Mol Biotechnol (2011) 49:48–55
DOI 10.1007/s12033-011-9377-7
RESEARCH
A Species-Specific Polymerase Chain Reaction Assay for Rapid
and Sensitive Detection of Colletotrichum capsici
C. Torres-Calzada • R. Tapia-Tussell • A. Quijano-Ramayo
R. Martin-Mex • R. Rojas-Herrera • I. Higuera-Ciapara •
D. Perez-Brito
Published online: 21 January 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Colletotrichum capsici is an important fungal
species that causes anthracnose in many genera of plants
causing severe economic losses worldwide. A primer set
was designed based on the sequences of the ribosomal
internal transcribed spacer (ITS1 and ITS2) regions for use
in a conventional PCR assay. The primer set (CcapF/
CcapR) amplified a single product of 394 bp with DNA
extracted from 20 Mexican isolates of C. capsici. The
specificity of primers was confirmed by the absence of
amplified product with DNA of four other Colletotrichum
species and eleven different fungal genera. This primer set
is capable of amplifying only C. capsici from different
contaminated tissues or fungal structures, thereby facili-
tating rapid diagnoses as there is no need to isolate and
cultivate the fungus in order to identify it. The sensitivity
of detection with this PCR method was 10 pg of genomic
DNA from the pathogen. This is the first report of a
C. capsici-specific primer set. It allows rapid pathogen
detection and provides growers with a powerful tool for a
rational selection of fungicides to control anthracnose in
different crops and in the post-harvest stage.
C. Torres-Calzada  R. Tapia-Tussell  A. Quijano-Ramayo 
R. Martin-Mex  D. Perez-Brito (&)
Laboratorio GeMBio, Centro de Investigación Cientı́fica de
Yucatán A.C, Calle 43 # 130, Col. Chuburná de Hidalgo,
Mérida, Yucatán 97200, México
e-mail: daisypb@cicy.mx
I. Higuera-Ciapara
Centro de Investigación Cientı́fica de Yucatán A.C, Calle 43 #
130, Col. Chuburná de Hidalgo, Mérida, Yucatán 97200, México
R. Rojas-Herrera
Facultad de Ingenierı́a Quı́mica, UADY, Campus de Ingenierı́as
y Ciencias Exactas, Periférico Norte km 33.5, Tablaje Catastral
13615, Col. Chuburná de Hidalgo Inn C. P, 97203 Mérida,
Yucatán, México
123
•
Keywords Colletorichum capsici  Diagnostics  ITS
sequences  Anthracnose  Molecular detection
Introduction
Colletotrichum species are among the most important plant
pathogens worldwide. They cause significant economic
damage to crops in tropical, subtropical, and temperate
regions [1]. Cereals, legumes, ornamentals, vegetables, and
fruit trees may be seriously affected by these pathogens [2].
Colletotrichum capsici is an important anamorphic species
that causes anthracnose in 121-host genera from 45 plant
families [3]. It is known to cause anthracnose in pepper
(Capsicum spp.) [4–6], and has been associated with
anthracnose in papaya (Carica papaya) in Mexico and
USA [7, 8].
Traditionally, identification and classification of Collet-
otrichum species have been based on the morphological
characters, such as conidial shape and size, pathogenicity
tests, and biochemical approaches [9, 10]. Although valu-
able, detection of pathogens by these methods is laborious,
time-consuming, and requires extensive knowledge of fun-
gal taxonomy.
Molecular biology techniques have proven to be effec-
tive for identification [11–13] and characterization [14–16]
of Colletotrichum species. The nuclear rDNA region,
b-tubulin gene, and an ascomycete mating-type gene are
the most common DNA sequences used for these purposes
as their characteristics allow the identification of pathogens
at species level. The ITS region is the most widely
sequenced region and has been used to design species-
specific primers to unequivocally detect the presence of the
species of interest, as reported for some fungal species
Mol Biotechnol (2011) 49:48–55
[17–20] as well as Colletotrichum acutatum, C. gloeospo-
rioides, and C. coccodes [21, 22]. In this study, species-
specific primers were designed with the aim of developing
a reliable, rapid, and sensitive method to specifically detect
C. capsici.
Materials and Methods
Fungal Isolation
Colletotrichum isolates were obtained from lesions on leaf,
petiole, and fruit from diseased plants sampled from dif-
ferent locations in the Yucatan Peninsula and Chiapas
State, in Mexico. Reference isolates Alternaria sp., Athelia
rolfsii, Bipolaris sp., Cochliobolus lunatus, Corynespora
cassiicola, Fusarium oxysporum, Phanerochaete chrysos-
porium, Rhyzoctonia sp., and Trametes hirsuta were pre-
viously identified and characterized in our laboratory by
ITS nucleotide sequence analysis, other reference on
Colletotrichum spp. isolates were obtained from ATCC and
CBS Fungal Collection (Table 1). Pure cultures were
obtained by single spore isolation and maintained on
Richard’s V8 (RV8) plates, subjected to natural illumina-
tion at 28°C for 7 days, before observation of cultural and
morphological characteristics [23].
Genomic DNA Extraction
For genomic DNA extraction, 10 pieces of agar culture
(1 9 1 cm) obtained from the 7-day-old colonies grown on
RV8 were transferred into 250-ml Erlenmeyer flasks con-
taining 50 ml of nutrient broth (NB). After incubation at
28°C on an orbital shaker (100 rpm) for 7 days, the
mycelia were collected, frozen at -80°C for 2 h and
lyophilized (LABCONCO 77530) until use.
The total genomic DNA was extracted from mycelia
according to the method described previously [24] and
diluted to a final concentration of 50 ng/ll.
PCR Amplification
Colletotrichum isolates were further characterized by
nucleotide sequence analysis. The 5.8-ITS regions were
amplified with the universal primers ITS1 and ITS4 [25].
PCR reactions were performed in reaction volumes of
25 ll containing 100 ng of genomic DNA, 19 PCR buffer
(109 : 20 mM Tris–HCl, 500 mM KCl, pH 8.4; Promega),
0.20 mM of each dNTP (Invitrogen), 2.5 mM MgCl2
(Promega), 1 lM of each primer, and 1.25 U GoTaqÒ Flexi
DNA polymerase (Promega). DNA amplification was
performed in a GeneAmp 9700 DNA Thermal Cycles
49
(Perkin-Elmer) and the program consisted of an initial
denaturing step at 95°C for 1 min, followed by 30 cycles of
1 min at 94°C, 1 min at 58°C and 1 min at 72°C, and a
final extension step of 7 min at 72°C. PCR products were
separated by electrophoresis in 1.5% (w/v) agarose gels
and visualized by ethidium bromide staining. PCR products
were purified and sequenced by Macrogen Inc. Korea.
Alignment and edition were carried out with the BioEdit
Sequence Alignment program [26] and visually corrected.
Sequences were then compared with those available in the
GenBank database.
Design of C. capsici-Specific PCR Primers
In order to design specific primers for the detection of
C. capsici, the internal transcribed spacer regions (ITS1
and ITS2) of the rDNA gene from 17 Colletotrichum
species were obtained from the GenBank database
(C. acutatum, C. ampelinum, C. boninense, C. capsici,
C. cereale, C. dematium, C. destructivum, C. fragariae,
C. fuscum, C. gloeosporioides, C. graminicola, C. linicola,
C. lupini, C. musae, C. ricini, C. sublineolum, and
C. truncatum). A multiple sequence alignment was made
for these sequences using the BioEdit Sequence Alignment
program [26]. The alignment was analyzed for divergences
among the sequences. These divergences led to the
development of primers that specifically amplified
C. capsici ITS region using the Primer3 v. 0.4.0 software
[27]. A specific primer pair CcapF (50 -GTA GGC GTC
CCC TAA AAA GG-30 ) and CcapR (50 -CCC AAT GCG
AGA CGA AAT G-30 ) was designed for the specific
detection of C. capsici among the other pathogens, and the
expected amplicon size for the PCR primers was 394 bp.
The specificity of the designed primer sequences were
confirmed before synthesis following BLAST database
searches of DNA sequences.
Primers CcapF/CcapR Amplification
The primers CcapF/CcapR were used for rDNA amplifi-
cation of samples. The PCR reaction contained 50 ng of
DNA, 19 PCR buffer (109: 20 mM Tris–HCl, 500 mM
KCl, pH 8.4; Promega), 0.20 mM of each dNTP (Invitro-
gen), 2.5 mM MgCl2 (Promega), 1 lM Primers, and 1.25
U Go Taq Flexi DNA polymerase (Promega) at volume of
25 ll. DNA amplification was performed in a GeneAmp
9700 thermal cycler (Perkin-Elmer) and consisted of an
initial denaturing step at 95°C for 5 min, followed by 25
cycles of 30 s at 94°C, 30 s at 62°C, and 2 min at 72°C,
and a final extension step of 5 min at 72°C. PCR products
were visualized by electrophoresis in 1.5% (w/v) agarose
gel run in 19 Tris–Borate-EDTA [TBE] buffer and stained
with ethidium bromide.
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50 Mol Biotechnol (2011) 49:48–55

Table 1 Isolates of different fungi used to screen the primers specificity

Species Accession number Host Origin PCR amplification
ITS1/ITS4 CcapF/CcapR

C. capsici ATCC 48574 Capsicum annuum 1 1

C. capsici HM450127.1 Tithonia rotundifolia Chocholá, Yucatán 1 1
C. capsici HM450126.1 Jatropha curcas Muna, Yucatán 1 1
C. capsici HM450128.1 Carica papaya José M. M., Q. Roo 1 1
C. capsici HM450129.1 Carica papaya José M. M., Q. Roo 1 1
C. capsici HM450130.1 Carica papaya José M. M., Q. Roo 1 1
C. capsici HM450131.1 Capsicum annuum Peto, Yucatán 1 1
C. capsici HM450132.1 Capsicum annuum Ticul, Yucatán 1 1
C. capsici HM562705.1 Citrus limonium Tizimı́n, Yucatán 1 1
C. capsici HM562706.1 Capsicum annuum Tekax, Yucatán 1 1
C. capsici HM562707.1 Capsicum annuum Tekax, Yucatán 1 1
C. capsici HM562708.1 Jatropha curcas Umán, Yucatán 1 1
C. capsici HM562709.1 Carica papaya Valladolid, Yucatán 1 1
C. capsici HM015005.1 Carica papaya Cacalchen, Yucatán 1 1
C. capsici – Carica papaya Cacalchén, Yucatán 1 1
C. capsici – Carica papaya Campeche, Campeche 1 1
C. capsici – Carica papaya Chiapas 1 1
C. capsici – Carica papaya Chiapas 1 1
C. capsici – Carica papaya José M. M., Q. Roo 1 1
C. capsici – Carica papaya José M. M., Q. Roo 1 1
C. capsici – Hylocereus undatus Tetiz, Yucatán 1 1
C. gloeosporioides HM562710.1 Carica papaya José M. M., Q. Roo 1 –
C. gloeosporioides HM562711.1 Carica papaya José M. M., Q. Roo 1 –
C. gloeosporioides HM562712.1 Mangifera indica Merida, Yucatán 1 –
C. gloeosporioides HM562713.1 Persea americana Merida, Yucatán 1 –
C. dematium ATCC 200634 Rubus strigosus 1 –
C. lindemuthianum CBS 131.57 Phaseolus vulgaris 1 –
C. graminicola CBS 113173 Zea mays 1 –
Corynespora cassiicola GU461301 Carica papaya 1 –
C. cassiicola GU461299 Carica papaya 1 –
Trametes hirsuta GQ280373 Wood wastes 1 –
Phanerochaete chrysosporium GQ280374 Wood wastes 1 –
Cochliobolus lunatus GQ280375 Agave fourcroydes 1 –
Bipolaris sp GQ280376 Agave fourcroydes 1 –
Athelia rolfsii – Agave fourcroydes 1 –
Alternaria sp – Tagetes erecta 1 –
Rhizoctonia solani – Solanum tuberosum 1 –
Fusarium oxysporum – Zea mays 1 –

Specificity of Primers and Sensitivity of PCR
The specificity of the primers was tested against genomic
DNA from 21 C. capsici isolates, other Colletotrichum
spp. (C. gloeosporioides, C. dematium, C. lindemuthianum,
and C. graminicola), and isolates from different fungal
species including ascomycetes, basidiomycetes, and
deuteromycetes (Table 1). To exclude false negative
results with C. capsici-specific primers, all template
DNA samples were tested for PCR amplification using
universal primers ITS1/ITS4 for fungal 18S and 28S
rDNA [25].
PCR sensitivity was tested to detect C. capsici using
DNA templates at decimally diluted concentrations from
100 ng/ll to 10 fg/ll. PCR reactions were performed as
previously described with C. capsici-specific primers.

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Mol Biotechnol (2011) 49:48–55 51

Fig. 1 DNA sequence comparison of the internal transcribed spacer (ITS) region between C. capsici (HM015005 and EU056740) and
C. gloeosporioides (HM562710). The location of the species-specific primer sequences are delimited in boxes

Detection of C. capsici from Naturally Infected Papaya Results

DNA was extracted from leaves, petioles, and fruits of nine
infected papaya plants showing characteristic symptoms of
early anthracnose. About 50 mg of fresh tissue from
infected lesion was processed according to the method
described previously [28].
In order to detect C. capsici, PCR reactions were
carried out with the C. capsici-specific primers developed
in this study and under the reaction conditions described
for this primer set. DNA from pure culture of C. capsici
ATCC 48574 was used as a positive control, whereas
tissues of healthy papaya fruits were used as negative
control.
Sequence Variation in the ITS Region and C. capsici-
Specific Primers Design
The primers ITS1/ITS4 were used to amplify the region of
partial 18S rDNA, the ITS1, 5.8S, ITS2, and partial 28S
rDNA. The size of PCR products for all Colletotrichum
spp. isolates was 590 bp. After alignment of the ITS
sequences with other previously characterized isolates of
Colletotrichum spp., representing 17 species, one pair of
primers (CcapF/CcapR) was designed to sequence within
the ITS1/ITS2 regions of C. capsici isolates (Fig. 1).
Comparisons between primer sequences CcapF and CcapR

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Fig. 2 Specificity of PCR with

the primer pair CcapF/CcapR
for detection of C. capsici. Lane
M, DNA marker (1 kb DNA
ladder); lanes 1 and 19,
C. capsici ATCC 48574; lanes
2–17 and 20–24, C. capsici
isolates, lanes 25–28
C. gloeosporioides isolates, lane
29, C. dematium ATCC 200634;
lane 30, C. lindemuthianum
CBS 131.57; lane 31,
C. graminicola CBS 113173;
lanes 32–33, C. cassiicola; lanes
34–41, Trametes hirsuta,
Phanerochaete chrysosporium,
Cochliobolus lunatus, Athelia
rolfsii, Bipolaris sp., Alternaria
sp., Rhyzoctonia solani,
Fusarium oxysporum; lanes 18
and 42, negative control

the CcapF/CcapR primers. The expected specific band of

394 bp was detected (Fig. 5). This PCR method was suc-
cessful in amplifying PCR products from all diseased plant
tissues (leaves, fruits, and petioles).

Discussion

The objective of this study was to develop a sensitive and

Fig. 3 PCR sensitivity assay using the primer pairs CcapF/CcapR.
Lane M, DNA marker (100 bp DNA ladder); lanes 1–8, amplified
products using genomic DNA at concentration of 100 ng/ll, 10 ng/ll,
1 ng/ll, 100 pg/ll, 10 pg/ll, 1 pg/ll, 100 fg/ll, 10 fg/ll; lane 9,
negative control without DNA template
with DNA database sequences (GenBank) showed 100% of
identity with the sequences of C. capsici.
Specificity and Sensitivity of the CcapF/CcapR Primers
All isolates tested had a positive PCR reaction using
the ITS universal primers (Table 1). The specificity of the
primers CcapF/CcapR was demonstrated when only
the C. capsici isolates showed a 394 bp DNA band while
the remaining seventeen isolates of other species had no
PCR products with these primers (Fig. 2).
The sensitivity of the CcapF/CcapR primers was 10 pg
of genomic DNA in a 25 ll PCR reaction (Fig. 3).
Detection of Fungi Infected Papaya Tissues
After total DNA extraction from naturally infected papaya
tissues (Fig. 4), PCR amplification was performed using
accurate method based on conventional PCR to identify
and detect C. capsici. It was based on the design of two
C. capsici-specific primers. Amplification with the CcapF/
CcapR primers resulted in a product of approximately
394 bp only for the 21 isolates of C. capsici.
Traditionally, the identification of C. capsici from dis-
eased plant tissues is time-consuming and not applicable to
a large number of samples. In addition, it requires con-
siderable expertise to differentiate Colletotrichum species
based on morphology due to the complexity of morpho-
logical characteristics among different species [5]. On the
other hand, specificity is crucial for the success of the PCR
detection methods. The PCR primers CcapF/CcapR can
provide a rapid identification of C. capsici among other
pathogens, thereby facilitating an accurate diagnosis and
observation of the pathogen from different hosts and
locations.
To our knowledge, there are no previous reports of
C. capsici-specific primers; therefore, the development of
this set of primers is of great importance not only for
diagnostic laboratories but also for further research in this
field. The early and accurate diagnosis of diseased plants
would facilitate pathogen identification and lead to more
effective control measures for both pre and post-harvest

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Fig. 4 Anthracnose symptoms
from naturally infected papaya
a leaves, b petioles, and c fruits
Fig. 5 Detection of infected papaya tissues. Lane M, DNA marker
(100 bp DNA ladder); lane 1, C. capsici ATCC 48574; lanes 2–4,
infected papaya fruits; lanes 5–7, infected papaya petioles; lanes
8–10, infected papaya leaves; lane 11, healthy papaya fruit tissue
crops, since the different species of Colletotrichum
respond in different ways to the same control strategy
[29]. Furthermore, until 2008, anthracnose in papaya was
associated with C. gloeosporioides, but findings, first in
Mexico and later in other countries [7, 8], revealed that
C. capsici also causes anthracnose in this crop. These
findings reinforce the importance of this specific primers
set in diagnostics.
The primers CcapF/CcapR were developed based on
ITS regions and 5.8S rDNA gene. These regions are useful
in measuring close genealogical relationships, because they
exhibit great interspecies differences. Several species-
specific primers have been designed, therefore, to identify
different Colletotrichum species [30, 31] and other patho-
gens [32] based on the ITS sequences.
53
For our system, in a 25-ll PCR reaction volume, the
sensitivity of detection for C. capsici was as low as 10 pg
of pure genomic DNA by the conventional PCR. The fact
that the C. capsici-specific primer set is capable of
amplifying only this pathogen from fungal structures or
different contaminated tissues at early stages of infection
when first symptoms appear is also indicative of its sen-
sitivity and accuracy. All these strengths facilitate a fast
diagnosis since there is no need to isolate and cultivate the
fungus in order to identify it.
In some papaya production areas, problems caused by
anthracnose have been maximized by the development of
pathogen resistance to fungicides [33], this also occurs in
other crops [34] and hence a rapid identification and
monitoring of different Colletotrichum spp populations at
several stages of crop production process would facilitate
to recognize the prevalent species and the application of
effective control methods.
This research is the first report of C. capsici-specific
PCR detection, and the development of this PCR method
has significant practical applications. This rapid detection
assay would provide the agricultural industry with a tool
enabling growers to avoid high-risk situations and apply a
rational selection of fungicides to control anthracnose in
different crops while permitting a more detailed monitoring
of this fungus.
Acknowledgments We thank Angel Nexticapan-Garcez for his
help in samples collection. We are also grateful to Alberto Cortes-
Velazquez and Anuar A. Magaña-Alvarez for their excellent technical
assistance.
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