Morphological and Molecular Identification of Colletotrichum acutatum from Tomato Fruit

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INTRODUCTION
The genus Colletotrichum (teleomorph Glomerella) contains an extremely diverse number of fungi including both plant pathogens and saprophytes.Plant pathogenic species are important worldwide, causing diseases commonly known as anthracnose of grasses, legumes, vegetables, fruits, and perennial tree crops.The disease can occur on leaves, stems, and fruits of host plant (Sutton, 1992).Anthracnose diseases appear in both developing and mature plant tissues.Two distinct types of diseases are: those affecting developing fruit in the field (preharvest) and those damaging mature fruit during storage (postharvest).The ability to cause latent or quiescent infections has grouped Colletotrichum among the most important postharvest pathogens (Bailey et al., 1992).
In Serbia, the occurrence of anthracnose on tomato fruit was recorded in 2007 and 2008 (Živković et al., 2008).Typical symptoms include dark, sunken, and circular lesion that produces mucilaginous, orange conidial masses.Economic losses caused by the disease are mainly attributed to lower fruit quality and marketability.
Differentiation between Colletotrichum species based on host range or host of origin may not be a reliable criterion for fungi of this genus, since taxa such as C. gloeosporioides, C. dematium, C. acutatum, and others infect a broad range of host plants.Some taxa appear to be restricted to host families, genera or species within those families, or even cultivars, whereas others have more extensive host ranges (Freeman et al., 1998).Identification of Colletotrichum spp. is therefore a fundamental criterion in the development of more efficient control measures.Traditional identification and characterization of Colletotrichum species has relied primarily on differences in morphological features such as colony colour, size and shape of conidia and appressoria, growth rate, presence or absence of setae, and existence of the Glomerella teleomorph (Smith and Black, 1990;Gunnell and Gubler, 1992;Sutton, 1992).However, due to their morphological variability, the ample range of hosting crops, and wide variety of isolates, they are partially difficult to identify by traditional taxonomic methods, which must be complemented with molecular techniques (Whitelaw-Weckert et al., 2007).Construction of species-specific primers, especially from the ribosomal DNA internal transcribed spacer (ITS) region, has been proposed as the most efficient and reliable system for detection and differentiation of Colletotrichum spp.(Sreenivasaprasad et al., 1996;Freeman et al., 2000).
The objectives of the presented study were to substantiate the results obtained from previous investigations describing the symptoms, and identifying the species of Colletotrichum causing the anthracnose on tomato fruit using both classical and molecular techniques.

Pathogen isolation and maintenance
Tomato fruit samples with typical anthracnose symptoms were collected from the markets during 2007 and 2008.Pieces of the diseased tissues were sterilized in 3% NaOCl for 3 min, followed by several rinses with sterile distilled water, and placed on water agar (WA) in Petri plates at 25 o C for 5 days.Monoconidial cultures were produced for each isolate and maintained on potato dextrose agar (PDA) slants at 4°C.The reference isolates of C. acutatum (CBS 294.67) and C. gloeosporioides (CBS 516.97) were obtained from the Fungal Biodiversity Centre, Netherlands.

Pathogenicity test
Pathogenicity tests with representative isolates were conducted on symptomless, detached tomato fruits.The fruits were surface-sterilized with ethanol (70%), wounded with sterile needle, and inoculated with 20 µl of the conidial suspension (10 6 onidia/ml).Control fruit was inoculated with 20 µl of sterile distilled water.The fruits were then incubated in a plastic container at 25°C and >95% relative humidity, and examined for lesion development 7 days after inoculation.After 14 days, spores from diseased fruits were aseptically transferred and subcultured onto PDA plates, which were incubated at 25°C in darkness.The resultant cultures were checked for colony and spore morphology to confirm Koch's postulates.

Morphological and cultural characteristics
The isolates were cultured on potato dextrose agar PDA in darkness at 25 o C. The appearance of the colonies, the occurrence of sectors, and the vegetative and reproductive structures were described after 10 days of incubation.The conidia were taken from actively growing colonies and suspended in sterile water.Length and width were measured for 100 conidia, and conidial shape was recorded using the light and scanning electronic microscopy.Appressoria were produced using a slide culture technique, in which 10 mm 2 squares of PDA were placed in an empty Petri plate.The edge of the agar was inoculated with spores taken from a sporulating culture, and a sterile cover slip was placed over the inoculated agar (Johnston and Jones, 1997).After 5 days, the shape and size of the 100 appressoria formed across the underside of the cover slip were examined microscopically.Morphological characteristics of conidia and appressoria of tomato isolates were compared with reference isolates of C. acutatum and C. gloeosporioides.

DNA extraction
Total genomic DNA was extracted from mycelium obtained from cultures grown on PDA for 7 days at 25°C.Aerial mycelium was removed from each culture using a sterile transfer needle and placed in a sterile 1.5ml microcentrifuge tube containing 300 μl of extraction 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 5 min and then cooled to 25°C; 200 μl of phenol that was equilibrated with extraction buffer (vol/vol), and 200 μl of chloroform were added.The tubes were vortexed for 4 min, and then centrifuged at 12.000 rpm for 5 min.The supernatant was pipetted to a new sterile 1.5 ml tube and 200 μl of chloroform was added; the mixture was vortexed for 30 s and then centrifuged at 12.000 rpm for 15 min.The supernatant was again transferred to a new 1.5 ml tube and 200 μl of isopropanol was added; the capped tube was inverted several times to adequately mix and precipitate DNA and then centrifuged at 12.000 rpm for 15 min.The supernatant was discarded, and the nucleic acid pellet was washed in 400 μl of 70% ethanol and centrifuged at 12.000 rpm for 5 min.Again, the supernatant was discarded, and the nucleic acid pellet was air-dried for 10 min, resuspended in 50 μl of low-TE buffer (10 mM Tris-HCl and 0.1 mM EDTA, pH 8.5), and gently agitated to dissolve the DNA.DNA was treated with Ribonuclease A for a final concentration of 10 μg of RNase/ml for 1 h at 37°C.

Disease symptoms
Fruit symptoms begin as small, dark, sunken lesions that have a water-soaked appearance, which increase in diameter and coalesce, leaving a large sunken soft area.Under favourable temperatures, lesions on ripe fruit become visible within 5 to 6 days after infection.Orange conidial masses may occur scattered or in concentric rings on the lesion (Figure 1a).Black acervuli are produced just beneath the skin of the infected fruit (Figure 1b).

Pathogenicity test
All tested isolates caused anthracnose lesions on tomato fruit after 7 days of incubation (Figure 2a).No lesions developed on fruit inoculated with water (Figure 2b).Koch's postulates were fulfilled by reisolation from inoculated tomato fruits.Spore shape, size, and colony morphology were identical for the original and recovered isolates.

Morphological and cultural characteristics
Colonies of tomato isolates were dense aerial, initially white or cream white, becoming gray and then turning dark gray, as the cultures aged on PDA (Figure 3a).Colony reverse was white to white gray.Bright orange spore masses were produced outward from the center of the colony.The cultures developed black acervuli around the centre of the colony (Figure 3b and 4d).No setae were observed.Dark structures, which resembled perithecia but did not produce asci, were often observed throughout.The cultures did not form sectors after 10 days of incubation.
Mycelia were branched, septate, and hyaline.Conidia were hyaline, aseptate, and fusiform or rarely cylindrical with obtuse apices and tapering basis (Figure 4a  and 4b).Appressoria were observed on the underside of sterile covers slips arising from vegetative hyphae.
They were smooth, simple, clavate to ovate, and varied from light to dark brown (Figure 4c).Conidial and appressorial shape and size of tomato isolates, and reference isolates of C. acutatum and C. gloeosporioides are shown in Table1.

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

DISSCUSION
Anthracnose caused by the fungi C. acutatum and C. gloeosporioides is an important disease in Serbia.Several host species can be affected, including sour cherry (Arsenijević, 1984;Ivanović and Ivanović, 1992), apple (Trkulja, 2003), strawberry (Ivanović et al., 2007), and pear fruit (Živković et al., 2009).Anthracnose on tomato fruit was observed during the last several years.The objective of this study was to identify the causing agent of tomato anthracnose using morphological and molecular analysis.
Morphological identification of tomato isolates was based on phenotypic traits such as colony appearance, and character of vegetative and reproductive structures.The colour of cultures may vary considerably within and between species of C. acutatum and C. gloeosporioides.Colonies of C. gloeosporioides were usually gray in appearance, while C. acutatum colonies had a chromogenic (pink) or nonchromogenic (white to gray) phenotype (Baxter et al., 1983;Freeman et al., 1998;Lardner et al., 1999;Forster and Adaskaveg, 1999).The results of cultures studies showed no distinct differences in characteristics among the tomato isolates.All isolates were nonchromogenic.The colours of colonies were white and white gray to gray.Tomato isolates were demonstrated to be pathogenic on wounded fruit, and were reisolated, fulfilling Koch's postulates.
Conidial size of C. acutatum was described variably as 8. 3-14.4 × 2.5-4 μm (Simmonds, 1965), 8-16 × 2.5-4 μm (Dyko and Mordue, 1979), 12.3-14.7 × 4.6-5.3 µm (Smith and Black, 1990), and 12.5-20 × 3-5 μm (Gunnell and Gubler, 1992).Conidia of our isolates from tomato were compared with conidia of reference isolates of C. acutatum, and they were found to be similar in size.These results were almost fully consistent with description of Smith and Black (1990).In general, conidia of C. acutatum are elliptic-fusiform in shape; whereas conidia of C. gloeosporioides are cylindrical with obtuse ends (Dyko and Mordue, 1979;Baxter, et.al., 1983;Smith and Black, 1990).The conidial shape of most tomato isolates was fusiform, but conidia of PC-4 and PC-6 isolates were cylindrical with tapering end.The shape and size of appressoria have also been used for taxonomy of the genus Colletotrichum.Isolates from tomato fruits showed slightly smaller appressoria than reference isolate of C. gloeosporioides.These results correspond to description of Sutton (1992).The shape of appressoria of C. acutatum from tomato was clavate or ovate, and appressoria of C. gloeosporioides isolate CBS 516.97 were variable, irregular or ovate.Accordingly, appressoria of pathogens and reference isolates of C. acutatum and C. gloeosporioides differed in shape and size.Sanders andKorsten, (2003), andThan et al., (2008) also found that appressorial morphology was unreliable in distinguishing Colletotrichum species.However, a definite identification of Colletotrichum species based on morphology is difficult because isolates have overlapping ranges of conidial and colony characteristics, and because the variation in morphology is accepted for isolates within a species (Sutton, 1992).
Based on morphological descriptions, many diseases reported before 1965 to be caused by C. gloeosporioides (or one of its synonyms) could have been caused by C. acutatum (Baxter, et al., 1983).C. acutatum represents a species that encompasses a wide range of morphological and genetic diversity.Characterization of C. acutatum has been enhanced by the use of molecular markers, which have identified genetically distinct and perhaps biologically discrete groups among morphologically similar isolates (Johnston and Jones 1997;Forster and Adaskaveg, 1999;Freeman et al., 2001).
Morphological characteristics of isolates from tomato fruit indicated that the causal agent could be C. acutatum, but culture morphology, and conidial and appressorial characteristics were insufficient to separate C. acutatum from C. gloeosporioides.However, PCR with primers specific for both species, followed by nucleotide sequencing of the amplicons, demonstrated that the causal agent of tomato anthracnose was C. acutatum.A PCRamplified fragment of 490 bp was evident in all isolates from tomato fruits and C. acutatum CBS 294.67, but not in C. gloeosporioides CBS 516.97 isolate.C. gloeosporioides was not detected among the tomato isolates in this study.These results demonstrate that rDNA analysis is a reliable method for taxonomic species identification (Sreenivasaprasad, et al., 1996;Freeman, et al., 2000).
Anthracnose caused by C. acutatum is an emerging disease that may threaten the profitability of tomato and other crops in areas where it becomes established.Additional studies of the etiology and epidemiology of anthracnose are needed to define further the disease management strategies.

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

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

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

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