrotecnol
31: 10001006.
282 Ishikawa etal.

Sutton BC. (1992) The genus Glomerella and its anamorph Colletotrichum. In: Bailey JA, Jeger MJ.
(eds) Colletotrichum: Biology, Pathology and Control. Wallingford, Great Britain, CAB
International,pp126.
Tucker SL, Talbot NJ. (2001) Surface attachment and pre-penetration stage development by plant
pathogenic fungi. Annu Rev Phytopathol 39:385417.
Veneault-Fourrey C, Lauge R, Langin T. (2005) Nonpathogenic strains of Colletotrichum
lindemuthianum trigger progressive bean defense responses during apressorium-mediated penetration.
Appl Environ Microbiol 71:47614770.