Pestic. Phytomed. (Belgrade), 25(3), 2010, 231-239 UDC: 632.4:635.64:58.

083
Pestic. fitomed. (Beograd), 25(3), 2010, 231-239 Scientific paper * Naučni rad
 DOI: 10.2298/PIF1003231Z

Morphological and Molecular

Identification of Colletotrichum acutatum
from Tomato Fruit
Svetlana Živković1, Saša Stojanović1, Žarko Ivanović1, Nenad Trkulja1,
Nenad Dolovac1, Goran Aleksić1 and Jelica Balaž2
1Institute for Plant Protection and Environment, Teodora Drajzera 9, 11000 Belgrade,

Serbia ([email protected])
2Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovića 8, 21000 Novi Sad,

Serbia
Received: April 9, 2010
Accepted: September 22, 2010

Summary
Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes, and
Colletotrichum dematium are the four main species of Colletotrichum that cause tomato
anthracnose. In Serbia, the occurrence of anthracnose on tomato fruit has been record-
ed during the last several years. Typical fruit symptoms include dark, sunken, and circu-
lar lesion with orange conidial masses. Pathogen isolates were obtained from a diseased
tomato fruits, on PDA medium forming a white to gray colonies. The cultures developed
black acervuli around the center of the colony. Conidia were hyaline, aseptate, and fusi-
form or rarely cylindrical. Appressoria were smooth, simple, clavate to ovate, and varied
from light to dark brown. Pathogenicity tests with representative isolates were conducted
on symptomless, detached tomato fruits. All tested isolates caused anthracnose lesions
on tomato fruit after 7 days of incubation. Koch’s postulates were fulfilled by reisolation
from inoculated tomato fruits. PCR analysis (using species-specific primer pair, CaInt2/
ITS4) of genomic DNA from tomato isolates resulted in an amplification product of 490
bp, specific for C. acutatum, further confirming the identity of the pathogen. Based on
morphological and molecular characteristics, the isolates from tomato fruit were deter-
mined as C. acutatum.
Keywords: Anthracnose; Tomato; Colletotrichum acutatum; Identification

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INTRODUCTION relied primarily on differences in morphological featu-

res such as colony colour, size and shape of conidia and
The genus Colletotrichum (teleomorph Glomerella) appressoria, growth rate, presence or absence of setae,
contains an extremely diverse number of fungi inclu- and existence of the Glomerella teleomorph (Smith and
ding both plant pathogens and saprophytes. Plant pa- Black, 1990; Gunnell and Gubler, 1992; Sutton, 1992).
thogenic species are important worldwide, causing di- However, due to their morphological variability, the
seases commonly known as anthracnose of grasses, le- ample range of hosting crops, and wide variety of iso-
gumes, vegetables, fruits, and perennial tree crops. The lates, they are partially difficult to identify by traditi-
disease can occur on leaves, stems, and fruits of host onal taxonomic methods, which must be complemen-
plant (Sutton, 1992). Anthracnose diseases appear in ted with molecular techniques (Whitelaw-Weckert et
both developing and mature plant tissues. Two distin- al., 2007). Construction of species-specific primers, es-
ct types of diseases are: those affecting developing fru- pecially from the ribosomal DNA internal transcribed
it in the field (preharvest) and those damaging matu- spacer (ITS) region, has been proposed as the most ef-
re fruit during storage (postharvest). The ability to ca- ficient and reliable system for detection and differen-
use latent or quiescent infections has grouped Colleto- tiation of Colletotrichum spp. (Sreenivasaprasad et al.,
trichum among the most important postharvest patho- 1996; Freeman et al., 2000).
gens (Bailey et al., 1992). The objectives of the presented study were to su-
Anthracnose disease caused by several Colletotri- bstantiate the results obtained from previous investiga-
chum spp. is a significant economic constraint on to- tions describing the symptoms, and identifying the spe-
mato (Lycopersicon esculentum Mill.) production worl- cies of Colletotrichum causing the anthracnose on toma-
dwide. Colletotrichum gloeosporioides (Penz.) Penz. & to fruit using both classical and molecular techniques.
Sacc., Colletotrichum coccodes (Wallr.) S.J. Hughes, and
Colletotrichum dematium (Pers. ex Fr.) Grove are three
main species of Colletotrichum that cause tomato ant- MATERIAL AND METHODS
hracnose in the United States (Dillard, 1989; Byrne et
al., 1997; Sanogo et al., 1997; LeBoeuf, 2007). In Bulga- Pathogen isolation and maintenance
ria, Colletotrichum acutatum J.H. Simmonds has been
reported as a causal agent of tomato fruit anthracnose Tomato fruit samples with typical anthracnose symp-
(Jelev et al., 2008). The corky root of tomato caused by toms were collected from the markets during 2007 and
Colletotrichum atramentarium (Bert. et Br.) Taubenh., 2008. Pieces of the diseased tissues were sterilized in
(synonym of C. coccodes) has also been found in Croa- 3% NaOCl for 3 min, followed by several rinses with
tia (Panjan and Lušin, 1963). sterile distilled water, and placed on water agar (WA)
In Serbia, the occurrence of anthracnose on tomato in Petri plates at 25oC for 5 days. Monoconidial cultu-
fruit was recorded in 2007 and 2008 (Živković et al., res were produced for each isolate and maintained on
2008). Typical symptoms include dark, sunken, and potato dextrose agar (PDA) slants at 4°C. The referen-
circular lesion that produces mucilaginous, orange co- ce isolates of C. acutatum (CBS 294.67) and C. gloeos-
nidial masses. Economic losses caused by the disease porioides (CBS 516.97) were obtained from the Fungal
are mainly attributed to lower fruit quality and mar- Biodiversity Centre, Netherlands.
ketability.
Differentiation between Colletotrichum species ba- Pathogenicity test
sed on host range or host of origin may not be a reli-
able criterion for fungi of this genus, since taxa such Pathogenicity tests with representative isolates were
as C. gloeosporioides, C. dematium, C. acutatum, and conducted on symptomless, detached tomato fruits. The
others infect a broad range of host plants. Some taxa fruits were surface-sterilized with ethanol (70%), woun-
appear to be restricted to host families, genera or spe- ded with sterile needle, and inoculated with 20 µl of the
cies within those families, or even cultivars, whereas conidial suspension (106 onidia/ml). Control fruit was
others have more extensive host ranges (Freeman et al., inoculated with 20 µl of sterile distilled water. The fru-
1998). Identification of Colletotrichum spp. is therefo- its were then incubated in a plastic container at 25°C
re a fundamental criterion in the development of mo- and >95% relative humidity, and examined for lesion de-
re efficient control measures. Traditional identificati- velopment 7 days after inoculation. After 14 days, spo-
on and characterization of Colletotrichum species has res from diseased fruits were aseptically transferred and

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subcultured onto PDA plates, which were incubated at supernatant was discarded, and the nucleic acid pellet
25°C in darkness. The resultant cultures were checked was washed in 400 μl of 70% ethanol and centrifuged
for colony and spore morphology to confirm Koch’s po- at 12.000 rpm for 5 min. Again, the supernatant was
stulates. discarded, and the nucleic acid pellet was air-dried for
10 min, resuspended in 50 μl of low-TE buffer (10 mM
Morphological and cultural Tris-HCl and 0.1 mM EDTA, pH 8.5), and gently agi-
characteristics tated to dissolve the DNA. DNA was treated with Ri-
bonuclease A for a final concentration of 10 μg of RNa-
The isolates were cultured on potato dextrose agar se/ml for 1 h at 37°C.
PDA in darkness at 25oC. The appearance of the colo-
nies, the occurrence of sectors, and the vegetative and PCR amplification
reproductive structures were described after 10 days
of incubation. The conidia were taken from actively Species-specific primers for C. gloeosporioides (CgInt;
growing colonies and suspended in sterile water. Len- 5’-GGCCTCCCGCCTCCGGGCGG- 3’), and C.
gth and width were measured for 100 conidia, and co- acutatum (CaInt2; 5’-GGCGCCGGCCCCGTCA-
nidial shape was recorded using the light and scanning CGGGGG-3’), from the ITS1 region of the riboso-
electronic microscopy. Appressoria were produced mal DNA gene were used in combination with the
using a slide culture technique, in which 10 mm2 squ- conserved primer ITS4. PCR amplification was per-
ares of PDA were placed in an empty Petri plate. The formed in a 25 μl reaction mixture containing 1.5 μl
edge of the agar was inoculated with spores taken from of DNA extract in low-TE buffer (10 mM Tris-HCl,
a sporulating culture, and a sterile cover slip was placed 1 mM EDTA; pH 8.0); 4 μl of 200 μM each of dATP,
over the inoculated agar (Johnston and Jones, 1997). dCTP, dGTP, and dTTP; 2.5 μl of 10× Taq reaction
After 5 days, the shape and size of the 100 appressoria buffer (500 mM KCl, 100 mM Tris-HCl pH 9.0, and
formed across the underside of the cover slip were exa- 1% Triton X-100); 0.5 μl of 100 μM MgCl 2; 1.0 μl of
mined microscopically. Morphological characteristi- 1 μM target primer; 1 μl of 1 μM ITS4 primer; 0.65
cs of conidia and appressoria of tomato isolates were U Taq DNA polymerase, and 14.85 μl of sterile water.
compared with reference isolates of C. acutatum and Amplifications were performed in Eppendorf Master
C. gloeosporioides. Cycler programmed for the following cycling condi-
tions: initial denaturation at 94°C for 5 min; 35 am-
DNA extraction plification cycles consisting of 1 min at 94°C, 2 min
at 59°C, 1 min of extension at 72°C, and final exten-
Total genomic DNA was extracted from mycelium sion at 72°C for 5 min. PCR products were separa-
obtained from cultures grown on PDA for 7 days at ted using electrophoresis in 1% agarose gels in TBE
25°C. Aerial mycelium was removed from each culture buffer. Gels were stained in dilute ethidium bromide
using a sterile transfer needle and placed in a sterile 1.5- (0.2µg/ml), visualized by UV transilluminator, and
ml microcentrifuge tube containing 300 μl of extracti- photographed.
on buffer (0.2 M Tris-HCl, 0.25 M NaCl, 25 mM ED-
TA, and 2% sodium dodecyl sulfate, pH 8.5). Tubes
were uncapped and placed in a boiling water bath for RESULTS
5 min and then cooled to 25°C; 200 μl of phenol that
was equilibrated with extraction buffer (vol/vol), and Disease symptoms
200 μl of chloroform were added. The tubes were vor-
texed for 4 min, and then centrifuged at 12.000 rpm Fruit symptoms begin as small, dark, sunken lesions
for 5 min. The supernatant was pipetted to a new ste- that have a water-soaked appearance, which increase
rile 1.5 ml tube and 200 μl of chloroform was added; in diameter and coalesce, leaving a large sunken soft
the mixture was vortexed for 30 s and then centrifu- area. Under favourable temperatures, lesions on ripe
ged at 12.000 rpm for 15 min. The supernatant was fruit become visible within 5 to 6 days after infecti-
again transferred to a new 1.5 ml tube and 200 μl of on. Orange conidial masses may occur scattered or in
isopropanol was added; the capped tube was inverted concentric rings on the lesion (Figure 1a). Black acer-
several times to adequately mix and precipitate DNA vuli are produced just beneath the skin of the infected
and then centrifuged at 12.000 rpm for 15 min. The fruit (Figure 1b).

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Svetlana Živković et al.

Figure 1. Anthracnose symptoms on tomato fruit: (a) sunken necrotic lesion with orange conidial masses; (b) black acervuli
on the infected fruit

Pathogenicity test developed on fruit inoculated with water (Figure 2b).

Koch’s postulates were fulfilled by reisolation from inocu-
All tested isolates caused anthracnose lesions on toma- lated tomato fruits. Spore shape, size, and colony morpho-
to fruit after 7 days of incubation (Figure 2a). No lesions logy were identical for the original and recovered isolates.

Figure 2. Pathogenicity test: (a) necrotic lesion on tomato fruit inoculated with isolate PC-3; (b) control fruit inoculated with
sterile water

Morphological and cultural characteristics were produced outward from the center of the colony. The
cultures developed black acervuli around the centre of the
Colonies of tomato isolates were dense aerial, initially colony (Figure 3b and 4d). No setae were observed. Dark
white or cream white, becoming gray and then turning structures, which resembled perithecia but did not produ-
dark gray, as the cultures aged on PDA (Figure 3a). Colony ce asci, were often observed throughout. The cultures did
reverse was white to white gray. Bright orange spore masses not form sectors after 10 days of incubation.

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Mycelia were branched, septate, and hyaline. Coni- They were smooth, simple, clavate to ovate, and vari-
dia were hyaline, aseptate, and fusiform or rarely cylin- ed from light to dark brown (Figure 4c). Conidial and
drical with obtuse apices and tapering basis (Figure 4a appressorial shape and size of tomato isolates, and re-
and 4b). Appressoria were observed on the underside ference isolates of C. acutatum and C. gloeosporioides
of sterile covers slips arising from vegetative hyphae. are shown in Table1.

Figure 3. Cultural appearance of C. acutatum, isolate PC-3 on PDA: (a) top of culture; (b) bottom of culture

Figure 4. Morphological characteristics of C. acutatum from tomato: (a) conidia of isolate PC-4 (light microscope, x1000);
(b) conidia of isolate PC-4 (scanning electronic microscope); (c) appressoria of isolate PC-6 (light microscope, x1000);
(d) microscopic section of acervuli, isolate PC-1 (light microscope, x600)

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Svetlana Živković et al.

Table 1. Morphology of conidium and appressorium of Colletotrichum isolates from tomato, and reference isolates of C.acutatum
and C. gloeosporioides

Conidium Appressorium

Size (µm) Size (µm)

Isolate
a Shape b Shape
Length x Width Length x Width
min-(mean)-max min-(mean)-max

PC-1 F, Ta 11.2-(14.3)-16.6 x 3.2-(3.6)-4.8 Cl 6.4-(7.2)- 9.6 x 4.8-(5.3)-6.4

PC-2 F, Ta 11.2-(13.9)-16.0 x 2.4-(3.7)-4.8 Cl 8.0-(8.5)-9.6 x 4.8-(5.5)-6.4

PC-3 F, Ta 11.2-(13.3)-15.2 x 2.4-(3.9)-4.8 Cl 6.4-(7.3)-9.6 x 5.6-(6.5)-7.2

PC-4 C, Ta 11.2-(13.7)-16.0 x 3.2-(3.7)-4.8 Ov 6.4-(7.3)-9.6 x 5.6-(6.2)-6.4

PC-5 F, Ta 12.8-(14.6)-17.6 x 2.4-(3.9)-4.8 Cl 8.0-(8.7)-10.4 x 5.6-(6.7)-7.2

PC-6 C, Ta 11.2-(13.7)-16.0 x 3.2-(3.9)-4.8 Ov 6.4-(7.2)-9.6 x 5.6-(6.2)-6.4

CBS 294.67 F, Ta 11.2-(13.2)-15.2 x 3.2-(3.9)-4.8 Cl 6.4-(8.1)-9.6 x 5.6-(5.8)-6.4

CBS 516.97 C, Ob 12.8-(17.9)-19.2 x 3.2-(3.7)-4.8 Ir, Ov 9.6-(12.3)-14.4 x 6.4-(7.9)-8.8

a Shape of conidium: F – fusiform; C – cylindrical; Ob – with obtuse ends; Ta – with tapering one end
b Shape of appressorium: Cl – clavate; Ir – irregular; Ov – ovate;

Molecular identification trast, DNA of the tomato isolates, as well as the refe-
rence C. acutatum, were not amplified by the species-
The species-specific primer CaInt2 in conjunction specific primer CgInt in conjunction with ITS4 primer,
with ITS4 primer amplified a 490 bp fragment from whereas a 450 bp fragment was amplified only from
genomic DNA of tomato isolates, and the reference iso- DNA of reference C. gloeosporioides CBS 516.97 (Fi-
late of C. acutatum CBS 294.67, but not from DNA of gure 5b). No PCR products were produced with water
C. gloeosporioides reference isolate (Figure 5a). In con- controls in any of the reactions.

Figure 5. Amplification of specific DNA fragments from Colletotrichum isolates: (a) Primer pairs CaInt2/ITS4 specific for
C. acutatum; (b) Primer pairs CgInt/ITS4 specific for C. gloeosporioides; reference isolate of C. acutatum CBS 294.67 (line 1);
reference isolate of C. gloeosporioides CBS 516.97 (line 2); isolates PC-1,PC-2, PC-3, PC-4, PC-4, PC-5, PC-6 (lines 3 to 8);
M = molecular size marker GeneRulerTM DNA Ladder Mix (100-10.000 bp); K = negative control (water)

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DISSCUSION Than et al., (2008) also found that appressorial morphol-

ogy was unreliable in distinguishing Colletotrichum spe-
Anthracnose caused by the fungi C. acutatum and cies. However, a definite identification of Colletotrichum
C. gloeosporioides is an important disease in Serbia. Sev- species based on morphology is difficult because isolates
eral host species can be affected, including sour cher- have overlapping ranges of conidial and colony character-
ry (Arsenijević, 1984; Ivanović and Ivanović, 1992), ap- istics, and because the variation in morphology is accept-
ple (Trkulja, 2003), strawberry (Ivanović et al., 2007), and ed for isolates within a species (Sutton, 1992).
pear fruit (Živković et al., 2009). Anthracnose on tomato Based on morphological descriptions, many diseases
fruit was observed during the last several years. The objec- reported before 1965 to be caused by C. gloeosporioides
tive of this study was to identify the causing agent of tomato (or one of its synonyms) could have been caused by C.
anthracnose using morphological and molecular analysis. acutatum (Baxter, et al., 1983). C. acutatum represents
Morphological identification of tomato isolates was a species that encompasses a wide range of morpholog-
based on phenotypic traits such as colony appearance, and ical and genetic diversity. Characterization of C. acu-
character of vegetative and reproductive structures. The tatum has been enhanced by the use of molecular mark-
colour of cultures may vary considerably within and be- ers, which have identified genetically distinct and per-
tween species of C. acutatum and C. gloeosporioides. Colo- haps biologically discrete groups among morphologi-
nies of C. gloeosporioides were usually gray in appearance, cally similar isolates (Johnston and Jones 1997; Forster
while C. acutatum colonies had a chromogenic (pink) or and Adaskaveg, 1999; Freeman et al., 2001).
nonchromogenic (white to gray) phenotype (Baxter et al., Morphological characteristics of isolates from toma-
1983; Freeman et al., 1998; Lardner et al., 1999; Forster and to fruit indicated that the causal agent could be C. acu-
Adaskaveg, 1999). The results of cultures studies showed tatum, but culture morphology, and conidial and appres-
no distinct differences in characteristics among the tomato sorial characteristics were insufficient to separate C. acu-
isolates. All isolates were nonchromogenic. The colours of tatum from C. gloeosporioides. However, PCR with prim-
colonies were white and white gray to gray. Tomato isolates ers specific for both species, followed by nucleotide se-
were demonstrated to be pathogenic on wounded fruit, and quencing of the amplicons, demonstrated that the causal
were reisolated, fulfilling Koch’s postulates. agent of tomato anthracnose was C. acutatum. A PCR-
Conidial size of C. acutatum was described variably amplified fragment of 490 bp was evident in all isolates
as 8.3-14.4 × 2.5-4 μm (Simmonds, 1965), 8-16 × 2.5-4 from tomato fruits and C. acutatum CBS 294.67, but not
μm (Dyko and Mordue, 1979), 12.3-14.7 × 4.6-5.3 µm in C. gloeosporioides CBS 516.97 isolate. C. gloeosporio-
(Smith and Black, 1990), and 12.5-20 × 3-5 μm (Gun- ides was not detected among the tomato isolates in this
nell and Gubler, 1992). Conidia of our isolates from to- study. These results demonstrate that rDNA analysis is
mato were compared with conidia of reference isolates of a reliable method for taxonomic species identification
C. acutatum, and they were found to be similar in size. (Sreenivasaprasad, et al., 1996; Freeman, et al., 2000).
These results were almost fully consistent with descrip- Anthracnose caused by C. acutatum is an emerging
tion of Smith and Black (1990). In general, conidia of C. disease that may threaten the profitability of tomato
acutatum are elliptic-fusiform in shape; whereas conid- and other crops in areas where it becomes established.
ia of C. gloeosporioides are cylindrical with obtuse ends Additional studies of the etiology and epidemiology of
(Dyko and Mordue, 1979; Baxter, et.al., 1983; Smith and anthracnose are needed to define further the disease
Black, 1990). The conidial shape of most tomato isolates management strategies.
was fusiform, but conidia of PC-4 and PC-6 isolates were
cylindrical with tapering end. The shape and size of ap-
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 Pestic. Phytomed. (Belgrade), 25(3), 2010, 231–239

Morfološka i molekularna
identifikacija Colletotrichum
acutatum sa ploda paradajza
Rezime
Četiri glavna prouzrokovača antraknoze paradajza iz roda Colletotrichum su: Colletotri-
chum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes i Colletotrichum
dematium. U Srbiji je tokom poslednjih godina zabeležena pojava antraknoze ploda para-
dajza. Tipični simptomi na plodu su tamne, ulegnute, kružne lezije sa masom narandžastih
konidija. Sa obolelih plodova paradajza dobijeni su izolati patogena koji na KDA podlozi
obrazuju bele do sivo obojene kolonije. Acervuli crne boje formiraju se u kulturi oko cen-
tra kolonije. Konidije su hialinske, neseptirane, fusiformne ili ređe cilindrične. Apresorije su
glatke, jednostavne, okruglastog ili oblika izdužene palice, svetlo do tamno braon boje. Test
patogenosti je obavljen sa reprezentativnim izolatima, na odabranim, zdravim plodovima.
Svi ispitivani izolati prouzrokuju antraknozne lezije na plodu paradajza 7 dana nakon inoku-
lacije. Kohovi postulati su zadovoljeni reizolacijama sa inokulisanih plodova paradajza. PCR
analiza (korišćenjem para prajmera specifičinog za vrstu, CaInt2/ITS4) iz genoma DNA izo-
lata sa paradajza rezultirala je amplifikacijom produkta od 490 bp, specifičnog za C. acuta-
tum, što je potvrdilo identitet patogena. Na osnovu morfoloških i molekularnih karakteristi-
ka izolati sa ploda paradajza determinisani su kao C. acutatum.
Ključne reči: Antraknoza; paradajz; Colletotrichum acutatum; identifikacija

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