J Gen Plant Pathol (2014) 80:15–23
DOI 10.1007/s10327-013-0492-0
REVIEW FOR THE 100TH ANNIVERSARY
Virulence factors of Botrytis cinerea
Masami Nakajima • Katsumi Akutsu
Received: 19 March 2013 / Accepted: 20 August 2013 / Published online: 10 October 2013
Ó The Phytopathological Society of Japan and Springer Japan 2013
Abstract Botrytis cinerea is responsible for gray mold
disease in more than 200 host plant species. The infection of
host plants is mediated by numerous extracellular enzymes,
proteins and metabolites. Each of these compounds may play a
role in different stages of the infection process. Cell wall-
degrading enzymes may facilitate the penetration into the host
surface, while toxins, oxalic acid and reactive oxygen species
may contribute to killing of the host cells. Cell wall-degrading
enzymes contribute to the conversion of host tissue into fungal
biomass. On the other hand, B. cinerea infection induces
biosynthesis of phytoalexins. Therefore, the ability to over-
come a wide spectrum of phytoalexins contributes to the
pathogenicity of the fungus with a broad host range. The
cloning of the corresponding genes has facilitated studies on
gene expression and targeted mutagenesis. This review gives
an overview of the research performed on virulence factors
that play the roles in pathogenesis.
Keywords Botrytis cinerea
Extracellular enzymes

Penetration
Phytotoxic activity
Programmed cell
death
Virulence factor
Introduction
Botrytis cinerea is an important plant pathogen with a wide
range of host plants, particularly in temperate regions of
the world. This fungus causes gray mold disease in over
200 plant species, including most vegetable and fruit crops,
trees and flowers. It can attack many organs, including
M. Nakajima (&)
K. Akutsu
Laboratory of Plant Pathology, College of Agriculture,
Ibaraki University, Ami-machi, Ibaraki 300-0393, Japan
e-mail: mnakaji@mx.ibaraki.ac.jp
leaves, stems and fruits, as a necrotroph, often causing
heavy losses after harvest. It is also a saprophyte on
senescent and dead plant materials. Chemical control
remains the primary way to reduce the incidence of grey
mold on major crops because resistant cultivars are not yet
available. However, due to the development of multidrug
resistance in field strains of the fungus, this strategy is only
partially successful. Due to the wide host range of this
pathogen and the severe damage it causes in agriculture,
efforts have been made to understand the mechanisms of
pathogenicity. The identified virulence factors that play the
roles in pathogenesis are listed in Table 1.
Penetration of the host surface
Conidia produced on infected plants or on infected plant
debris on germinating sclerotia are dispersed by air cur-
rents to new plants. The disease cycle starts with conidia
landing on the host surface. Upon attachment, they ger-
minate on the host surface and produce a germ tube that
develops into an appressorium that facilitates penetration
of the host surface. The first barrier to breach is the host
cuticle covering all aerial parts of the plants. The cuticle
consists primarily of cutin, an insoluble polyester of C16
and C18 hydroxy fatty acids, in many cases covered with a
hydrophobic wax layer. Physical damage or mechanical
penetration of the cuticle by B. cinerea is not usually
observed (Cole et al. 1996; Williamson et al. 1995), indi-
cating that enzymatic activity is involved in penetrating
intact host surfaces (Salinas and Verhoeff 1995).
Penetration of the wax layer
Waxes on leaf and fruit surfaces form a water-repellent
surface, thereby preventing the formation of a film of water
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Table 1 Virulence factors of Botrytis cinerea antibodies did not affect germination of the fungal conidia
Virulence factors References
nor did they inhibit the infection of wounded tissue, sug-
gesting a role for the lipase specifically during host surface
Penetration into the host surface penetration. A partial amino-acid sequence of the lipase
Lipase Comménil et al. (1998) was determined (Comménil et al. 1999). The correspond-
Cutinase Salinas (1992) ing gene lip1 was cloned and targeted mutants were made
Killing of host cells (Reis et al. 2005); the lip1-deficient mutants did not pro-
Botcinolide Cutler et al. (1993) duce extracellular lipase under inducing conditions, but
Sesquiterpenoid metabolites Durán-Patrón et al. (2000) remained able to infect intact primary leaves, indicating
Botrydial and related metabolites Colmenares et al. (2002) that the lipase is not essential for host surface penetration.
Botrydial derivatives Reino et al. (2004)
Botcinins E and F Tarn et al. (2006) Penetration of the cutin layer
Cu–Zn-superoxide dismutase Rolke et al. (2004)
NADPH oxidases Segmüller et al. (2008) Under the wax layer lies cutin, a complex mixture of
Endopolygalacturonase1 ten Have et al. (1998) polymeric fatty acids mainly interconnected by ester, per-
Xylanase Noda et al. (2010) oxide, and ether linkages, probably as a three-dimensional
Nep1-like proteins Schouten et al. (2008) network. Cutinases break cutin molecules and release
Cerato-platanin family protein Fnas et al. (2011) monomers as well as oligomers of the component fatty acid
Conversion of host tissue into derivatives from the insoluble cutin polymer. Cutinase has
fungal biomass been purified from B. cinerea and identified as an 18-kDa
Pectin methylesterase Valette-Collet et al. (2003) protein. The 18-kDa cutinase was suggested to play an
BcPGl ten Have et al. (1998) important role in penetrating host tissue by B. cinerea,
BcPG2 Kars et al. (2005a) based on the observation that treatment of gerbera flowers
Aspartic protease Movahedi and Heale (1990b) with monoclonal antibodies raised against the cutinase
Overcoming host defense reduced lesion formation by B. cinerea (Salinas 1992).
responses The 18-kDa cutinase-encoding gene cutA was cloned
BcBirl Shlezinger et al. (2011a, b) (van der Vlugt-Bergmans et al. 1997). The expression of
ATP-binding cassette Stefanato et al. (2009) cutA was analyzed using a cutA promoter-GUS reporter
transporters gene fusion. High GUS activity was detected from the
onset of conidial germination and during penetration into
epidermal cells (van Kan et al. 1997). Transformants
on which pathogens might be deposited and germinate or lacking a functional cutA gene were constructed by gene
multiply. Infection efficiency of conidia of B. squamosa on disruption, but their ability to infect gerbera flowers and
onion leaves was increased when the leaves were wiped to tomato fruits was unaltered compared to the wild-type
remove wax layer before inoculation (Sutton et al. 1984). strain. These results suggest that cutinase A is not essential
No relation was found between the mass of the wax layer for successful penetration of intact cuticle surfaces (van
and susceptibility of gerbera and rose petals to B. cinerea Kan et al. 1997).
(Kerssies and Frinking 1996). Botrytis cinerea may have
surfactants that reduce surface hydrophobicity and dissolve Penetration of the cell wall
the wax layer. When B. cinerea is grown in a medium
containing a fatty acid ester as an inducer, it produces an Botrytis cinerea secretes numerous cell wall-degrading
inducible extracellular lipase with a molecular mass of enzymes (CWDEs) to breach the plant cell wall and use it
60 kDa. This lipase has been found to degrade unsaturated as a source of nutrients. Penetration into the epidermis
long chain fatty acid esters of the type that are reported to often occurs at the anticlinal position and is frequently
be components of cutin and waxes (Comménil et al. 1995). accompanied by swelling of the epidermal cell wall as a
The lipase possesses cutinolytic activity, although its result of enzyme activity (Mansfield and Richardson 1981).
kinetic properties are distinct from those of a typical cu- Enzymes that attack pectic substances in the plant cell wall
tinase (Comménil et al. 1998). Studies with polyclonal have an important function in tissue degradation and hence
antibodies blocking the active site suggested that the lipase in pathogenesis (Clark and Lorbeer 1976; Cole et al. 1998;
plays an important role in the infection process. When Collmer and Keen 1986). Endopolygalacturonase activity
antibodies were applied to intact tomato leaves before was found before germination of B. cinerea conidia could
inoculation with conidia, the fungal germ tubes were be observed (Verhoeff and Warren 1972), and two polyg-
unable to penetrate the cuticle (Comménil et al. 1998). The alacturonases with a high isoelectric value were associated

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with the penetration stage of the infection process (van den
Heuvel and Waterreus 1985). Kapat et al. (1998), sug-
gesting that the early production of constitutive enzymes
probably enables fast penetration of the host tissue.
Killing of host cells
After cuticle penetration, B. cinerea kills epidermal and
mesophyll cells in advance of invasion of infection hyphae
(Clark and Lorbeer 1976). A number of metabolites and
proteins secreted by the fungus have been shown to cause
cell death when applied to plant tissues, and some also
induce symptoms of programmed cell death (PCD). The
induction of PCD facilitates B. cinerea invasion and may in
fact be essential for successful infection (Govrin and
Levine 2000).
Toxins
Several secondary metabolites showing phytotoxic activity
have been identified in culture filtrates of B. cinerea: bot-
cinolide, a highly substituted lactone (Cutler et al. 1993);
botrydial, a bicyclic sesquiterpene (Colmenares et al.
2002); and several compounds with lower toxicity that are
related to botcinolide and botrydial (Colmenares et al.
2002; Durán-Patrón et al. 2000). Botrydial is produced in
infected tissue (Deighton et al. 2001) and induces chlorosis
and cell collapse (Colmenares et al. 2002). An interesting
feature of botrydial is also the light dependence of its
toxicity (Colmenares et al. 2002). Bcbot1, encoding a P450
monooxygenase, is involved in botrydial biosynthesis.
Deletion of bcbot1 in three strains demonstrated that the
botrydial effect on virulence is strain-dependent (Siewers
et al. 2005). In addition to botrydial and its derivatives,
virulent B. cinerea strains also produce other phytotoxic
metabolites such as botcinic acid and related botcinins
(Reino et al. 2004; Tani et al. 2006).
Oxalic acid
The physiological roles of oxalic acid (OA) in pathogenesis
are numerous. OA enhances the activity of polygalacturon-
ases at low pH (Bateman and Beer 1965), inhibits plant
protective enzymes (Favaron et al. 2004; Marciano et al.
1983), suppresses the plant oxidative burst (Cessna et al.
2000), mediates pH signaling (Rollins 2003), and induces
PCD (Kim et al. 2008). OA is known to play a key role in
pathogenesis of Sclerotinia sclerotiorum, a close relative of
B. cinerea. Mutants of S. sclerotiorum that do not produce
OA were nonpathogenic on host plants including Arabi-
dopsis thaliana and the deficiency could be restored by
supplementing inocula with OA (Dickman and Mitra 1992;
Godoy et al. 1990). Botrytis cinerea produces OA both in
17
media (Gentile 1954) and in colonized plant tissue (Verhoeff
et al. 1988). The production of OA is regulated by ambient
pH and detectable when ambient pH exceeds 5.0 (Manteau
et al. 2003). Activity of secreted pathogenicity factors
(endopolygalacturonase, aspartic protease (AP) and laccase)
by B. cinerea was enhanced in low ambient pH through the
secretion of OA (Manteau et al. 2003; ten Have et al. 2002).
Reactive oxygen species
The generation of reactive oxygen species (ROS) is asso-
ciated with defense responses in plant–fungus interactions.
The oxidative burst is an early and universal plant response
following pathogen challenge. This rapid production of ROS
by the plant was originally identified as being required for
defense gene expression and the hypersensitive response
(HR), a type of PCD thought to limit access of the pathogen
to water and nutrients. In the case of a necrotroph such as B.
cinerea, plant cell death is beneficial to the pathogen and
leads to susceptibility (Govrin and Levine 2000). During the
course of the infection, mycelium growing in a lesion is
confronted with ROS (Govrin and Levine 2000; Schouten
et al. 2002a). Botrytis cinerea itself also generates ROS, and
a number of enzymes potentially contribute to H2O2 gen-
eration. A Cu–Zn-superoxide dismutase gene and a glucose
oxidase gene were cloned and characterized. Whereas
deletion of the glucose oxidase gene did not reduce viru-
lence on bean leaves, deletion of the superoxide dismutase
gene significantly reduced virulence (Rolke et al. 2004).
Botrytis cinerea actively triggers an oxidative burst during
cuticle penetration (Tenberge 2004) and primary lesion
formation (Lyon et al. 2004). Nicotinamide adenine dinu-
cleotide (NADPH) oxidases are involved in various differ-
entiation processes in fungi. Segmüller et al. (2008)
investigated the role of two NADPH oxidases (BcnoxA and
B) in B. cinerea. Mutants in both were impaired in viru-
lence, but to different extents: BcnoxB was important for
penetration, whereas BcnoxA was involved in lesion
expansion. A double mutant was almost avirulent. Plant cell
death also occurred through an oxidative burst produced by
the host in reaction to B. cinerea attack (Govrin and Levine
2000). Studies in A. thaliana and tobacco suggested that B.
cinerea may even require the HR to achieve full pathoge-
nicity (Dickman et al. 2001; Govrin and Levine 2000).
Endopolygalacturonase1 (BcPG1) of B. cinerea, first known
as a virulence factor (ten Have et al. 1998), elicits defense
responses in grape, including the production of ROS
(Poinssot et al. 2003; Vandelle et al. 2006), and xylanase
Xyn11A induces necrosis in the infected plant. A conserved
30-amino acid region on the enzyme surface, away from the
xylanase active site, is responsible for this effect and
mediates binding to plant cells (Noda et al. 2010). A number
of metabolites and proteins secreted by B. cinerea cause cell
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death when applied to plant tissues, and some also induce
symptoms of HR, such as botrydial (Rossi et al. 2011),
Nep1-like proteins (Schouten et al. 2008), and the cerato-
platanin protein family (Frias et al. 2011).
Conversion of host tissue into fungal biomass
The main function of the plant cell wall is to provide
mechanical strength and intercellular adhesion. Plant cell
walls consist primarily of polysaccharides, i.e., cellulose
fibers embedded in a matrix of hemicelluloses and pectin,
but structural proteins, in the form of glycoproteins, may
also form networks in the cell wall. From one cell to the
next, the following regions can often be distinguished: a
primary cell wall, the middle lamella and again a primary
cell wall. The primary cell wall of dicotyledons typically
consists of a cellulose-hemicellulose network embedded in
the pectin matrix. The middle lamella has a high content of
pectin, which forms a matrix. Many reports on B. cinerea
describe CWDEs, and they are presumably involved in all
steps of the infection process. When the fungus penetrates
the anticlinal epidermal cell wall, it subsequently grows
into and through the middle lamella. Pathogens secrete
numerous CWDEs to breach the plant cell wall and use it
as a source of nutrients.
Pectin methylesterase
Pectin methylesterases catalyze the demethylesterification
of homogalacturonan, thereby making the pectin available
for degradation by polygalacturonases and pectate lyases.
Reignault et al. (1994) purified pectin methylesterase con-
sisting in a single protein band of 42 kDa that corresponded
to two distinct isozymes of pI 7.1 and 7.4. Pectin methy-
lesterase activity was not induced by pectic derivatives and
not subject to catabolic repression. Disruption of the Bcpme1
gene encoding B. cinerea pectin methylesterase in strain
Bd90 revealed that this gene encoded the isozyme with pI
7.4. Pectin methylesterase activity was found to be 75 %
reduced in the Bcpme1 mutant. Moreover, the mutant was
less virulent on apple fruits, grapevines and A. thaliana
leaves (Valette-Collet et al. 2003). However, single and
double mutants in two Bcpme genes, including the same
Bcpme1 gene in a different strain, B05.10, were not affected
in their growth on highly methylated pectin, nor did they
show any reduction in virulence. It was therefore concluded
that B. cinerea strain B05.10 can degrade pectin without
prior demethylation (Kars et al. 2005b).
Endopolygalacturonases
Endopolygalacturonases are endo-acting enzymes that
catalyze the hydrolysis of the homogalacturonan domain of
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pectic polysaccharides, a linear chain of a-1,4-linked
galacturonosyl residues. These enzymes cannot cleave
highly methylated pectin, but first require the action of
pectin methylesterase to demethylate pectin to pectate. In
the late 1990s, the first B. cinerea polygalacturonase
(BcPG)-encoding genes were cloned and characterized.
The B. cinerea genome contains at least six Bcpg genes
(Wubben et al. 1999). The expression of a set of six distinct
genes (Bcpg1 through 6) was investigated by ten Have
et al. (2001) on four host plants: tomato, broad bean, apple
and zucchini. The pattern of expression of these Bcpg
genes showed that they are differentially expressed,
depending on the stage of infection and the host. Whereas
Bcpg1 was expressed in all tissues tested, Bcpg3 and
Bcpg5 were expressed in apple tissue only. On the other
hand, Bcpg2 expression was detected early in the infection
of all plants except apple fruit. The availability of the Bcpg
gene facilitated the analysis of function of individual iso-
zymes in pathogenesis. Disruption of Bcpg1 resulted in a
reduction in virulence on tomato leaves and fruit, as well as
on apple (ten Have et al. 1998). Deletion of Bcpg2 resulted
in a strong reduction in virulence on tomato and broad
bean. Primary lesion formation was delayed, and the lesion
expansion rate was reduced by 50–80 %. These data indi-
cated that BcPG2 is important for B. cinerea pathogenicity
(Kars et al. 2005a).
Exopolygalacturonases
Exopolygalacturonases catalyze the hydrolytic cleavage of
one galacturonic acid residue from the non-reducing end of
galacturonan, thereby providing the fungus with low
molecular mass compounds that can easily be taken up.
Johnston and Williamson (1992) were the first to purify
two exopolygalacturonases from B. cinerea with molecular
weights of 65 and 70 kDa. The same two exopolygalac-
turonases were detected in B. cinerea cultures containing
citrus pectin as carbon sources and were induced by po-
lygalacturonic acid as well as its monomer, galacturonic
acid, in vitro. By immunohistochemical analysis, secreted
exopolygaracturonases from hyphae were detected in
cucumber leaves at 9 h after inoculation with conidia of
B. cinerea (Rha et al. 2001). Tobias et al. (1993) detected
four exopolygalacturonase isozymes from culture filtrate of
B. cinerea grown on apple pectin.
Cellulases
Cellulases catalyze the degradation of the b-1,4 glycoside
bonds in the cellulose polymer. Botrytis cinerea was found
to produce one extracellular b-glucosidase in liquid culture
(Sasaki and Nagayama 1994), and three intracellular
b-glucosidases (Gueguen et al. 1995). The extracellular
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b-glucosidase and one of the intracellular b-glucosidases
were purified and characterized, but only the extracellular
b-glucosidase was suggested to be involved in plant cell
wall degradation (Sasaki and Nagayama 1996). Espino
et al. (2005) isolated the endo-b-1,4-glucanase encoded by
the gene cel5A. The levels of cel5A transcript and extra-
cellular b-1,4-glucanase activity were regulated by the
carbon source in the same way: both were induced by
carboxymethylcellulose and repressed by glucose. cel5A
mRNA could also be detected during the infection of
tomato leaves. Disruption of cel5A results in the generation
of a strain with virulence identical to the wild type on
tomato leaves or gerbera petals, with no significant
reduction in the extracellular b-1,4-glucanase activity
(Espino et al. 2005).
Xylanases
Endo-b-1,4-xylanases hydrolyze the b-1,4-linked polysac-
charide backbone of xylan, which forms the major hemi-
cellulose component of the plant cell wall. Urbanek and
Zalewska-Sobczak (1984) reported that B. cinerea secreted
three xylanases during apple cell wall degradation. Brito
et al. (2006) isolated and characterized the xyn11A gene,
which encodes an endo-b-1,4-xylanase and is induced by
xylan, repressed by glucose, and expressed in planta. A
mutation of xyn11A had a strong effect on pathogenicity,
reducing the average lesion size by more than 70 %.
Xyn11A was required for full virulence of B. cinerea. In
addition, Noda et al. (2010) demonstrated that the xylan-
hydrolyzing activity of Xyn11A does not contribute to the
infection process, since reintroducing in the xyn11A mutant
altered variants of the xyn11A gene that code for enzymes
lacking xylanase activity also restores the less-virulent
phenotype back to the wild type, as does the wild-type
xyn11A gene.
Aspartic proteases
Movahedi and Heale (1990a) demonstrated that B. cinerea
produced aspartic protease (AP) in both liquid culture and
infected carrot tissues. AP activity was detected in unger-
minated conidia as well as during germination, before the
appearance of pectinase activity. When the inoculum was
supplemented with the specific AP inhibitor pepstatin, AP
activity was blocked, and infection was strongly reduced.
These results suggested that AP might be involved in
pathogenesis (Movahedi and Heale 1990b). However, this
study did not permit distinction between proteases derived
from the pathogen and host. A tomato AP was shown to be
systemically induced in tomato leaves by wounding (Sch-
aller and Ryan 1996), and B. cinerea infection induced the
wound signaling response of tomato (Diaz et al. 2002).
19
Five AP-encoding genes were cloned and denoted Bcap1
through 5. BcAP2 is a vacuolar AP, while BcAP1 and
BcAP5 are considered to be secreted enzymes. BcAP3 and
BcAP4 were predicted to remain attached to the plasma
membrane by means of a glycosylphosphatidylinositol
anchor. All were expressed in vitro as well as in planta (ten
Have et al. 2004). Scrutiny of the B. cinerea genome
revealed the presence of nine additional Bcap genes,
denoted Bcap6 through 14. Bcap8-deficient mutants
secreted 70–80 % less AP activity but were just as virulent
as the wild-type strain (ten Have et al. 2010).
Laccases
Laccases are in the family of blue multi-copper oxidases
that catalyze the monoelectronic oxidation of a broad
spectrum of substrates, e.g., ortho- and para-diphenols,
polyphenols, aminophenols, and aromatic or aliphatic
amines coupled with a full, four-electron reduction of O2 to
H2O (Riva 2006). Secondary metabolites from the Cucur-
bitaceae, known as cucurbitacins, protect cucumber fruits
and cabbage leaves from infection by B. cinerea (Bar-Nun
and Mayer 1990). Laccase activity in a B. cinerea culture
was repressed by cucurbitacins, while the activity of other
fungal enzymes was not affected (Viterbo et al. 1992),
leading to the hypothesis that laccases play an important
role in pathogenesis (Staples and Mayer 1995; Viterbo et al.
1992). Cucurbitacin led to lower induction of laccase
activity (Viterbo et al. 1994) as a consequence of repression
at the mRNA level (Gonen et al. 1996). Three laccase genes
from B. cinerea have been characterized, Bclcc1, Bclcc2
and Bclcc3. Only Bclcc2 was strongly expressed in liquid
cultures in the presence of either resveratrol or tannins. The
grapevine secondary metabolite resveratrol is considered a
phytoalexin that protects the plant from B. cinerea infec-
tion. Laccase activity displayed by the fungus is assumed to
detoxify resveratrol and to facilitate colonization of grape.
Bclcc2 replacement mutants were incapable of converting
either resveratrol or tannins. When grown on resveratrol,
both the wild type and the Bclcc1 replacement mutant show
inhibited growth, whereas Bclcc2 replacement mutants are
unaffected. BcLCC2 does not detoxify resveratrol, but
rather converts it into compounds that are more toxic to the
fungus (Schouten et al. 2002b).
Overcoming host defense responses
Botrytis cinerea infection induces biosynthesis of phyto-
alexins (Bennett et al. 1994; Kliebenstein et al. 2005;
Mansfield et al. 1980; van Baarlen et al. 2007) and accu-
mulation of pathogenesis-related proteins (Benito et al.
1998; Diaz et al. 2002; Ferrari et al. 2003). These defense
responses are presumably local, being restricted to the
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infection site(s). The total spectrum of defense responses
results in a primary necrotic lesion in which the fungus is
effectively restricted. A proportion of the primary lesions
eventually develop into aggressive, expanding lesions
(Benito et al. 1998). Arabidopsis thaliana is known to
produce the phytoalexin camalexin. Studies of A. thaliana
mutants affected in their capacity to produce camalexin
upon B. cinerea challenge revealed that camalexin plays a
role in resistance to the fungus. Camalexin treatment
induces fungal apoptotic-like PCD, and a transgenic strain
with enhanced anti-apoptotic capacity is less susceptible to
camalexin (Shlezinger et al. 2011a). In planta, camalexin
might thus induce fungal PCD, limiting the spread of
lesions during the early infection stage, while the fungal
anti-apoptotic machinery would allow the fungus to
recover and subsequently establish an infection (Shlezinger
et al. 2011a, b). Moreover, B. cinerea possesses ATP-
binding cassette (ABC) transporters that are involved in
limiting the sensitivity of the fungus to phytoalexins and
fungicides (Hayashi et al. 2002; Nakajima et al. 2001;
Schoonbeek et al. 2002; Vermeulen et al. 2001). Exposure
of this fungus to camalexin also induces the expression of
BcatrB, an ABC transporter that has an efflux function,
acting as a protective mechanism against the fungitoxic
effect of camalexin (Stefanato et al. 2009). We isolated an
avirulent monoascosporic strain of B. cinerea that had lost
pathogenicity in several host plants, including tomato.
Conidial germination, appressorium formation and infec-
tion hypha formation were investigated using onion epi-
dermis. Conidia of the avirulent strain germinated well, and
appressoria and infection hyphae were similar to those of
the wild type. The avirulent strain was arrested by a
hypersensitive-like necrosis of tomato leaf tissue. Because
ROS production is one of the earliest defense responses in
plant–pathogen interactions, we examined H2O2 accumu-
lation at the inoculation site using DAB staining. Micro-
scopic observation confirmed DAB staining in epidermal
cells penetrated by the avirulent strain. The growth rate of
the avirulent strain declined with increasing H2O2 con-
centrations compared with wild type (unpublished data).
Conclusion
Botrytis cinerea synthesizes a large number of enzymes
and metabolites that enable the pathogen to infect a broad
spectrum of host plants. Each of these compounds may
play a role in different stages of the infection process. In
addition, the ability to overcome the plant defenses con-
tributes to the pathogenicity of this fungus. Targeted
mutagenesis is a powerful research tool to study the role of
specific gene products. A number of genes have been
functionally analyzed by targeted-inactivation approaches
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J Gen Plant Pathol (2014) 80:15–23
in B. cinerea. However, the majority of the knock-out
mutants were not obviously altered in virulence because
there are functional overlaps between different types of
virulence factors and there is redundancy within a given
family of isozymes. In addition, the strain dependency of a
virulence factor has been reported. Deletion of a pectin
methylesterase gene (bcpme1) in strain Bd90 resulted in
reduced virulence (Valette-Collet et al. 2003), whereas a
deletion mutant of the same gene in strain B05.10 was fully
pathogenic (Kars et al. 2005b). The generation of multiple
knock-out mutants from different field isolates will provide
a better understanding of the complex role of the different
virulence factors during the infection process.
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