FEMS Microbiology Letters 213 (2002) 33^39
www.fems-microbiology.org
Engineering a genetic transformation system for
Colletotrichum acutatum, the causal fungus of lime anthracnose
and postbloom fruit drop of citrus
Kuang-Ren Chung , Turksen Shilts, Wei Li, L.W. Timmer
University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA
Received 10 May 2002; received in revised form 14 May 2002; accepted 16 May 2002
First published online 9 June 2002
Abstract
Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small
fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican
lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including
hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance
to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was
tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments
(0^17.9 per Wg circular DNA using 107 protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was
randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with
homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the
transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were
unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic
manipulation of C. acutatum. ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights
reserved.
Keywords : Hormone; Pathogen; Sulfonylurea ; Sweet orange
1. Introduction
Infection of citrus by Colletotrichum species can result in
Key lime anthracnose (KLA), postbloom fruit drop
(PFD), or postharvest anthracnose [1]. Both PFD and
KLA are caused by C. acutatum [2]. PFD and KLA isolates are nearly identical in morphology, but the two isolates can be di¡erentiated pathogenically and genetically
[3]. PFD isolates infect £ower petals of most citrus cultivars, inducing abscission of small fruit and resulting in
yield loss. PFD isolates are weakly pathogenic on the
leaves and fruit of lime. In contrast, KLA isolates cause
all symptoms of PFD on sweet orange £owers and also
cause anthracnose on Mexican lime.
* Corresponding author. Tel. : +1 (863) 956-1151 ext. 369;
Fax : +1 (863) 956-4631.
E-mail address : [email protected]£.edu (K.-R. Chung).
PFD of citrus was ¢rst reported in 1979 in Belize [4].
Since then, PFD has been reported in many citrus-growing
areas [5]. The fungus infects petals of open and unopen
£owers and results in brownish necrosis. PFD usually
causes drop of fruitlets, formation of persistent calyces
(known as buttons), and leaf distortion, suggesting that
phytohormones might be involved in symptom development [1]. PFD occurs endemically in high-rainfall areas
such as southern Mexico and Central America, and becomes a limiting factor for citrus production in many
areas [1,4]. PFD-a¡ected trees usually produce less fruit
and yield loss can be up to 100% without fungicide control.
The mechanisms by which C. acutatum infects citrus
petals resulting in young fruit drop are not known due
to a lack of understanding of speci¢c host/pathogen interactions and the lack of molecular tools available to characterize fungal pathogenicity or virulence factors. Thus, it
is important to develop a gene transfer system that will
0378-1097 / 02 / $22.00 ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 5 7 - 7
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K.-R. Chung et al. / FEMS Microbiology Letters 213 (2002) 33^39
provide an e⁄cient means for gene manipulation in the
species to elucidate these mechanisms and to conduct molecular genetic analyses such as plasmid insertional mutagenesis, restriction enzyme-mediated mutagenesis (REMI)
[6], and gene disruption. Although gene transfer systems
have been developed for many Colletotrichum species
[7^10], systems developed for other species are not suitable
for citrus isolates. Both KLA and PFD isolates of
C. acutatum are highly resistant to compounds such as
hygromycin, geneticin/G418, bialaphos, and benomyl
([11]; this study) that have been widely used for transformation selection in many other fungi [7^10,12,13].
Here we report the genetic transformation of citrus isolates of C. acutatum to sulfonylurea resistance by transferring plasmids that contain a sur gene cassette from
Magnaporthe grisea [14]. The sur gene encodes acetolactate
synthase involved in isoleucine and valine biosynthesis.
Sulfonylurea inhibits the activity of acetolactate synthase.
Overexpression of the sur gene in a fungus results in sulfonylurea resistance [14]. In this study we have identi¢ed
stable transformants from 11 isolates of C. acutatum. Molecular analyses of transforming DNA revealed that the
plasmid DNA was integrated successfully into the fungal
genome with multiple copies. The described protocol will
be useful for identi¢cation and analysis of fungal genes
required for pathogenicity or virulence in C. acutatum.
2. Materials and methods
2.1. Fungal isolates, cultural conditions and sensitivity tests
Isolates of C. acutatum for the DNA recipient hosts
were collected from infected Key lime leaves or sweet
orange petals from geographically di¡erent origins as indicated in Table 1. All isolates were cultured from surfacesterilized leaves or petals of infected plants and were subsequently single-spore isolated. An isolate of Colletotrichum gloeosporioides causing only postharvest anthracnose
of fruit was cultured from infected citrus fruit in Florida.
For protoplast preparation and inoculation experiments,
fungal cultures were maintained routinely on potato dextrose agar (PDA, Difco, Detroit, MI, USA). The stability
of transformants was assessed by transferring sulfonylurea-resistant transformants once a week on a non-selective medium for ¢ve sequential weeks, followed by transfer
back to the medium containing 50 Wg ml31 sulfonylurea.
The sensitivity of C. acutatum to various compounds
was determined to identify a positive marker for transformation selection. Approximately 104 per ml protoplasts
were regenerated at 28‡C in a regeneration medium [1 mg
ml31 Ca(NO3 )2 W4H20 , 0.2 mg ml31 KH2 PO4 , 0.25 mg ml31
MgSO4 W7H2 O, 0.15 mg ml31 NaCl, 1% glucose, and 1 M
sucrose] containing various concentrations of selection
agents as indicated in Table 2. Hygromycin (Roche
Applied Science, Indianapolis, IN, USA), geneticin/G418
(Sigma, St. Louis, MO, USA) and pestanal (glufosinate
ammonium, the active ingredient of bialaphos) (Fluka,
Milwaukee, WI, USA) were dissolved in water. Sulfonylurea (chlorimuron ethyl, the active ingredient of the herbicide Classicreg ) (Chem Service, West Chester, PA, USA)
was dissolved in dimethylformamide (DMF). Fungal colonies were scored after 7 days.
2.2. Nucleic acid manipulation
Plasmid vectors pCB1551 (chloramphenicol resistance)
and pCB1532 (ampicillin resistance) used for fungal transformation in this study were obtained from the Fungal
Genetic Stock Center (Kansas City, KS, USA) [14] (Fig.
1). Both plasmids carrying a sur cassette have been shown
to be able to overcome sulfonylurea toxicity, and have
been used for transformation selection in M. grisea [14].
The plasmid pCREC02 vector harboring a homologous
DNA fragment of C. acutatum was constructed as follows.
The DNA fragment (PKA) containing the regulatory subunit of protein kinase A was ampli¢ed by PCR using a
high-¢delity DNA polymerase system (Roche) from genomic DNA of C. acutatum isolate KLA 207 using degenerate primers [sense: 5P-gg(a/c/g/t) tac tt(c/t) ta(c/t) gt-3P
and antisense: 5P-cg(a/t) ag(c/t) tc(a/g) ccg aa(a/g/c/t)
ga-3P] and cloned into pGEM-T easy vector (Promega,
Table 1
Colletotrichum isolates used in this study and their transformation frequenciesa
Isolate
C. acutatum
Host species
Origin (country)
Transformants per Wg DNA
KLA 207
Homestead
MGG-1
M3
Orosi
ADS
KLA-2
IM-2CS
Navel
SM3
SRL-FTP
KLA
KLA
KLA
KLA
KLA
KLA
KLA
PFD
PFD
PFD
PFD
Key lime
Key lime
Key lime
Key lime
Key lime
Key lime
Key lime
Navel orange
Navel orange
Valencia orange
Valencia orange
Florida, USA
Florida, USA
Brazil
Mexico
Costa Rica
Belize
Dominican Republic
Florida, USA
Florida, USA
Florida, USA
Florida, USA
1.4^7.7
0.2^0.9
1.7^8.1
0^0.2
15.3^17.6
0.8^0.9
1.7^17.9
4.5
0^5.2
7.0
4.3
a
The numbers of transformants were obtained from at least three di¡erent transformation experiments except for isolates IM-2CS, SM-3 and SRL-FTP.
FEMSLE 10540 12-7-02
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35
gtc-3P). Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The conditions for PCR ampli¢cation and labeling were
performed as described previously [15]. Fungal DNA
was puri¢ed using a DNeasy1 Plant Mini kit (Qiagen,
Valencia, CA, USA). After enzyme digestion, DNA was
blotted to positively charged nylon membranes (Osmonics,
Westborough, MA, USA). The hybridization to the sur
gene probe was performed in aqueous solution at 65‡C.
Hybridization probe was detected immunochemically using CSPD1 ready-to-use chemiluminescent substrate
(Roche) as recommended by the manufacturer.
Fig. 1. Maps of the constructs used for transformation of C. acutatum.
Plasmids pCB1532 and pCB1551 containing the acetolactate synthase
gene (sur) cassette from M. grisea conferred resistance to chlorimuron
ethyl (a sulfonylurea herbicide) [14] and were used for transformation
selection in C. acutatum. Plasmid pCREC02 was constructed by fusing
a DNA fragment of the regulatory subunit of protein kinase A (PKA)
from C. acutatum into pCB1532 at the EcoRI site. The position of the
sur gene promoter and its direction for expression is indicated by the arrow. The ampicillin (ampr ), chloramphenicol (cm) resistance genes, and
plasmid origin (colE1) and f1 phage origin of replication are also indicated.
2.3. Preparation of protoplasts
For protoplast isolation, fungal isolates were grown in
50 ml of potato dextrose broth (PDB) for 5 days, ground
with a sterile blender, then mixed with 200 ml fresh PDB,
and grown for an additional 18 h at 28‡C. The young
hyphae were harvested using low-speed centrifugation
(5000Ug for 10 min), washed twice with solution containing 1 M NaCl and 10 mM CaCl2 . The resulting fungal
tissues were re-suspended in osmotic bu¡er (10 mM
Na2 HPO4 , pH 5.8, 20 mM CaCl2 , 1.2 M NaCl) containing
a mixture of cell wall-degrading enzymes [8 mg ml31
L-D-glucanase (InterSpex, San Mateo, CA, USA), 5 mg
ml31 driselase (InterSpex), 1050 U ml31 L-glucuronidase
type H2 (Sigma), 81.25 U ml31 lyticase (Sigma)]. To prepare the enzyme solution, driselase was ¢rst dissolved in
the osmotic bu¡er, and the solution was centrifuged at
65 000Ug for 10 min to remove starch carriers [16], then
other enzymes were added at the indicated amounts, and
sterilized by ¢ltration. Each gram of fungal tissue was
mixed with 20 ml of enzyme solution, and incubated at
Madison, WI, USA) to produce pCREC01. The 2.0-kb
EcoRI fragment of pCREC01 was subcloned into
pCB1532 producing pCREC02. Plasmid DNA was manipulated in competent Escherichia coli DH5K cells (Invitrogen, Gaithersburg, MD, USA) and puri¢ed using a Wizard DNA kit (Promega). Standard procedures were used
for endonuclease digestion of DNA, electrophoresis, and
Southern blotting. The hybridization probe for the sur
gene was labeled with digoxigenin-11-dUTP (Roche) by
PCR using Taq DNA polymerase and primers (sense
5P-gtatgcacggttcggcttat-3P; antisense 5P-acaagttctgccattgg-
Table 2
Sensitivity of C. acutatum KLA 207 isolate protoplasts to antibiotics and compounds that are commonly used for fungal transformation
Concentration (Wg ml31 )
0
0.1% DMFa
2
4
6
8
10
15
20
25
50
100
150
200
300
400
Hygromycin
Pestanal (bialaphos)
Geneticin/G418
Sulfonylurea
7 days
7 days
7 days
7 days
14 days
21 days
+
+
nd
nd
nd
nd
nd
nd
nd
nd
nd
+
+
+
+
+
+
+
nd
nd
nd
nd
nd
nd
nd
nd
nd
+
+
+
+
+
+
+
nd
nd
nd
nd
+
nd
nd
nd
+
+
nd
+
+
nd
+
+
3
3
3
3
3
3
3
3
+
+
+
+
3
3
3
3
3
3
nd
nd
nd
nd
nd
nd
+
+
+
+
+
+
+
+
3
3
+ indicates regeneration of fungal protoplasts; 3 indicates no fungal growth. nd, not done.
Sulfonylurea was dissolved in dimethylformamide (DMF), other chemicals were dissolved in water, sterilized by ¢ltration, and added to medium at appropriate concentrations. Each experiment was conducted at least twice using di¡erent batches of protoplasts.
a
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30‡C with gentle shaking (100 rpm). After a 2-h digestion,
the solution was passed through cheesecloth and glass
wool in a 50-ml syringe. Fungal protoplasts were harvested by centrifugation at 65 000Ug for 10 min, and
washed with STC bu¡er (1.2 M sorbitol, 10 mM Tris^
HCl, pH 7.5, 10 mM CaCl2 ). The protoplasts were dissolved in four parts of STC and one part 50% PEG (polyethylene glycol 3350, 10 mM Tris^HCl, pH 7.5, 10 mM
CaCl2 ), dispensed into a small volume, and stored at
380‡C.
2.4. Fungal transformation
Fungal transformation was performed as described
[13,17] with modi¢cations. Approximately 1U107 protoplasts in 100 Wl STC/PEG solution were mixed gently
with 10^20 Wg DNA of pCB1551, pCB1532, or pCREC02
(Fig. 1), and placed on ice for 30 min. For REMI transformation, 20 U EcoRI or BamHI was mixed with
pCB1532 and added to the transformation reaction. The
mixture was then mixed with 1 ml of 50% PEG solution,
incubated at room temperature for 30 min, and mixed
with 3 ml of liquid regeneration medium. After incubation
at 28‡C for 2 h with gentle shaking, protoplasts were
plated on regeneration agar medium with 10 Wg ml31 sulfonylurea. The colonies of transformants that appeared
between 5 and 7 days were selected and transferred to
PDA containing 50 Wg ml31 sulfonylurea.
2.5. Pathogenicity tests
C. acutatum KLA 207 isolate and its derived transformants were inoculated onto immature detached Key lime
leaves to assess fungal pathogenicity. Fungal cultures
were grown on PDA under continuous £uorescent light
for 5 days, and conidia were washed o¡ with sterile water.
After washing once with water, conidia were harvested by
centrifugation at low speed, and the concentration was
adjusted to 5U104 conidia per ml. 10 Wl of conidial suspension with at least four replicates for each isolate was
placed onto the undersides of detached leaves (5^7 days
after £ushing and approximately 5 cm in length). The inoculated leaves were kept in a moist chamber under £uorescent light at room temperature. Necrotic lesions usually
appeared in 7^10 days. An untransformed KLA 207 isolate and C. gloeosporioides were used as a positive and
negative control, respectively.
3. Results and discussion
3.1. Sensitivity of C. acutatum to various selection
compounds
A transformation system requires a selective compound
that can be e¡ectively used for di¡erentiation of trans-
FEMSLE 10540 12-7-02
Fig. 2. Southern blot analysis of genomic DNA from the untransformed
KLA 207 isolate (lane 1) and the pCB1532-transformed isolates (lanes
2^11) of C. acutatum. Fungal DNA was puri¢ed, digested with EcoRI
and EcoRV, electrophoresed, blotted onto a nylon membrane, hybridized to a PCR-generated sur probe, and washed at high stringency.
C. acutatum contains a cross-hybridizing band (V13 kb in size, indicated by arrow) to the sur gene probe of M. grisea. The isolate in lane
11 containing a similar hybridizing band as that of wild-type was sensitive to sulfonylurea after serial transfer on the non-selective medium.
The position of the 5.2-kb fragment of pCB1532 is indicated by the
arrowhead. Size markers were obtained from bacteriophage V DNA
digested with HindIII, and their sizes are indicated in kb.
formed and untransformed isolates. To identify useful
markers for the selection of transformants, we ¢rst determined the sensitivity of C. acutatum to antibiotics and
compounds commonly used for other fungal species. Recently, the availability of Novozym 234 commonly used
for the isolation of protoplasts for many fungi has become
problematic. In this study, we have used combinations of
available enzymes including L-D-glucanase, L-glucuronidase, lyticase, and driselase to produce protoplasts from
C. acutatum. In general, over 108 protoplasts were obtained easily from 250 ml of germinating mycelia. Of
those, V80% could regenerate on an osmotic medium,
indicating that the quality of protoplasts was suitable for
transformation. Protoplasts from isolates KLA 207 and
Orosi (KLA isolate), Navel and Pocora-6 (PFD isolates)
were regenerated in media containing various concentrations of antibiotics or herbicides as indicated in Table 2.
The results indicated that protoplasts from both KLA and
PFD isolates displayed insensitivity to high concentrations
of hygromycin (up to 400 Wg ml31 ), pestanal (up to 400 Wg
ml31 ), or geneticin (up to 300 Wg ml31 ). Resistance of
fungal protoplasts to these antibiotics or compounds precludes their use as a selective agent in C. acutatum. Further testing of protoplast regeneration in a medium containing sulfonylurea revealed that C. acutatum was highly
sensitive to sulfonylurea at concentrations as low as 2 Wg
ml31 (Table 2). Both KLA and PFD isolates exhibited a
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K.-R. Chung et al. / FEMS Microbiology Letters 213 (2002) 33^39
similar level of sensitivity to sulfonylurea (data not
shown). However, many small colonies appeared after
prolonged incubation, suggesting that spontaneous resistance to sulfonylurea occurred, likely due to the presence
of a sur gene homologue in C. acutatum. We therefore
tested whether or not sulfonylurea could serve as a useful
marker for transformation selection in C. acutatum.
37
were also tested in 10 other KLA and PFD isolates. The
transformation frequencies varied greatly among di¡erent
isolates regardless of their geographical origins (Table 1).
Among them, KLA isolates Orosi, KLA-2, MGG-1, and
four PFD isolates were easily transformed using the plasmid pCB1551 or pCB1532. Three KLA isolates (Homestead, M3 and ADS) were transformed only at low frequencies.
3.2. Transformation of C. acutatum using sulfonylurea as a
selection compound
3.3. Analyses of transformants
Both plasmid vectors pCB1551 and pCB1532 carrying
the M. grisea gene encoding acetolactate synthase were
used for transformation. Plasmid DNA was transformed
into protoplasts of C. acutatum isolate KLA 207 using the
PEG/CaCl2 method, and fungal transformants were selected initially in a medium containing 10 Wg ml31 sulfonylurea. Compared to the no DNA control, putatively
transformed and resistant colonies to sulfonylurea appeared in 5^7 days. The transformants were transferred
onto media amended with 50 Wg ml31 sulfonylurea. Only
colonies that grew at a high dosage of sulfonylurea were
considered transformants. Using plasmid pCB1551, the
transformation frequency in isolate KLA 207 was about
1.4^7.7 transformants per Wg DNA using 107 protoplasts.
The transformation frequency (10^12.3 transformants per
Wg circular DNA) was increased slightly using a higher
concentration (5U107 ) of protoplasts in the transformation reaction. There were no signi¢cant di¡erences between the transformation frequencies using plasmid
pCB1551 or pCB1532 (data not shown).
Transformations using sulfonylurea as a selective agent
Southern blot analysis using a sur gene probe was used
to examine the integrity and copy numbers of plasmid
DNA in the transformants (Fig. 2). The results indicated
that the untransformed KLA isolate contained a crosshybridizing, high-molecular band of size 13 kb (lane 1,
indicated by arrow) to the sur probe, suggesting that
C. acutatum harbors a sur gene homologue. In contrast,
identi¢cation of di¡erent banding patterns of nine transformants (lanes 2^10) indicated that integration had
occurred at di¡erent sites in the C. acutatum genome.
All transformants contained a common 5.2-kb band (indicated by arrowhead). Since there are EcoRI and EcoRV
sites in pCB1532, each insertion should result in a 5.2-kb
band if no rearrangement has occurred. In all cases, many
hybridizing bands larger or smaller than 5.2 kb were also
identi¢ed, strongly suggesting that gene rearrangement or
deletion had occurred at some integration sites. Multiple
integrations of the plasmid after transformation were also
observed in the other KLA and PFD isolates when two
transformants from each isolate were examined (data not
shown).
Fig. 3. Southern blot analyses of C. acutatum transformants after transformation using a prelinearized pCB1532 with additional 20 U EcoRI (lanes 1^
4), or BamHI (lanes 5^7) (A), or after transformation using the pCREC02 construct containing a homologous fragment of protein kinase A subunit
(B). Fungal genomic DNA was puri¢ed, digested with EcoRI and EcoRV, electrophoresed, blotted onto a nylon membrane, hybridized to a sur gene
probe, and washed at high stringency. The positions of molecular mass markers and sizes in kb are indicated.
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K.-R. Chung et al. / FEMS Microbiology Letters 213 (2002) 33^39
Fig. 4. Inoculation of C. acutatum (C. a.) wild-type KLA 207 and its transforming derivatives onto the detached Key lime leaves. C. gloeosporioides
(C. g.), a non-pathogen of Key lime was used as a negative control. Conidial inoculum (5U104 conidia per ml) was prepared from 5-day-old cultures.
10 Wl of conidial suspension with at least four replicates from each transformant was placed onto the undersides of the detached Key lime leaves. The
inoculated leaves were kept in a moist chamber and incubated under £uorescent light at room temperature. Disease symptoms with necrotic lesions appeared in 7^10 days. Some transformants completely or partially lost their virulence on the detached Key lime leaves (for example #9).
Stability tests among 40 randomly selected transformants from isolate KLA 207 revealed that one isolate was
unable to re-grow on sulfonylurea-containing medium
after subculturing on the non-selective condition for
5 weeks. Loss of the plasmid in the isolate was con¢rmed
by Southern blot analysis since the only high-molecular
band of size 13 kb identi¢ed was that of the untransformed parent (Fig. 2, lanes 1 and 11). The results indicated that the majority of transformants expressing the sur
gene were mitotically stable for sulfonylurea resistance.
In other fungal transformations, restriction enzymes can
increase transformation frequency and promote singlecopy integration events [6,18,19]. Transformants obtained
from the transformation experiments using prelinearized
plasmid pCB1532, or with addition of EcoRI, BamHI
did not have a signi¢cant e¡ect on transformation frequency (data not shown). Addition of enzymes, however,
resulted in fewer copies of the plasmid as assessed by
Southern blot analysis (Fig. 3A), suggesting that the re-
FEMSLE 10540 12-7-02
striction enzyme tended to reduce the number of integrations in C. acutatum. The e¡ect of homologous sequences
on chromosomal integration was also determined. Southern blot analysis of genomic DNA from pCREC02 transformants identi¢ed a uniform pattern of hybridizing bands
among transformants (Fig. 3B), suggesting that integration was predominately at homologous sites. These observations raise the possibility that the transformation system
will facilitate screening for mutants derived from insertional mutagenesis and will be useful for molecular analyses of
fungal pathogenicity.
Transformants derived from isolate KLA 207 were inoculated onto host plant leaves to test whether the transformation and plasmid integration had caused changes in
fungal pathogenicity. Using the detached Key lime leaves,
necrotic lesions could be observed in 7^10 days after inoculation with the wild-type isolate (Fig. 4). No lesion was
produced following inoculation with C. gloeosporioides.
The wild-type KLA 207 isolate caused obvious brownish
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K.-R. Chung et al. / FEMS Microbiology Letters 213 (2002) 33^39
necrosis (a typical symptom of lime anthracnose) on the
detached Key lime leaves. All transformants produced
conidia normally in culture. Over 300 KLA-derived transformants from REMI and non-REMI were tested, and
most of them caused necrotic lesions similar to those of
the wild-type isolate. Three transformants (one from nonREMI and two from REMI experiments) apparently lost
or had reduced virulence on the detached Key lime leaves
(Fig. 4). The non-pathogenic phenotype of transformants
was tested at least three times. Whether or not these transformants were non-pathogenic mutants resulting from
plasmid integration remains to be determined.
In conclusion, this paper describes the ¢rst successful
and reproducible transformation system for the PFD
pathogen C. acutatum of citrus. We have streamlined the
process by which sulfonylurea was utilized as a selective
marker for identifying stable transformants from 11 C.
acutatum isolates. The transformation procedure developed in this study is simple and straightforward, and
will be very useful for conducting genetic and molecular
analyses of fungal pathogenicity or virulence in C. acutatum.
Acknowledgements
The authors are indebted to Drs. K.S. Derrick and J.W.
Grosser for their critical review of the manuscript. This
research was supported by the Florida Citrus Production
Research Advisory Council (FCPRAC) Grant 012-04P,
and approved for publication as Journal Series No.
R-08805.
References
[1] Timmer, L.W. and Brown, G.E. (2000) Biology and control of anthracnose diseases of citrus. In: Colletotrichum : Host Speci¢city,
Pathology, and Host-Pathogen Interaction (Prusky, D., Freeman,
S. and Dickman, M.B., Eds.), pp. 300^316. APS Press, St. Paul, MN.
[2] Brown, A.E., Sreenivasaprasad, S. and Timmer, L.W. (1996) Molecular characterization of slow-growing orange and Key lime anthracnose strains of Colletotrichum from citrus as C. acutatum. Phytopathology 86, 523^527.
[3] Agostini, J.P., Timmer, L.W. and Mitchell, D.J. (1992) Morphological and pathological characteristics of strains of Colletotrichum
gloeosporioides from citrus. Phytopathology 82, 1377^1382.
FEMSLE 10540 12-7-02
39
[4] Fagan, H.J. (1979) Postbloom fruit drop, a new disease of citrus
associated with a form of Colletotrichum gloeosporioides. Ann.
Appl. Biol. 91, 13^20.
[5] Timmer, L.W., Agostini, J.P., Zitko, S.E. and Zul¢qar, M. (1994)
Postbloom fruit drop, an increasingly prevalent disease of citrus in
the America’s. Plant Dis. 78, 329^334.
[6] Maier, F.J. and Scha«fer, W. (1999) Mutagenesis via insertion- or
restriction enzyme-mediated-integration (REMI) as a tool to tag
pathogenicity related genes in plant pathogenic fungi. Biol. Chem.
380, 855^864.
[7] Panaccione, D.G., McKiernan, M. and Hanau, R.M. (1988) Colletotrichum graminicola transformed with homologous and heterologous
benomyl-resistance genes retains expected pathogenicity to corn. Mol.
Plant-Microbe Interact. 3, 113^120.
[8] Dickman, M.B. (1988) Whole cell transformation of the alfalfa fungal
pathogen Colletotrichum trifolii. Curr. Genet. 14, 241^246.
[9] Redman, R.S. and Rodriguez, R.J. (1994) Factors which a¡ect e⁄cient transformation of Colletotrichum species. Exp. Mycol. 18, 230^
246.
[10] Robinson, M. and Sharon, A. (1999) Transformation of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene by electroporation of germinated conidia. Curr. Genet. 36, 98^104.
[11] Sonoda, R.M. and Pelosi, R.R. (1988) Outbreak of citrus postbloom
fruit drop caused by Colletotrichum gloeosporioides from lesions on
citrus blooms in the Indian River of Florida. Proc. Florida State
Hortic. Soc. 101, 36^38.
[12] Churchill, A.C.L., Ciu¡etti, L.M., Hansen, D.R., Van Etten, H.D.
and Van Alfen, N.K. (1990) Transformation of the fungal pathogen
Cryphonectria parasitica with a variety of heterologous plasmids.
Curr. Genet. 17, 25^31.
[13] Ehrenshaft, M., Jenns, A.E. and Daub, M.E. (1995) Targeted gene
disruption of carotenoid biosynthesis in Cercospora nicotianae reveals
no role for carotenoids in photosensitizer resistance. Mol. Plant-Microbe Interact. 8, 569^575.
[14] Sweigard, J.A., Chumley, F.C., Carroll, A.M., Farrall, L. and Valent,
B. (1997) A series of vectors for fungal transformation. Fungal
Genet. Newsl. 44, 52^53.
[15] Chung, K.R., Leuchtmann, A. and Schardl, C.L. (1996) Inheritance
of mitochondrial DNA and plasmids in the ascomycetous fungus,
Epichloe« typhina. Genetics 142, 259^265.
[16] Oakley, B.R., Rinehart, J.E., Mitchell, B.L., Oakley, C.E., Carmona,
C., Gray, G.L. and May, G.S. (1987) Cloning, mapping and molecular analysis of the pyrG (orotidine-5P-phosphate decarboxylase) gene
of Aspergillus nidulans. Gene 61, 385^399.
[17] Ehrenshaft, M., Jenns, A.E., Chung, K.R. and Daub, M.E. (1998)
SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms.
Mol. Cell 1, 603^609.
[18] Brown, J.S. and Holden, D.W. (1998) Insertion mutagenesis of
pathogenic fungi. Curr. Opin. Microbiol. 1, 390^394.
[19] Riggle, P.J. and Kumamoto, C.A. (1998) Genetic analysis in fungi
using restriction-enzyme-mediated integration. Curr. Opin. Microbiol. 1, 395^399.
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