Fungal Genetics and Biology 27, 186198 (1999)

Article ID fgbi.1999.1143, available online at http://www.idealibrary.com on

REVIEW

Colletotrichum: A Model Genus for Studies on

Pathology and FungalPlant Interactions

Sarah E. Perfect,* H. Bleddyn Hughes,* Richard J. OConnell,

and Jonathan R. Green*,1
*School of Biological Sciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom;
and IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol,
Long Ashton, Bristol, BS41 9AF, United Kingdom

Accepted for publication April 19, 1999

Index Descriptors: adhesion; appressoria; biotrophy;

fungal differentiation; fungal genes; fungalplant interactions; necrotrophy; spores.

Perfect, S. E., Hughes, H. B., OConnell, R. J., and Green,

J. R. 1999. Colletotrichum: A model genus for studies on
pathology and fungalplant interactions. Fungal Genetics
and Biology 27, 186198. Species of Colletotrichum use
diverse strategies for invading host tissue, ranging from
intracellular hemibiotrophy to subcuticular intramural
necrotrophy. In addition, these pathogens develop a series
of specialized infection structures, including germ tubes,
appressoria, intracellular hyphae, and secondary necrotrophic hyphae. Colletotrichum species provide excellent
models for studying the molecular basis of infection structure differentiation and fungalplant interactions. In this
review we cover the various stages of the infection processes of Colletotrichum species, including spore adhesion and germination, germ tube and appressorium differentiation and functions, and biotrophic and necrotrophic
development. The contribution of molecular, biochemical,
and immunological approaches to the identification of
genes and proteins relevant to each stage of fungal
development will be considered. As well as reviewing
results from several groups, we also describe our own
work on the hemibiotrophic pathogen, C. lindemuthianum. r 1999 Academic Press

Colletotrichum is a large genus of Ascomycete fungi,

containing many species which cause anthracnose or blight
on a wide range of important crop and ornamental plants
(Bailey and Jeger, 1992). Species from this genus have
been used for many years in studies concerning fungal
differentiation and fungalplant interactions. During the
colonization of plant hosts, fungal pathogens exhibit two
main modes of nutrition: biotrophy, where nutrients are
obtained from living host cells, and necrotrophy, where
nutrients are obtained from dead host cells which have
been killed by the fungus (Thrower, 1966). Both of these
nutritional strategies are exhibited by species from the
genus Colletotrichum. In addition, these fungi develop a
series of specialized infection structures including germ
tubes, appressoria, intracellular hyphae (IH), and secondary necrotrophic hyphae. Colletotrichum species therefore
provide excellent models for studying the molecular and
cellular bases of fungal pathogenicity (Bailey et al., 1992).
In this review we will outline each of the main infection
strategies in more detail, concentrating on the initial stages
of colonization. We will then review some of the results
gained from investigations using a wide range of approaches on species from the genus Colletotrichum. Each

1 To whom correspondence should be addressed. School of Biological

Sciences, University of Birmingham, Birmingham, B15 2TT, UK. Fax:
(0)121 414 5925. E-mail: j.r.green@bham.ac.uk.

186

1087-1845/99 $30.00
Copyright r 1999 by Academic Press
All rights of reproduction in any form reserved.

Colletotrichum: Pathology and FungalPlant Interactions

of the major stages of infection will be considered in

sequence, alongside proteins and genes of interest which
have been linked to each stage.

INFECTION STRATEGIES OF
COLLETOTRICHUM SPECIES
Colletotrichum species utilize two main infection strategies: intracellular colonization or subcuticular intramural
colonization (Table 1; Bailey et al., 1992). The initial stages
of infection are very similar for both groups of pathogens;
conidia adhere to, and germinate on, plant surfaces,
produce germ tubes, and then go on to form appressoria
which penetrate the cuticle directly. Following penetration, subcuticular intramural pathogens develop beneath
the cuticle by forming an intramural network of hyphae,
before spreading rapidly throughout the tissue with both
inter- and intracellular hyphae, killing in advance of

TABLE 1
Infection Strategies of Colletotrichum Species
Colletotrichum species
Intracellular hemibiotrophic pathogens
C. lindemuthianum
C. graminicola
C. truncatum
C. gloeosporioides

C. orbiculare (C. lagenarium)

C. destructivum1
C. trifolii2
C. sublineolum3
Subcuticular intramural pathogens
C. capsici
C. circinans
C. musae4
C. gloeosporioides

Pathogens exhibiting both infection strategies

C. gloeosporioides

Plant host
Phaseolus vulgaris
Zea mays
Pisum sativum
Medicago sativa
Stylosanthes guianensis
Stylosanthes scabra
Populus tremuloides
Cucumis sativus
Vigna unguiculata
Medicago sativa
Sorghum bicolor
Gossypium hirsutum
Vigna unguiculata
Allium cepa
Musa spp.
Carica papaya
Muscadinia rotundifolia
Persea americana5
Citrus spp.
Stylosanthes spp.

Note. References on the above can be found in Fig 5.1. in Bailey et al.
(1992).
1 Latunde-Dada et al. (1996), 2 Mould et al. (1991), 3 Wharton and
Julian (1996), 4 Swinburne and Brown (1983), 5 Prusky et al. (1991).

187

mycelial spread (Fig. 1a; Bailey et al., 1992). There is no

detectable biotrophic stage in these interactions. Examples
of subcuticular intramural pathogens include C. capsici on
cowpea (Vigna unguiculata: Bailey et al., 1992; Pring et al.,
1995) and cotton (Gossypium hirsutum L.; Roberts and
Snow, 1984), and C. circinans on onion (Walker, 1921).
The majority of Colletotrichum species exhibit the
second type of infection strategy, i.e., intracellular colonization, although the length of the biotrophic stage is variable
and is absent in many interactions. This is exemplified by
the infection process of C. lindemuthianum on bean
(Phaseolus vulgaris) illustrated in Fig. 1b. Following penetration, fungal hyphae grow within the cell lumen without
penetrating host protoplasts, i.e., they grow between plant
plasma membranes and plant cell walls. After colonizing
one or more host cells, the IH, which are biotrophic,
subsequently give rise to secondary necrotrophic hyphae
(OConnell et al., 1985; Bailey et al., 1992; Latunde-Dada
et al., 1996). These fungi which initially feed on living host
cells before switching to necrotrophy are considered to be
hemibiotrophic or facultative biotrophs. In many Colletotrichum interactions it is still not known whether a biotrophic stage is present. However, it is significant that during
initial intracellular colonization by the fungus, whether
biotrophic or not, the host plant does not appear to
recognize the pathogen and there is no specific resistance
response. Examples where distinct biotrophic stages are
well-characterized include the interactions between C.
lindemuthianum and bean (OConnell et al., 1985; OConnell,
1987), C. truncatum and pea (OConnell et al., 1993), C.
destructivum and cowpea (Latunde-Dada et al., 1996) and C.
sublineolum and sorghum (Wharton and Julian, 1996).
Much of our own work has focused on the bean
anthracnose fungus, C. lindemuthianum. This is one of the
best-studied species of the genus due to its economic
importance (Allen, 1983), interesting infection strategy
(OConnell et al., 1985), the ease with which it can be
cultured in vitro (Mathur et al., 1950), and the availability
of a reproducible and efficient transformation system
(Rodriguez and Redman, 1992). As such, it forms a good
model system for the study of several aspects of plant
fungal interactions, including plant defence responses
(Mahe` et al., 1993; Bergmann et al., 1994; Wojtaszek et al.,
1995; Nuss et al., 1996), phytoalexins (Bailey et al., 1980;
Dixon et al., 1986), fungal cell wall degrading enzymes
(Wijesundera et al., 1984, 1989), and the differentiation of
fungal infection structures (OConnell and Bailey, 1988;
Green et al., 1995). In contrast to the hemibiotrophic C.

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

188

Perfect et al.

lindemuthianum, most obligate biotrophs cannot be cultured axenically, and consequently they are recalcitrant to
transformation. Therefore, C. lindemuthianum provides a
model system in which to study biotrophy using techniques
that are not generally applicable to obligate biotrophs.

APPROACHES USED TO IDENTIFY

GENES AND PROTEINS INVOLVED IN
COLLETOTRICHUM DIFFERENTIATION
AND PATHOGENICITY

FIG. 1. Two species of Colletotrichum which exhibit different infection

strategies (the diagrams are not to scale). Diagram (a): C. capsici, a subcuticular
intramural pathogen. Spores (S) germinate to form domed, melanized appressoria (A) from which penetration hyphae develop. (1) The host cuticle is
penetrated and fungal hyphae spread subcuticularly within the walls of the host
epidermal cells (E) which subsequently die. (2) During the later stages of
infection fungal hyphae penetrate plant epidermal and mesophyll cells (M)
causing further host cell death. (b): The hemibiotrophic C. lindemuthianum
bean interaction. Spores (S) germinate to form domed, melanized appressoria
(A) from which penetration hyphae develop. (1) Upon entering host epidermal
cells (E), penetration hyphae swell to form infection vesicles (IV) and broad
primary hyphae (PH), around which the plant plasma membrane invaginates.
Both IVs and PHs are surrounded by an interfacial matrix (illustrated by
diagonal hatching). The host protoplast remains alive during this biotrophic
stage of the interaction. (2) Primary hyphae progressively colonize new
epidermal and mesophyll cells (MS), becoming constricted as they pass
through cell walls. The interfacial matrix separating the plant plasma membrane from the fungal cell wall is maintained and newly infected cells remain
viable for some time. (3) Approximately 48 h after initial penetration secondary
necrotrophic hyphae (SH) are formed, which are not surrounded by a matrix
layer. In cells containing primary and secondary hyphae the plant plasma
membrane and matrix surrounding primary hyphae begin to disintegrate. (4)
The narrow secondary hyphae secrete cell wall degrading enzymes in advance
of their spread and create rapidly expanding necrotic lesions.

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

A number of different approaches have been employed

to study events involved in the differentiation of Colletotrichum infection structures and Colletotrichumplant interactions. Subtractive hybridization, differential screening,
and differential display reverse transcriptasePCR have
been used to identify genes which are differentially expressed during infection. For example, these techniques
have been used to clone several genes expressed during
appressorium formation by C. gloeosporioides (Hwang et
al., 1995). Differential screening of a cDNA library prepared from nitrogen-starved axenic cultures of C. gloeosporioides led to the isolation of a glutamine synthetase gene
that is upregulated during infection of Stylosanthes guianensis (Stephenson et al., 1997).
Insertional mutagenesis was used to identify a serine/
threonine kinase gene (clk1) expressed by C. lindemuthianum which is required for pathogenicity on P. vulgaris
(Dufresne et al., 1998). The gene is constitutively expressed, but the mutant seems to be blocked at the
penetration stage of infection. Restriction enzymemediated integration (REMI) has recently been used to
identify mutants in C. graminicola that have unpigmented
spores or weakened cell walls (Epstein et al., 1998). The
genes discussed in some detail in this review are listed in
Table 2. Monoclonal antibodies (MAbs) have also been
used to identify and characterize developmentally regulated proteins in C. lindemuthianum (Green et al., 1995).
MAbs were raised either to homogenates of germlings
grown in liquid culture (Pain et al., 1992) or to infection
structures isolated from bean leaves by isopycnic centrifugation (Pain et al., 1994a, 1994b). Figure 2 illustrates the
cell type-specific binding of these MAbs. Recently, one of
these MAbs (UB25) was used for expression cloning to
isolate a gene encoding a fungal glycoprotein present at the
biotrophic interface in host cells (Perfect et al., 1998).

Colletotrichum: Pathology and FungalPlant Interactions

TABLE 2
Colletotrichum Genes
Stage of interaction
at which gene is
expressed/function

Colletotrichum
species from which
gene was isolated

Gene
name

Spore contact with

substratum

C. gloeosporioides

chip1

Ubiquitin-conjugating
enzyme1

Appressorium formation

C. gloeosporioides

cap3

Metallothionin-like2

C. gloeosporioides
C. gloeosporioides

cap5
cap20

C. gloeosporioides

cap22

Gene product

C. gloeosporioides

CUT1

Metallothionin-like2
Appressorial cell wall
protein3
Appressorial cell wall
protein2
Cutinase4

Melanin synthesis

C. lagenarium
C. lagenarium
C. lagenarium

SCD1
PKS1
THR1

Scytalone dehydratase5
Polyketide synthase6
1,3,8-THN reductase7

Biotrophy

C. lindemuthianum

CIH1

Biotrophy-related protein8

Necrotrophy

C. gloeosporioides
C. gloeosporioides
C. lindemuthianum

pnlA
pel
Clpg1

C. lindemuthianum

Clpg2

Pectin lyase9
Pectate lyase10
Endo-polygalacturonase11
Endo-polygalacturonase12

C. trifolii

TB3

C. gloeosporioides
C. gloeosporioides

cam
CaMK

Signalling/pathogenicity

C. lindemuthianum

clk1

Serine/threonine
kinase16

Housekeeping
genes

C. lindemuthianium

gpd

C. gloeosporioides
C. gloeosporioides

TUB1
TUB2

Glyceraldehyde-3phosphate dehydrogenase17
-tubulin18
-tubulin19

C. gloeosporioides

gln

Signalling

Nutrition

Serine/threonine
kinase13
Calmodulin14
Calmodulin kinase15

Glutamine synthetase20

Note. 1 Liu and Kolattukudy (1998), 2 Hwang and Kolattukudy (1995),

Hwang et al. (1995), 4 Ettinger et al. (1987), 5 Kubo et al. (1996),
6 Takano et al. (1995), 7 Perpetua et al. (1996), 8 Perfect et al. (1998),
9 Templeton et al. (1994), 10 Wattad et al. (1997), 11 Centis et al. (1996),
12 Centis et al. (1997), 13 Buhr et al. (1996), 14 Kim et al. (1998), 15 Kim et al.
(1998), 16 Dufresne et al. (1998), 17 Templeton et al. (1992), 18 Buhr and
Dickman (1993), 19 Buhr and Dickman (1994), 20 Stephenson et al. (1997).
3

ADHESION
Adhesion of spores to host plants is essential for the
initiation of disease in fungalplant interactions. The

189

spores (conidia) of Colletotrichum species are dispersed by

rain splash and they rapidly adhere to the aerial parts of
host plants (Nicholson, 1992, 1996; Mercure et al., 1994a).
Conidia are produced in acervuli embedded in a watersoluble mucilage composed of high-molecular-weight glycoproteins, some of which are proline-rich (Nicholson,
1992). This mucilage contains germination inhibitors as
well as a variety of enzymes and may function to protect
conidia from dessication and toxic plant metabolites (Nicholson, 1992). However, at least in the case of C.
graminicola, the acervular mucilage appears to play no part
in spore adhesion (Mercure et al., 1994b).
So far, no adhesive glycoproteins have been cloned from
any Colletotrichum species. However, some progress has
been made toward the identification of candidate molecules. The initial attachment of Colletotrichum conidia to
the host cuticle involves hydrophobic interactions, as is the
case for many other plant pathogenic fungi (Nicholson and
Epstein, 1991; Mercure et al., 1994a). Conidial adhesion in
C. musae and C. graminicola was inhibited after treatment
with a proteolytic enzyme, suggesting that for these fungi
preformed proteins on the surface of spores are required
for their initial attachment. In addition to passive hydrophobic interactions, which provide rapid adhesion to the host,
spore adhesion in C. lindemuthianum, C. musae, and C.
graminicola appears to require active metabolism, including protein synthesis (Young and Kauss, 1984; SelaBuurlage et al., 1991; Mercure et al., 1994b). It is
suggested that these active processes are associated with a
second phase of adhesion, which may serve to consolidate
the initial hydrophobic attachment. In some Colletotrichum species, this second phase of adhesion is correlated
with the release of a protein exudate, which spreads
outward from the spore as a thin film over the substratum
or leaf surface (Mercure et al., 1994b). However, some
species, e.g., C. lindemuthianum, do not appear to release
any protein exudates (OConnell et al., 1996).
In C. lindemuthianum, there is no evidence for the
secretion of material prior to germ tube emergence,
suggesting that any spore adhesive molecules are preformed at the spore surface (OConnell et al., 1996).
Recent observations have shown that the surface of C.
lindemuthianum conidia, prepared by cryofixation and
freeze-substitution, is covered by a brush-like layer of
fibrillar material arranged perpendicular to the cell wall
with a reticular appearance in oblique section and approximately 60 nm thick (OConnell et al., 1996). This layer,
termed the spore coat, is similar to structures that have
been observed on the conidia of C. truncatum and the

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

190

Perfect et al.

FIG. 2. Binding characteristics of MAbs that recognize cell surfaces of C. lindemuthianum conidia and infection structures. UB20 (Pain et al., 1992);
UB26 (Pain et al., 1996); UB31 (OConnell et al., 1996); UB27 (Pain et al., 1995); UB25 (Pain et al., 1994b).

yeast form of the human pathogen Candida albicans (Van

Dyke and Mims, 1993; Bobichon et al., 1994). In C.
albicans, the cell coat was found to contain mannan,
suggesting the presence of mannoproteins (Bobichon et
al., 1994) and is thought to be the layer of the cell wall
which controls adhesion of this fungus. In C. lindemuthianum, the spore coat reacted positively with the carbohydrate-specific PA-TCH-SP stain and could be removed by
pronase, suggesting the presence of glycoproteins
(OConnell et al., 1996; Hughes et al., 1999). However, the
spore coat lacks the major cell wall polysaccharides chitin
and 1-3 glucans.
We have used an antibody approach to identify candidate adhesive molecules on the surface of C. lindemuthianum spores. The MAb UB20 labels the surface of C.
lindemuthianum spores much more intensely than germ
tubes or appressoria (Pain et al., 1992). EM-immunogold
labeling shows that UB20 binds to the outer layer of the
spore coat and the cell wall/plasma membrane interface.
Preincubation of spores with UB20 completely inhibits
adhesion of these spores to polystyrene, which suggests
that the antibody binds to surface components involved in
adhesion (Hughes et al., 1999). The spore coat of C.
lindemuthianum can be completely removed from intact
spores by extraction with hot water or 0.2% SDS using
methods based on those developed for C. albicans. Western blots with UB20 showed that a small number of
glycoproteins were extracted by these procedures, including major bands at 110 and 220 kDa and minor bands at

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

45 and 155 kDa. UB20 recognizes a carbohydrate epitope

on N-linked side chains of these glycoproteins. Colored
polystyrene microspheres bind to the 110-kDa glycoprotein in Western blots. Taken together, this evidence
suggests that the 110-kDa glycoprotein contributes to the
hydrophobicity of the spore surface and could be involved
in the initial rapid attachment of conidia to the plant
surface.
Adhesion of germ tubes and appressoria to surfaces is
generally stronger than spore adhesion. Appressoria adhesion is particularly strong to provide a firm base for
penetration, which is demonstrated by the observation that
fragments of the appressorial cell wall remain attached to
the substrate after sonication (Pain et al., 1996). Hair-like
fimbriae (up to 6 m long) are present at the surfaces of
germ tubes and appressoria of Colletotrichum species, and
it is thought that components in the extracellular matrix
(ECM) secreted by these cells are responsible for adhesion. We have identified glycoproteins that have adhesive
properties in the ECM produced by C. lindemuthianum
germ tubes and appressoria. MAbs UB26 and UB31
recognize high-molecular-weight glycoproteins that remain attached to the substratum after sonication of adhering Colletotrichum infection structures (Pain et al., 1996;
OConnell et al., 1996). Evidence also suggests that some
of these glycoproteins become insolubilized after secretion, possibly by cross-linking to other components of the
ECM/cell wall or by polymerization. Recently, the ECM
released onto hydrophobic surfaces by conidia and germ-

Colletotrichum: Pathology and FungalPlant Interactions

lings of C. graminicola was analyzed using lectins, and the

results showed that high-molecular-weight glycoproteins
(200 kDa) were attached to the substratum (Sugui et al.,
1998).

GERMINATION AND APPRESSORIUM

FORMATION
Colletotrichum spores sense both physical and chemical
signals from the plant surface to trigger germination and
differentiation into appressoria. Examples of chemical
inducers are provided by work on C. gloeosporioides
infecting avocado and C. musae infecting banana (Podila et
al., 1993; Kolattukudy et al., 1995). Germination of C.
gloeosporioides spores and appressorium formation are
selectively triggered by the surface wax of its host avocado.
The signal is specific since the waxes of other plants cannot
substitute for the avocado wax. Ethylene, the fruit-ripening
hormone, induces germination and appressorial formation
in both C. gloeosporioides and C. musae, pathogens that
attack climacteric fruits, but not other Colletotrichum
species that normally infect nonclimacteric fruits. Thus,
spores landing on developing fruit germinate and penetrate after triggering by the wax signal, but remain latent
until the fruit ripens, when infection can then occur
(Kolattukudy et al., 1995). For C. gloeosporioides, contact
with a hard surface is necessary for the chemical signals,
i.e., host surface wax and ethylene, to induce appressorium
formation (Liu and Kolattukudy, 1998). Genes expressed
in C. gloeosporioides conidia during contact with a hard
surface were isolated by differential display. One of the
genes (chip1) encodes a ubiquitin-conjugating enzyme
with high homology to the (UBC4-UBC5) enzyme in
Saccharomyces cerevisiae (Liu and Kolattukudy, 1998).
Expression of this gene may mediate ubiquitin-dependent
protein degradation associated with germination and appressorium formation.
Cell signalling pathways operating in spore germination
and appressorium formation in Colletotrichum have been
studied by using inhibitors or by monitoring the expression
of genes encoding putative signalling components, e.g.,
calmodulin (CaM) and protein kinases. In the C. gloeosporioides system described above, the protein kinase inhibitors H7 and genistein inhibit ethylene-induced appressorium formation and phosphorylation of proteins, suggesting
a possible role for the latter in differentiation (Flaishman et
al., 1995). Ethylene and avocado wax caused phosphorylation of proteins at 29 and 43 kDa. A putative CaM kinase

191

(CaMK) cDNA has been cloned from C. gloeosporioides

and inhibition of this enzyme by KN93 reduced germination, appressorium formation, and melanisation (Kim et al.,
1998).
Inhibitor studies (e.g., using KT5720) on C. trifolii have
shown that cAMP generation and PKA (cAMP dependant
protein kinase) are needed for germination and appressorium formation (Yang and Dickman, 1997). In addition,
cAMP induced appressorium formation on noninductive
surfaces under conditions of nutrient starvation (Yang and
Dickman, 1997). These results suggest similarities with
appressorium development in Magnaporthe grisea, where
it has been established that cAMP and PKA are prerequisites for appressorium development (Dean, 1997). Transcription of the signal-transduction pathway genes encoding protein kinase C, a serinethreonine kinase and CaM,
was found to occur at high levels in conidia of C. trifolii
prior to, and during, germination (Buhr and Dickman,
1997). Calmodulin transcription was also induced in C.
gloeosporioides by hard surface contact (Kim et al., 1998).
Overall, the evidence suggests that signalling pathways
involving cAMP, protein kinases, and CaM are operating
during differentiation of Colletotrichum infection structures. However, the precise sequence of signalling events is
still not known.

APPRESSORIUM DIFFERENTIATION
AND DEVELOPMENT
Appressoria of Colletotrichum species differentiate on
an inductive surface when apical growth of the conidial
germ tube stops and the tip swells and becomes delimited
by a septum. Maturation of the appressorium involves
formation of a penetration pore in the base of the cell,
deposition of new wall layers, and secretion of ECM
materials. Melanin is subsequently deposited in a layer of
the cell wall close to the plasma membrane (Bailey et al.,
1992). In some species, e.g., C. lindemuthianum, the
penetration pore becomes surrounded by a funnel-shaped
elaboration of the inner wall layer, termed the appressorial
cone (Fig. 1b). This structure does not contain chitin or
melanin and is continuous with the wall of the penetration
peg. In other species, appressorial cones are not present
but the cell wall forms a thickened ring around the
penetration pore. Apical growth resumes with the emergence of the penetration peg through the pore, and
penetration of the plant cuticle and cell wall probably

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

192

involves a combination of mechanical force, produced by

high turgor pressure, and enzymatic degradation.
It is clear from the above description that the mature
appressorium is a highly assymetric, polarized cell with an
upper domed region and a flattened basal region containing the pore apparatus. Using the MAb UB27, we have
identified a glycoprotein in C. lindemuthianum that is
specifically located in appressoria (Pain et al., 1995).
Within appressoria this glycoprotein is restricted to the
plasma membrane of the domed region of the cell, being
absent from a circular region surrounding the basal penetration pore. This shows that the plasma membrane of
appressoria is differentiated into two domains. Specialization of the plasma membrane in the pore region could be
an important preparation for subsequent penetration and
possibly for the establishment of biotrophy. UB27 recognizes a 48-kDa glycoprotein that has properties characteristic of an integral membrane protein, and evidence
suggests that it is linked to the appressorial cell wall or
cytoskeleton, which may be important in restricting the
protein to the domed region of the appressorium (Pain et
al., 1995).
Other studies have identified genes switched on during
appressorium formation in C. gloeosporioides. A subtracted cDNA library was constructed using mRNA from
nongerminating conidia and appressorium-forming conidia to enrich the cDNAs associated with appressorium
formation. Differential screening yielded four cDNA clones
representing transcripts found only in appressoriumforming spores (Hwang et al., 1995; Hwang and Kolattukudy, 1995). Two of these clones, cap3 and cap5, encode
26- and 27-amino-acid cysteine-rich polypeptides, respectively, which show some homology to metallothionins. Two
further genes are uniquely expressed during appressorium
formation after 4 h exposure of spores to wax. Cap22
encodes a 22-kDa protein which is probably glycosylated,
since an antibody raised against the E. coli expressed
protein recognises a 43-kDa protein. Cap20 encodes a
20-kDa protein which may have a role in appressorial
function, since a mutant with a disrupted cap20 gene
germinated and produced appressoria of normal morphology, but failed to produce lesions on avocado and tomato.
Immunogold labeling with antibodies against cap20 and
cap22 proteins showed that both of these gene products
were localized in the appressorial wall (Hwang et al., 1995;
Hwang and Kolattukudy, 1995).
Melanisation is thought to be involved in the generation
of appressorial turgor and is required for mechanical
penetration by C. lagenarium and C. lindemuthianum

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

Perfect et al.

(Kubo and Furusawa, 1991). Melanin biosynthesis begins

with the synthesis of pentaketide and the formation of
scytalone. Polyketide synthase, encoded by PKS1, is involved in this early step, which is followed by two dehydrations and a reduction step: the dehydrations of scytalone to
1,3,8-trihydroxynaphthalene (1,3,8-THN) and vermalone
to 1,8- dihydroxynaphthalene are performed by scytalone
dehydratase (encoded by SCD1). Reduction of 1,3,8-THN
to vermalone is performed by 1,3,8-THN reductase (encoded by THR1). 1,8-Dihydroxynaphthalene is then polymerized and oxidized to melanin. By generating melanindeficient mutants by treatment of conidia with N-methylN8-nitro-N-nitrosoguanidine and using homologues of
known genes, three melanin biosynthesis genes (PKS1,
SCD1, and THR1) have been cloned and analyzed from C.
lagenarium (Kubo et al., 1991; Takano et al., 1995; Kubo et
al., 1996; Perpetua et al., 1996). All three genes are
required for melanin synthesis and disruptants are unable
to effectively penetrate or infect plants. During appressorium differentiation, de novo transcripts of these three
genes accumulated 12 h after the start of conidial
incubation and the amount of transcripts decreased after 6
h (Takano et al., 1997). Appressorium formation is repressed by complex nutrients (e.g., yeast extract and
tryptone) and expression of these three genes was also
undetectable under these conditions. Similar expression
studies have been repeated with C. trifolii and showed that
THR1 and SCD1 were also expressed prior to, and during,
appressorium formation using inductive conditions (26 h;
Buhr and Dickman, 1997).
The role of cutinase in penetration of the host cuticle has
been examined for several Colletotrichum species. Several
cutinase-deficient mutants of C. gloeosporiodes were compared with respect to their pathogenicity on intact or
mechanically wounded papaya fruits (Dickman and Patil,
1986). The mutants were highly pathogenic when papaya
fruit surfaces were either artificially wounded or treated
with purified C. gloeosporioides cutinase prior to inoculation, but disease did not occur on intact fruit surfaces
where a relatively thick cuticle had to be penetrated.
Pathogenicity could be restored with exogenous cutinase,
and cutinase-specific antibodies were also shown to protect
papaya fruits from disease (Dickman et al., 1982). Results
on C. graminicola also suggest a role for cutinase in
penetration and the infection process (Pascholati et al.,
1993), but work by Bonnen and Hammerschnidt (1993)
suggested that cutinolytic enzymes were less important for
infection of cucumber by C. lagenarium. Cutinase genes

Colletotrichum: Pathology and FungalPlant Interactions

have been isolated and sequenced from C. gloeosporiodes

and C. capsici (Ettinger et al., 1987).

BIOTROPHIC DEVELOPMENT
The economic importance of biotrophic plant pathogens
means that the molecular basis of biotrophy is a key area of
research. Recently, several glycoproteins specific to haustoria formed in the pea powdery mildew interaction were
identified using MAbs (Green et al., 1995). Using a
differential screening approach, a putative amino acid
permease specifically located in the haustorial plasma
membrane of the rust fungus Uromyces fabae has been
cloned (Hahn et al., 1997). In contrast to these obligate
biotrophs, the facultative biotroph C. lindemuthianum can
be grown axenically and transformed, allowing gene function to be tested (Rodriguez and Redman, 1992). We have
been utilizing these techniques in our investigations of the
biotrophic stage of the interaction between C. lindemuthianum and bean.
In contrast to the wealth of information on Colletotrichum prepenetration events, very little is known about the
biotrophic stage of the interaction, especially at the molecular level. During the biotrophic phase of the interaction,
the plant plasma membrane invaginates around IH, a
situation also observed in interactions involving obligate
biotrophs where haustoria are formed. However, in the
case of haustorial pathogens, the invaginated plant plasma
membrane becomes specialized, whereas this does not
appear to happen in the C. lindemuthianumbean interaction (OConnell, 1987; Green et al., 1995). A feature in
common between haustoria and IH is the interfacial matrix
separating the fungal cell wall from the invaginated plant
plasma membrane (OConnell, 1987; Green et al., 1995).
This interface forms the region of most intimate contact
between the plant and fungus, and as such it may play
important roles in the establishment and maintenance of
biotrophy, and the avoidance or suppression of host
defences.
In previous studies, MAbs were raised against C. lindemuthianum infection structures isolated from bean leaves
to identify novel proteins specifically associated with IH
(Pain et al., 1994a, 1994b; Green et al., 1995; OConnell et
al., 1996). One of these MAbs, UB25, recognized a protein
epitope in a set of N-linked glycoproteins that are expressed only during biotrophic growth inside living host
cells. The glycoproteins are multimers of 40.5-kDa sub-

193

units, as shown by Western blotting, and are secreted to

the cell wall of IH and the interfacial matrix which
surrounds them (Pain et al., 1994b; Perfect et al., 1998).
These glycoproteins are not present in germ tubes, appressoria, or secondary necrotrophic hyphae.
In recent work, the MAb UB25 has been used to
immunoscreen a cDNA library prepared from infected
bean tissue, allowing the sequence of the corresponding
antigen to be elucidated (Perfect et al., 1998). The cloned
gene has been designated CIH1 (Colletotrichum Intracellular Hypha 1). Southern analysis shows that CIH1 is a
fungal gene, present as a single copy within the genome of
C. lindemuthianum. The DNA sequence of CIH1 predicts
a protein with 230 amino acid residues and a molecular
weight of approximately 25 kDa. A putative signal peptide
at the N-terminus, corroborates EM evidence that the
glycoprotein recognized by UB25 is secreted through the
wall of the IH into the biotrophic fungalplant interface.
The predicted amino acid sequence appears to be made
up of two domains. The first domain is very rich in proline
residues, which account for 30% of the amino acid content
in this region. Within this proline-rich region there are a
series of short repetitive motifs of three types, namely
LPEP, YKPK, and VEGPYKPK. Proline-rich proteins are
important structural components of plant cell walls, but
few such proteins have been identified in fungi, although
recently, a gene encoding a putative proline-rich protein
was isolated from another biotrophic fungus, E. graminis
(Justesen et al., 1996). The second domain of the CIH1
protein contains two partial C-terminal repeats which
appear to contain a consensus sequence found in certain
phage and bacterial secreted proteins such as bacteriophage lyzozymes and P60, a pathogenicity-related protein
from Listeria monocytogenes (Birkeland, 1994). It has
been hypothesized that these repeats are involved in the
recognition and binding of carbohydrate moieties.
A key feature of intracellular biotrophic interactions is
that the fungal parasite avoids triggering, or suppresses,
host defence responses such as hypersensitive cell death
and callose deposition (Heath and Skalamera, 1997). The
CIH1 glycoprotein seems to form a major structural
component of the interface formed by C. lindemuthianum
with bean and, as such, may provide a buffer zone,
physically separating the plant plasma membrane from the
fungal cell wall. This may delay recognition of the fungus
by acting as a diffusion barrier to soluble fungal elicitors,
enzymes, or toxins. The CIH1 glycoprotein could also act
as a barrier to host defence molecules and have a protective function. The sequence of CIH1 indicates some level

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

194

of similarity between CIH1 and structural plant cellwall

proteins. By mimicking such proteins the fungus may
present a pseudo plant cell wall to the host, so delaying or
preventing recognition. Thus, the CIH1 glycoprotein is the
first fungal component of a biotrophic interfacial matrix to
be identified and characterized. It may play roles in the
establishment and maintenance of the biotrophic stage of
the C. lindemuthianumbean interaction.

NECROTROPHIC DEVELOPMENT
The necrotrophic stage of development of Colletotrichum species has been studied mainly using the C.
lindemuthianumbean interaction. Necrotrophy is clearly
linked to the increased expression of plant cell walldegrading enzymes such as endo-polygalacturonases (endoPG) and pectin lyases. The secretion of these enzymes by
C. lindemuthianum both in culture and during pathogenesis has been investigated by several groups. Wijesundera
et al. (1984, 1989) showed that endo-PG, two forms of
pectin lyase, - and -galactopyranosidase, -arabinofuranosidase, and a protease are secreted into culture medium
containing polypectate or bean cell walls. Pectin lyase
activity was first observable 4 days after inoculation of
beans with C. lindemuthianum, rising to maximum activity
at 7 days, after which activity declined (Wijesundera et al.,
1989). Thus, the expression of pectin lyase activity correlates well with the onset of necrotrophy and the subsequent development of lesions. Efforts are now underway to
characterize the pectin lyase- and pectate lyase-encoding
gene families of G. cingulata (anamorph C. gloeosporioides). The cloning of the first gene in this family, pnlA, has
been achieved by degenerate PCR, and its identity has
been confirmed by sequence similarity to other cloned
pectin lyases (Templeton et al., 1994). Targeted gene
disruption of pnlA did not affect pathogenesis of G.
cingulata on Capsicum or apple, indicating that the pnlA
enzyme may not be essential for pathogenicity (Bowen et
al., 1995). However, this could be due to the fact that pnlA
comes from a multigene family. Another gene (pel) encoding a pectate lyase from C. gloeosporioides that infects
avocado has recently been isolated (Wattad et al., 1997).
Much recent interest has centred on the endo-PGs
expressed by C. lindemuthianum (Benhamou et al., 1991;
Lafitte et al., 1993; Centis et al., 1996, 1997). The presence
of these enzymes in culture medium of C. lindemuthianum
shows that this fungus has the potential to secrete these

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

Perfect et al.

enzymes during pathogenesis (Wijesundera et al., 1989).

Enzyme accumulation has also been visualized in infected
tissue using the PG-inhibiting protein (Benhamou et al.,
1991).
Endo-PG-encoding enzymes have now been isolated
from several fungal species. Southern analysis using C.
lindemuthianum genomic DNA and the endo-PG from
Cochliobolus carbonum as a probe showed that at least two
endo-PG-encoding genes are present in the genome of C.
lindemuthianum (Centis et al., 1997). To elucidate further
the control of endo-PG expression by C. lindemuthianum
during pathogenesis, these genes (designated Clpg1 and
Clpg2) have been cloned using degenerate PCR (Centis et
al., 1996, 1997). They show 61% identity to each other and
the expression of both genes can be induced in culture,
although Clpg1 appears to show prolonged expression,
while that of Clpg2 is transient. RT-PCR revealed that
Clpg1, but not Clpg2, is expressed during the necrotrophic
phase of the infection of bean hypocotyls by C. lindemuthianum and that this expression is initiated at the onset of
necrotrophy. Antibodies raised to Clpg1 showed that this
enzyme is associated with the extensive degradation of host
cell walls during necrosis. The above studies provide
evidence for the tightly controlled differential expression
of cell wall degrading enzymes by C. lindemuthianum
during host infection. The evidence also points to cell
wall-degrading enzymes playing a major role in the necrotrophic stage of the interaction and the onset of symptoms.
The nature of the switch from biotrophy to necrotrophy
in hemibiotrophic interactions has been debated for many
years. There are several possible events which may signal
the start of the destructive necrotrophic phase. The
hostpathogen interface formed in the C. lindemuthianum
bean interaction is relatively unspecialized when compared
to the interfaces formed by obligate biotrophs (Green et
al., 1995). This may limit the effectiveness of IH at
acquiring nutrients or maintaining host cell viability, thereby
inducing necrotrophy after a period of time to increase the
available nutrient pool. In fact, nutrients have been linked
to the control of expression of an endo-PG expressed by C.
lindemuthianum during the necrotrophic phase of infection of bean. It has been shown that two sugars, -Larabinose and -L-rhamnose, both of which are present in
plant cell wall pectic polysaccharides, increase endo-PG
expression in vitro (Hugouvieux et al., 1997). The release
of these sugars from plant cell walls by other fungal
enzymes at the end of the biotrophic phase may then
induce endo-PG activity, which in turn significantly contributes to necrotrophy.

195

Colletotrichum: Pathology and FungalPlant Interactions

CONCLUDING REMARKS

REFERENCES

The work discussed in this review has demonstrated the

progress made in the identification of genes and proteins
relevant to the different stages of fungal development and
fungalplant interactions for a range of Colletotrichum
species. Several genes and proteins which are specifically
expressed at different stages of the Colletotrichum life
cycle have been identified and some of their functions have
been established. However, relatively few genes have been
identified that can be defined as pathogenicity determinants. To date, two genes, clk1 which encodes a serine/
threonine kinase in C. lindemuthianum (Dufresne et al.,
1998), and cap20 which encodes a wall glycoprotein of C.
gloeosporioides appressoria (Hwang et al., 1995), have
been shown to have a role in pathogenicity and virulence,
respectively. Clearly there is a need to search for further
genes involved in pathogenicity and the production of
mutants continues to be a promising approach (Epstein et
al., 1998).
Although prepenetration events of Colletotrichum species appear similar, there are differences between species
in the mechanisms of spore adhesion and the relative
importance of melanization and cutinases for penetration
of the plant cuticle by the appressorium. Thus, it is difficult
to generalize between infection processes of different
species at the molecular level. Most of the studies on
postpenetration events have used the hemibiotroph C.
lindemuthianum. This will continue to be a useful system
for studying biotrophy in particular, until good transformation systems are developed for obligate biotrophs. However, there are clear differences between IH and haustoria
and it will be important to establish whether these infection structures do perform similar functions. The signalling
pathways involved in spore germination and appressorium
differentiation are being established and there are clearly
some parallels with pathways operating in M. grisea. One
of the challenges for the future will be to identify signals
involved in establishing biotrophic development in the host
and the transition to necrotrophy.

Allen, D. J. 1983. The Pathology of Tropical Food Legumes: Disease

Resistance in Crop Improvement, pp. 150187. Wiley, New York.
Bailey, J. A., and Jeger, M. J. 1992. Colletotrichum: Biology, Pathology
and Control. CAB International, Wallingford.
Bailey, J. A., Rowell, P. M., and Arnold, G. M. 1980. The temporal
relationship between host cell death, phytoalexin accumulation and
fungal inhibition during hypersensitive reactions of Phaseolus vulgaris
to Colletotrichum lindemuthianum. Physiol. Plant Pathol. 17: 329339.
Bailey, J. A., OConnell, R. J., Pring, R. J., and Nash, C. 1992. Infection
strategies of Colletotrichum species. In Colletotrichum: Biology, Pathology and Control (J. A. Bailey, and M. J. Jeger, Eds), pp. 88120. CAB
International, Wallingford.
Benhamou, N., Lafitte, C., Barthe, J-P., and Esquerre-Tugaye, M-T. 1991.
Cell surface interactions between bean leaf cells and Colletotrichum
lindemuthianum. Plant Physiol. 97: 234244.
Bergmann, C. W., Ito, Y., Singer, D., Albersheim, P., Darvill, A. G.,
Benhamou, N., Nuss, L., Salvi, G., Cervone, F., and De Lorenzo, G.
1994. Polygalacturonase-inhibiting protein accumulates in Phaseolus
vulgaris L. in response to wounding, elicitors and fungal infection.
Plant J. 5: 625634.
Birkeland, N-K. 1994. Cloning, molecular characterisation, and expression of the genes encoding lytic functions of lactococcal bacteriophage
LC3: A dual lysis system of modular design. Can. J. Microbiol. 40:
658665.
Bobichon, H., Gache, D., and Bouchet, P. 1994. Ultrarapid cryofixation of
Candida albicans: Evidence for a fibrillar reticulated external layer and
mannan channels within the cell wall. Cryoletters 15: 161172.
Bonnen, A. M., and Hammerschmidt, R. 1989. Role of cutinolytic
enzymes in infection of cucumber by Colletotrichum lagenarium.
Physiol. Mol. Plant Pathol. 35: 475481.
Bowen, J. K., Templeton, M. D., Sharrock, K. R., Crowhurst, R. N., and
Rikkerink, E. H. A. 1995. Gene inactivation in the plant pathogen
Glomerella cingulata: Three strategies for the disruption of the pectin
lyase gene pnlA. Mol. Gen. Genet. 246: 196205.
Buhr, T. L., and Dickman, M. B. 1993. Isolation and characterisation of a
-tubulin-encoding gene from Colletotrichum gloeosporioides f. sp.
aeschynomene. Gene 124: 121125.
Buhr, T. L., and Dickman, M. B. 1994. Isolation, characterisation, and
expression of a second -tubulin-encoding gene from Colletotrichum
gloeosporioides f. sp. aeschynomene. Appl. Environ. Microbiol. 60:
41554159.
Buhr, T. L., and Dickman, M. B. 1997. Gene expression analysis during
conidial germ-tube and appressorium development in Colletotrichum
trifolii. Appl. Env. Microbiol. 63: 23782383.
Buhr, T. L., Oved, S., Truesdell, G. M., Huang, C., Yarden, O., and
Dickman, M. B. 1996. A kinase-encoding gene from C. trifolii
complements a colonial growth mutant of Neurospora crassa. Mol.
Gen. Genet. 251: 565572.
Centis, S., Dumas, B., Fournier, J., Marolda, M., and Esquerre-Tugaye,
M-T. 1996. Isolation and sequence analysis of CLPG1, a gene coding
for an endopolygalacturonase of the phytopathogenic fungis Colletotrichum lindemuthianum. Gene 170: 125129.

ACKNOWLEDGMENTS
The work of Drs. J. R. Green and R. J. OConnell has been supported
by grants and studentships from the Biotechnology and Biological
Sciences Research Council of the United Kingdom. IACR-Long Ashton
receives grant-aided support from the BBSRC, United Kingdom. The
authors thank Dr. Phillip Wharton for help with preparation of Fig. 2.

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

196
Centis, S., Guillas, I., Sejalon, N., Esquerre-Tugaye, M-T., and Dumas, B.
1997. Endopolygalacturonase genes from Colletotrichum lindemuthianum: Cloning of CLPG2 and comparison of its expression to that of
CLPG1 during saprophytic and parasitic growth of the fungus. Mol.
Plant Microbe Interact. 10: 769775.
Dean, R. A. 1997. Signal pathways and appressorium morphogenesis.
Annu. Rev. Phytopathol. 35: 211234.
Dickman, M. B., Patil, S. S., and Kolattukudy, P. E. 1982. Purification,
characterisation and role in infection of an extracellular cutinolytic
enzyme from Colletotrichum gloeosporioides Penz. on Carica papya L.
Physiol. Mol. Plant Pathol. 20: 333347.
Dickman, M. B., and Patil, S. S. 1986. Cutinase deficient mutants of
Colletotrichum gloeosporioides are nonpathogenic to papaya fruit.
Physiol. Mol. Plant Pathol. 28: 235242.
Dixon, R. A., Bailey, J. A., Bell, J. N., Bolwell, G. P., Cramer, C. L.,
Edwards, K., Hamdan, M. A. M. S., Lamb, C. J., Robbins, M. P., Ryder,
T. B., and Schuch, W. 1986. Rapid changes in gene expression in
response to microbial elicitation. Philos. Trans. R. Soc. London B314:
411426.
Dufresne, M., Bailey, J. A., Dron, M., and Langin, T. 1998. Clk1, a
serine/threonine protein kinase-encoding gene, is involved in pathogenicity of C. lindemuthianum on common bean. Mol. Plant Microbe
Interact. 11: 99108.
Epstein, L., Lusnak, K., and Kaur, S. 1998. Transformation-mediated
developmental mutants of Glomerella graminicola (Colletotrichum
graminicola). Fungal Genet. Biol. 23: 189203.
Ettinger, W. F., Thukral, S. K., and Kolattukudy, P. E. 1987. Structure of
cutinase gene, cDNA and the derived amino acid sequence from
phytopathogenic fungi. Biochemistry. 26: 78837892.
Flaishman, M. A., Hwang, C-S., and Kolattukudy, P. E. 1995. Involvement of protein phosphorylation in the induction of appressorium
formation in Colletotrichum gloeosporioides by its host surface wax and
ethylene. Physiol. Mol. Plant Pathol. 47: 103117.
Green, J. R., Pain, N. A., Cannell, M. E., Jones, G. L., Leckie, C. L.,
McCready, S., Mendgen, K., Mitchell, A. J., Callow, J. A., and
OConnell, R. J. 1995. Analysis of differentiation and development of
the specialised infection structures formed by biotrophic fungal plant
pathogens using monoclonal antibodies. Can. J. Bot. 73 (Suppl.):
S408S417.
Hahn, M., Neef, U., Struck, C., Gottfert, M., and Mendgen, K. 1997. A
putative amino acid transporter is specifically expressed in haustoria of
the rust fungus Uromyces fabae. Mol. Plant Microbe Interact. 10:
438445.
Heath, M. C., and Skalamera, D. 1997. Cellular interactions between
plants and biotrophic fungal parasites. Adv. Bot. Res. 24: 196225.
Hughes, H. B., Carzaniga, R., Rawlings, S. L., Green, J. R., and
OConnell, R. J. 1999. Spore surface glycoproteins of Colletotrichum
lindemuthianum are recognized by a monoclonal antibody which
inhibits adhesion to polystyrene. Microbiol., in press.
Hugovieux, V., Centis, S., Lafitte, C., and Esquerre-Tugaye, M-T. 1997.
Induction by -L-arabinose and -L-rhamnose of endopolygalacturonase gene expression in Colletotrichum lindemuthianum. Appl. Env.
Microbiol. 63: 22872292.
Hwang, C-H., and Kolattukudy, P. E. 1995. Isolation and characterisation
of genes expressed uniquely during appressorium formation by Collet-

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

Perfect et al.

otrichum gloeosporioides conidia induced by the host surface wax. Mol.

Gen. Genet. 247: 282294.
Hwang, C-H., Flaishman, M. A., and Kolattukudy, P. E. 1995. Cloning of
a gene expressed during appressorium formation by Colletotrichum
gloeosporioides and a marked decrease in virulence by disruption of
this gene. Plant Cell 7: 183193.
Justesen, A., Somerville, S., Christiansen, S., and Giese, H. 1996.
Isolation and characterisation of two novel genes expressed in germinating conidia of the obligate biotroph Erysiphe graminis f.sp. hordei.
Gene 170: 131135.
Kim, Y-K., Li, D., and Kolattukudy, P. E. 1998. Induction of Ca2calmodulin signaling by hard-surface contact primes Colletotrichum
gloeosporioides conidia to germinate and form appressoria. J. Bacteriol.
180: 51445150.
Kolattukudy, P. E., Rogers, L. M., Li, D., Hwang, C-S., and Flaishman,
M. A. 1995. Surface signalling in pathogenesis. Proc. Natl. Acad. Sci.
USA 92: 40804087.
Kubo, Y., and Furusawa, I. 1991. Melanin biosynthesis: Pre-requisite for
successful invasion of the plant host by appressoria of Colletotrichum
and Pyricularia. In The Fungal Spore and Disease Initiation in Plants
and Animals (G. T. Cole and H. C. Hoch, Eds), pp. 205218. Plenum,
New York.
Kubo, Y., Nakamura, H., Kobayashi, K., Okuno, T., and Furusawa, I.
1991. Cloning of a melanin biosynthetic gene essential for appressorial
penetration of Colletotrichum lagenarium. Mol. Plant Microbe Interact. 5: 440445.
Kubo, Y., Takano, N., Endo, N., Yasuda, N., Tajima, S., and Furusawa, I.
1996. Cloning and structural analysis of the melanin biosynthesis gene
encoding scytalone dehydratase in Colletotrichum lagenarium. Appl.
Environ. Microbiol. 62: 43404344.
Lafitte, C., Barthe, J-P., Gansel, X., Dechamp-Guillaume, G., Faucher,
C., Mazau, D., and Esquerre-Tugaye, M-T. 1993. Differential induction by endopolygalacturonase of -1,3-glucanases in Phaseolus vulgaris isolines susceptible and resistant to Colletotrichum lindemuthianum race . Mol. Plant Microbe Interact. 6: 628634.
Latunde-Dada, A. O., OConnell, R. J., Nash, C., Pring, R. J., Lucas, J. A.,
and Bailey, J. A. 1996. Infection process and identity of the hemibiotrophic anthracnose fungus (Colletotrichum destructivum OGara) from
cowpea (Vigna unguiculata (L.) Walp.) Mycol. Res. 100: 11331141.
Liu, Z-M., and Kolattukudy, P. E. 1998. Identification of a gene product
induced by hard-surface contact of Colletotrichum gloeosporioides
conidia as a ubiquitin-conjugating enzyme by yeast complementation.
J. Bacteriol. 180: 35923597.
Mahe`, A., Grisvard, J., and Dron, M. 1993. Two avirulent races of
Colletotrichum lindemuthianum trigger different time courses of plant
defense reactions in bean. Mol. Plant Microbe Interact. 6: 423428.
Mathur, R. S., Barnett, H. L., and Lilly, V. G. 1950. Sporulation of
Colletotrichum lindemuthianum in culture. Phytopathology 40: 104
114.
Mercure, E. W., Leite, B., and Nicholson, R. L. 1994a. Adhesion of
ungerminated conidia of Colletotrichum graminicola to artificial hydrophobic surfaces. Physiol. Mol. Plant Pathol. 45: 421440.
Mercure, E. W., Kunoh, H., and Nicholson, R. L. 1994b. Adhesion of
Colletotrichum graminicola conidia to corn leaves: A requirement for
disease development. Physiol. Mol. Plant Pathol. 45: 407420.

Colletotrichum: Pathology and FungalPlant Interactions

Mould, M. J. R., Boland, G. J., and Robb, J. 1991. Ultrastructure of the

Colletotrichum trifolii-Medicago sativa pathosystem. 2. Post-penetration events. Physiol. Mol. Plant Pathol. 38: 195210.
Nicholson, R. L. 1992. Colletotrichum graminicola and the anthracnose
disease of corn and sorghum. In Colletotrichum: Biology, Pathology
and Control (J. A. Bailey, and M. J. Jeger, Eds), pp. 186202. CAB
International, Wallingford.
Nicholson, R. L. 1996. Adhesion of fungal propagules. In Histology,
Ultrastructure and Molecular Cytology of PlantMicroorganism Interactions (Nicole, M. and Gianinazzi-Pearson, V., Eds.), pp. 117134.
Kluwer Academic Publishers, Dordrecht/Norwell, MA.
Nuss, L., Mahe`, A., Clark, A. J., Grisvard, J., Dron, M., Cervone, F., and
De Lorenzo, G. 1996. Differential accumulation of PGIP (polygalacturonase-inhibiting protein) mRNA in two near-isogenic lines of Phaseolus vulgaris L. upon infection with Colletotrichum lindemuthianum.
Physiol. Mol. Plant Pathol. 48: 8389.
OConnell, R. J. 1987. Absence of a specialised interface between
infection hyphae of Colletotrichum lindemuthianum and Phaseolus
vulgaris. New Phytol. 107: 725734.
OConnell, R. J., Bailey, J. A., and Richmond, D. V. 1985. Cytology and
physiology of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum. Physiol. Plant Pathol. 27: 7598.
OConnell, R. J., and Bailey, J. A. 1988. Differences in the extent of fungal
development, host cell necrosis and symptom expression during
race-cultivar interactions between Phaseolus vulgaris and Colletotrichum lindemuthianum. Plant Pathol. 37: 351362.
OConnell, R. J., Uronu, A. B., Waksman, G., Nash, C., Keon, J. P. R., and
Bailey, J. A. 1993. Hemibiotrophic infection of Pisum sativum by
Colletotrichum truncatum. Plant Pathol. 42: 774783.
OConnell, R. J., Pain, N. A., Hutchison, K. A., Jones, G. L., and Green, J.
R. 1996. Ultrastructure and composition of the cell surfaces of infection
structures formed by the fungal plant pathogen Colletotrichum lindemuthianum. J. Microsc. 181: 204212.
Pain, N. A., OConnell, R. J., Bailey, J. A., and Green, J. R. 1992.
Monoclonal antibodies which show restricted binding to four Colletotrichum species: C. lindemuthianum, C. malvarum, C. orbiculare and
C. trifolii. Physiol. Mol. Plant Pathol. 40: 111126.
Pain, N. A., Green, J. R., Gammie, F., and OConnell, R. J. 1994a.
Immunomagnetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum from infected bean leaves using a monoclonal
antibody. New Phytol. 127: 223232.
Pain, N. A., OConnell, R. J., Mendgen, K., and Green, J. R. 1994b.
Identification of glycoproteins specific to biotrophic intracellular
hyphae formed in the Colletotrichumbean interaction. New Phytol.
127: 233242.
Pain, N. A., OConnell, R. J., and Green, J. R. 1995. A plasma
membrane-associated protein is a marker for differentiation and
polarisation of Colletotrichum lindemuthianum appressoria. Protoplasma 188: 111.
Pain, N. A., Green, J. R., Jones, G. L., and OConnell, R. J. 1996.
Composition and organisation of extracellular matrices around germ
tubes and appressoria of Colletotrichum lindemuthianum. Protoplasma
190: 119130.
Pascholati, S. F., Deising, H., Leite, B., Anderson, D., and Nicholson, R.
L. 1993. Cutinase and non-specific esterase activities in the conidial

197
mucilage of Colletotrichum graminicola. Physiol. Mol. Plant Pathol. 42:
3751.
Perfect, S. E., OConnell, R. J., Green, E. F., Doering-Saad, C., and
Green, J. R. 1998. Expression cloning of a fungal proline-rich glycoprotein specific to the biotrophic interface formed in the Colleotrichum
bean interaction. Plant J. 15: 273279.
Perpetua, N. S., Kubo, Y., Yasuda, N., Takano, Y., and Furusawa, I. 1996.
Cloning and characterisation of a melanin biosynthetic THR1 reductase
gene essential for appressorial penetration of Colletotrichum lagenarium. Mol. Plant Microbe Interact. 9: 323329.
Podila, G. K., Rogers, L. M., and Kolattukudy, P. E. 1993. Chemical
signals from avocado surface wax trigger germination and appressorium
formation in Colletotrichum gloeosporioides. Plant Physiol. 103: 267
272.
Pring, R. J., Nash, C., Zakaria, M., and Bailey, J. A. 1995. Infection
processes and host-range of Colletotrichum capsici. Physiol. Mol. Plant
Pathol. 46: 137152.
Prusky, D., Plumbley, R. A., and Kobiler, I. 1991. The relationship
between antifungal diene levels and fungal inhibition during quiescent
infection of unripe avocado fruits by Colletotrichum gloeosporioides.
Plant Pathol. 40: 4552.
Roberts, R. G., and Snow, J. P. 1984. Histopathology of cotton boll rot
caused by Colletotrichum capsici. Phytopathology 74: 390397.
Rodriguez, R. J., and Redman, R. S. 1992. Molecular transformation and
genome analysis of Colletotrichum species. In Colletotrichum: Biology,
Pathology and Control (J. A. Bailey and M. J. Jeger, Eds), pp. 4776.
CAB, Oxford.
Sela-Buurlage, M. B., Epstein, L., and Rodriguez, R. J. 1991. Adhesion of
ungerminated Colletotrichum musae conidia. Physiol Mol. Plant Pathol.
39: 345352.
Stephenson, S-A., Green, J. R., Manners, J. M., and Maclean, D. J. 1997.
Cloning and characterisation of glutamine synthetase from Colletotrichum gloeosporioides and demonstration of elevated expression during
pathogenesis on Stylosanthes guianensis. Curr. Genet. 31: 447454.
Sugui, J. A., Leite, B., and Nicholson, R. L. 1998. Partial characterisation
of the extracellular matrix released onto hydrophobic surfaces by
conidia and conidial germlings of Colletotrichum graminicola. Physiol.
Mol. Plant Pathol. 52: 411425.
Swinburne, T. R., and Brown, A. E. 1983. Appressoria development and
quiescent infections of banana fruit by Colletotrichum musae. Trans.
Brit. Mycol. Soc. 80: 176178.
Takano, Y., Kubo, Y., Shimizu, K., Mise, T., Okuno, T., and Furusawa, I.
1995. Structural analysis of PKS1, a polyketide synthase gene involved
in melanin biosynthesis of Colletotrichum lagenarium. Mol. Gen.
Genet. 249: 162167.
Takano, Y., Kubo, Y., Kuroda, I., and Furusawa, I. 1997. Temporal
transcriptional pattern of three melanin biosynthesis genes, PKS1,
SCD1 and THR1, in appressorium-differentiating and nondifferentiating conidia of Colletotrichum lagenarium. Appl. Env. Microbiol. 63:
351354.
Templeton, M. D., Rikkerink, E. H. A., Solon, S. L., and Crowhurst, R. N.
1992. The cloning and molecular characterisation of the glyceraldehyde3-phosphate dehydrogenase-encoding gene from Glomerella cingulata.
Gene 142: 141146.
Templeton, M. D., Sharrock, K. R., Bowen, J. K., Crowhurst, R. N., and
Rikkerink, E. H. A. 1994. The pectin lyase-encoding gene (pnl) family

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

198
from Glomerella cingulata: Characterisation of pnlA and its expression
in yeast. Gene 142: 141146.
Thrower, L. B. 1966. Terminology for plant parasites. Phytopathol. Zeit.
56: 258259.
Van Dyke, C. G., and Mims, C. W. 1991. Ultrastructure of conidia,
conidium germination and appressorium development in the plant
pathogenic fungus Colletotrichum truncatum. Can. J. Bot. 69: 2445
2467.
Walker, J. C. 1921. Onion smudge. J. Agric. Res. 20: 685721.
Wattad, C., Kobiler, D., Dinoor, A., and Prusky, D. 1997. Pectate lyase of
C. gloeosporioides attacking avocado fruit: cDNA cloning and involvement in pathogenicity. Physiol. Mol. Plant Pathol. 50: 197212.
Wharton, P. S., and Julian, A. M. 1996. A cytological study of compatible
and incompatible interactions between Sorgum bicolor and Colletotrichum sublineolum. New Phytol. 14: 2534.
Wijesundera, R. L. C., Bailey, J. A., and Byrde, R. J. W. 1984. Production
of pectin lyase by Colletotrichum lindemuthianum in culture and

Copyright r 1999 by Academic Press

All rights of reproduction in any form reserved.

Perfect et al.

infected bean (Phaseolus vulgaris) tissue. J. Gen. Microbiol. 130:

285290.
Wijesundera, R. L. C., Bailey, J. A., Byrde, R. J. W., and Fielding, A. H.
1989. Cell wall degrading enzymes of Colletotrichum lindemuthianum:
Their role in the development of bean anthracnose. Physiol. Mol. Plant
Pathol. 34: 403413.
Wojtaszek, P., Trethowan, J., and Bolwell, G. P. 1995. Specificity in the
immobilisation of cell wall proteins in response to different elicitor
molecules in suspension-cultured cells of French bean (Phaseolus
vulgaris L.). Plant Mol. Biol. 28: 10751087.
Yang, Z., and Dickman, M. B. 1997. Regulation of cAMP and cAMP
dependant protein kinase during conidial germination and appressorium formation in Colletotrichum trifolii. Physiol. Mol. Plant Pathol.
50: 117127.
Young, D. H., and Kauss, H. 1984. Adhesion of Colletotrichum lindemuthianum spores to Phaseolus vulgaris hypocotyls and to polystyrene.
Appl. Env. Microbiol. 47: 616619.