Introduction

The mango (Mangifera indica L.) is an important fruit crop in Brazil and other tropical and subtropical countries of the world (Evans and Mendoza 2009). In Brazil, the main areas of cultivation are in the northeastern region and in these areas are mainly produced for export. In 2010, Brazil exported 124,694 tons of mangoes worth approximately US$ 119,929 million (Anuário Brasileiro de Fruticultura 2012). Mango is affected by a number of diseases at all stages of its development, from seedlings in the nursery, to the fruits in storage or transit (Ploetz 2003; Prakash 2004). Anthracnose, caused by species of Colletotrichum, is the most important disease of mango in Brazil; the disease limits productivity and reduces fruit quality, thus directly affecting fruit export (Santos Filho and Matos 2003). In India, losses due to anthracnose have been estimated to be 2–39 % (Prakash 2004).

The genus Colletotrichum includes a number of plant pathogens of major importance, causing diseases of a wide variety of woody and herbaceous plants (Hyde et al. 2009; Wikee et al. 2011; Cannon et al. 2012). Identification of Colletotrichum species was previously based on morphology and to a lesser extend host association (Hyde et al. 2009). A polyphasic approach, using combined sequence analysis of multiple loci, coupled with morphological data, is now recommended for accurate species identification in the genus (Cai et al. 2009, 2011). Ko Ko et al. (2011) noted the need to resurvey plant pathogens such as Colletotrichum with molecular based approaches, as previous data based on morphology was likely to be incorrect. The genus was recently voted the worlds eighth most important group of plant pathogenic fungi, based on perceived scientific and economic importance (Dean et al. 2012).

Anthracnose caused by C. gloeosporioides is the most important and widespread form of decay affecting mango fruit worldwide (Jeffries et al. 1990; Prusky and Keen 1993; Freeman et al. 1998; Ploetz 2003). However, following epitypification of C. gloeosporioides (Cannon et al. 2008), Phoulivong et al. (2010) were able to show that C. gloeosporioides sensu stricto was actually not a common pathogen on fruits in the tropics and it was not the cause of mango anthracnose in Laos and Thailand.

In Brazil, anthracnose of mango is attributed exclusively to C. gloeosporioides (Santos Filho and Matos 2003; Ribeiro 2005) and there are no studies on the molecular characterization of Colletotrichum species associated with mango. Therefore, the objective of this study was to characterize species of Colletotrichum from a large number of isolates associated with mango anthracnose in the northeastern Brazil based on DNA sequence data, morphology, and pathogenicity and virulence tests.

Material and methods

Sampling and fungal isolation

During 2010 and 2011, mango fruits showing anthracnose symptoms were collected in 10 orchards (70 samples per orchard) located in São Francisco Valley, Assú Valley and Zona da Mata Pernambucana, northeastern Brazil. The orchards from Zona da Mata Pernambucana did not receive any fungicide applications, and other two orchards received at least one spray with methyl benzimidazole carbamates (MBC), demethylation inhibitor (DMI) or other fungicides. Samples were recovered from the cultivar Tommy Atkins. Fruit tissues were surface disinfested in 70 % ethanol for 30 s and 1 % NaClO for 1 min. Samples were then rinsed in sterile distilled water for 30 s and dried before small pieces (4–5 mm) of tissue were taken from the margin between necrotic and apparently healthy tissue to be plated onto potato dextrose agar (PDA) (Acumedia, Lansing, USA) amended with 0.5 g l−1 streptomycin sulfate (PDAS). Plates were incubated at 25 °C with a 12–h photoperiod provided by fluorescent light for 5 to 7 days. The growing edges of any fungal hyphae developing from the tissues were then transferred aseptically to PDA. When direct examination showed that the fungus was sporulating on the mango fruits, spore masses were picked off with a sterilized wire loop and streaked on the surface of water agar. After incubation overnight (25 °C), single germinated spores were picked up with a sterilized needle and transferred to PDA (Abang 2003; Chomnunti et al. 2011). The fungi were identified following sporulation. One-hundred and forty-three isolates were morphologically identified as Colletotrichum spp. (Sutton 1980) and single spore cultures were obtained using the procedure described by Goh (1999). Pure cultures were stored in sterilized water in Eppendorf tubes at 5 °C and stock cultures were stored in PDA slants at 5 °C in the dark.

DNA extraction

Isolates were grown on PDA for 7 days at 25 °C with a 12-h photoperiod. Using a sterile 10 μl pipette tip, a small amount of aerial mycelia was scraped from the colony surface and genomic DNA was extracted using the AxyPrep™ Multisource Genomic DNA Miniprep Kit (Axygen Scientific Inc., Union City, USA) following the manufacturer’s instructions. DNA concentrations were estimated visually in agarose gel by comparing band intensity with a DNA ladder 1 kb (Axygen).

PCR amplification and DNA sequencing

All strains in this study were amplified for the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene, as an initial measure of genetic diversity. A subset of 47 isolates, selected to represent the range of genetic diversity and geographic origin (Table 1), were further sequenced for partial actin (ACT), β-tubulin (TUB2), calmodulin (CAL), glutamine synthetase (GS) genes and rDNA-ITS (ITS) region were amplified by polymerase chain reaction (PCR) reaction.

Table 1 Strains of Colletotrichum studied in this paper with details of culture collection, host, location and GenBank accessions of the sequences generated
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The PCR amplification for GPDH, CAL and GS were carried out using the primers-pair GDF1 and GDR1 (Templeton et al. 1992), CL1 and CL2 (O’Donnell et al. 2000), and GSF1 and GSR1 (Stephenson et al. 1997), respectively. The partial ACT, TUB2 and ITS region were amplified with the primers-pair ACT512F and ACT783R (Carbone and Kohn 1999), Bt2a and Bt2b (Glass and Donaldson 1995), and ITS 1 and ITS 4 (White et al. 1990), respectively.

Each 50-μl polymerase chain reaction (PCR) mixture included 21 μl of PCR-grade water, 1 μl of DNA template, 1.5 μM of each primer, and 1 μl of PCR Master Mix (2×) (0.05 u μl−1 de Taq DNA polymerase, reaction buffer, 4 mM MgCl2, 0.4 mM of each dNTP; Thermo Scientific, Waltham, USA). PCR reactions were carried out in a thermal cycler (Biocycler MJ 96; Applied Biosystems, Foster City, USA). The cycling parameters for GPDH and GS consisted of a denaturation step at 94 °C for 2 min, followed by 35 cycles at 94 °C for 45 s, 60 °C for 45 s, 72 °C for 1 min and a final cycle at 72 °C for 10 min. The cycling parameters for CAL were initiated at 94 °C for 2 min followed by 35 cycles at 94 °C for 1 min, 54 °C for 30 s, 72 °C for 30 s and a final step at 72 °C for 10 min. The cycling parameters for partial ACT, TUB2 and ITS region consisted of a 2 min denaturing step at 94 °C followed by 34 cycles at 94 °C for 1 min, 55 °C for 30 s, 72 °C for 1 min and a final cycle of 10 min at 72 °C.

The PCR amplification products were separated by electrophoresis in 1.5 % agarose gels in 1.0× Tris-acetate acid EDTA (TAE) buffer and were photographed under UV light after staining ethidium bromide (0.5 μg ml−1) for 1 min. PCR products were purified using the AxyPrep™ PCR Cleanup Kit (Axygen) following the manufacturer’s instructions. DNA sequencing for GPDH, ACT, CAL, GS, TUB2, CAL genes and ITS region were performed using a ABI PRISM® 3100-Avant Genetic Analyzer (Applied Biosystems) at the Sequencing Platform LABCEN/CCB in the Universidade Federal de Pernambuco (Recife, Brazil).

Phylogenetic analysis

The quality of the nucleotide sequences and the contig assembly were carried out using the Staden Package (Staden et al. 1998). Multiple sequence alignments of each gene used Clustal W as implemented in MEGA v.5 (Tamura et al. 2011), and manually adjusted to allow maximum sequence similarity.

Bayesian inference (BI) was used to reconstruct the phylogenetic trees using MrBayes v. 3.2.1 (Ronquist et al. 2012). Bayesian inference has significant advantages over other methods of analysis such as maximum likelihood and maximum parsimony (Archibald et al. 2003) and provides measures of clade support as posterior probabilities rather than random resampling bootstraps (Weir et al. 2012). jModelTest v. 0.1.1 (Posada 2008) was used to carry out the statistical selection of best-fit models of nucleotide substitution using the Bayesian Information Criterion (BIC) (Table 2). The nucleotide multiple sequence alignments of all genes were concatenated and the analysis on the full data set were run twice for 5 × 107 generations. Samples were taken from the posterior every 1,000 generations. Convergence of all parameters was checked using the Tracer program (Rambaut and Drummond 2007). Therefore, the first 25 % of generations were discarded as burn-in.

Table 2 Nucleotide substitution models used in phylogenetic analyses
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Sequences of Colletotrichum species obtained from GenBank were included in the analyses (Table 1). Seventy–one isolates were used in the initial BI (A) analysis using a concatenated alignment for six: ACT, CAL, GS, GPDH, TUB2 and ITS. Colletotrichum theobromicola was used as outgroup in this analysis. A second analysis of BI (B) was carried out to confirm the identity of two isolates of Colletotrichum karstii. The second analysis was based on a concatenated alignment of four: ACT, GPDH, ITS and TUB2. Colletotrichum gloeosporioides was used as outgroup in this analysis.

Sequences derived in this study are deposited in GenBank. Representative isolates of different Colletotrichum species obtained in this study were deposited in the Culture Collection of Phytopathogenic Fungi “Prof. Maria Menezes” (CMM) at the Universidade Federal Rural de Pernambuco, Brazil and MFLU, Mae Fah Luang University, Thailand.

Morphological studies of Colletotrichum species

Based on the fungal species that were identified based on phylogenetic analyses, 47 Colletotrichum isolates [C. asianum (13), C. fructicola (2), C. tropicale (6), C. karstii (2) and Colletotrichum sp. (24)] were further characterized by colony morphology and conidial characteristics. A 4–mm–diameter mycelial plug from the growing margin of a 5-day-old colony was placed in the centre of a 90–mm–diameter PDA plate, and four replicates of each isolate were incubated at 28 °C with a continuous fluorescent light (24–h). The colony colour and diameter from isolates were recorded during 7 days. The colony colour was evaluated using the mycological colour chart (Rayner 1970) and the colony diameter (mm) was measured in two perpendicular directions. The colony diameter data were used to calculate the mycelial growth rate (mm day−1). After 7 days, conidia were mounted in 100 % lactic acid and digital images recorded with a Sansung SDC-415 camera (Sansung Co., Seoul, Korea) on an Olympus BX41 microscope (Olympus Co., Tokyo, Japan). The length and width of 50 conidia per isolate were measured with the Motic Image Plus v. 2.0 image analysis software (Motic Group Co., Beijing, China). For the new Colletotrichum species, appressoria were induced using a slide culture technique, in which 10 mm2 squares of PDA were placed in an empty Petri dish. 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). The length and width of 50 appressoria per isolate were measured with the Motic Image Plus v. 2.0. Mean and standard errors of the conidial and appressoria measurements were calculated.

One–way analyses of variance (ANOVA) were conducted to determine the significance of differences in conidia dimensions and growth rates with Colletotrichum species, and means were compared by Fisher’s least significant difference (LSD) test at the 5 % significance level using STATISTIX v. 9.0 (Analytical Software, Tallahassee, USA).

Pathogenicity and virulence in fruits

The isolates used in morphological characterization were selected for pathogenicity and virulence tests on detached fruits of mango (cv. Tommy Atkins) under controlled conditions. The fungal inoculum was produced in Petri dishes containing PDA, which were incubated for 7 days at 28 °C under 12-h photoperiod. Spore suspensions were prepared by adding 20 mL of sterile distilled water to the surface of the cultures, brushing with a soft bristle brush, and filtering through a double layer of cheesecloth. Spore concentration was determined using a hemocytometer and adjusted to 106 conidia ml−1 with sterile water. Mango fruits at stage three of maturation (Assis 2004), without disease symptoms and which were not treated with fungicides, were washed in running water, surface disinfested in 70 % ethanol for 1 min and 1 % NaClO for 3 min, then rinsed in sterile distilled water. Since non-wounded treatment caused no lesions of Colletotrichum (non published data), after drying each fruit was wounded at the medium region by pushing the tip of four sterile pins through the surface of the skin to a depth of 3 mm. Six microliters of conidial suspension were dispensed at wound site on the fruits. Negative controls were fruits treated with sterile distilled water. Inoculated fruits were placed in large plastic containers. Before, the bottom of each container was lined with four paper layers wetted in distilled water to maintain humidity. Each fruit was put on a sterilized Petri dish to avoid direct contact with water. The plastic containers were partially sealed with plastic bags and incubated at 25 °C in the dark. The plastic bags and paper towels were removed after 24–h, and fruits were kept at the same temperature. Fruits were monitored for the onset of symptoms for 10 days. Isolates were considered pathogenic when the lesion area advanced beyond the 4–mm diameter initial injury. The virulence of the isolates was evaluated by measurement of the lesion length at 10 days after inoculation in two perpendicular directions on each fruit. The experiment was arranged in a completely randomized design with six replicates per treatment (isolate) and four fruits per replicate. The experiment was conducted twice. Difference in virulence caused by Colletotrichum species was determined by one-way ANOVA and means were compared by LSD test at the 5 % significance level using STATISTIX.

Results

Phylogenetic analyses

The initial analysis of the partial sequence GPDH gene of the 143 isolates revealed a total of nine haplotypes (H1–H9). Initial analysis of these haplotypes reveal that eight (H1–H8) belong to the C. gloeosporioides species complex, all of them within the “Musae clade” sensu Weir et al. (2012). The haplotype 9 (H9) GPDH sequence had a high degree of similarity (99 %) with sequences of type species of C. karstii deposited in the GenBank. Forty seven isolates representing all haplotypes were further used for multilocus analysis. Sequences of the ACT gene of the Colletotrichum species ranged from 249 to 297 bp. TUB2 gene ranged from 404 to 467 bp. Sequences of the CAL gene ranged from 628 to 707 bp. The sequences of the GPDH gene ranged from 203 to 279 bp. The GS gene ranged from 854 to 1,015 bp. The sequences of the ITS region ranged from 484 to 598 bp.

The Bayesian inference with the haplotypes that belong to the C. gloeosporioides complex that was performed with the combined datasets of partial ACT, TUB2, CAL, GS, GPDH and the ITS region comprised 3,220 characters after alignment. The gene boundaries in the alignment were: ACT: 1–293, BT2b: 294–734, CAL: 735–1424, GPDH: 1425–1729, GS: 1730–2647, ITS: 2648–3218. Sequences of Colletotrichum type species from the GenBank were included in the analysis (Table 1). The combined dataset resulted in four well supported clades. Three clades corresponded to species previously described: C. asianum (13), C. fructicola (2) and C. tropicale (6). The fourth clade containing the majority isolates (24) did not cluster with any known species, which indicate that these isolates belong to new species (Fig. 1). The BI analysis of the two isolates from haplotype 9 using the combined datasets of ACT, GPDH, ITS and TUB2 cluster these isolates with the type species of C. karstii with high posterior probability support (Fig. 2).

Fig. 1
figure 1

A Bayesian inference phylogenetic tree from 72 of Colletotrichum isolates using the combined partial sequence data of ACT, CAL, GPDH, GS, ITS and TUB2 genes. The tree shows the phylogenetic relationships of Colletotrichum species isolated from Mangifera indica and selected Colletotrichum species of the “Musae clade” sensu Weir et al. (2012). Bayesian posterior probability values ≥ 0.5 are shown in each node. Ex-type or ex-epitype sequences are emphasized in bold font. Culture accession numbers are listed. Colletotrichum theobromicola was used as outgroup. The scale bar indicates the number of expected changes per site

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Fig. 2
figure 2

A Bayesian inference phylogenetic tree of 30 isolates to confirm the identity of two isolates of Colletotrichum karstii. The tree was built using concatenated sequences of the partial ACT, GPDH, ITS and TUB2 genes each with a separate model of DNA evolution. Bayesian posterior probability values ≥ 0.5 are shown in each node. Ex-type or ex-epitype sequences are emphasized in bold font. Culture accession numbers are listed. Colletotrichum gloeosporioides was used as outgroup. The scale bar indicates the number of expected changes per site

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The diversity analyses from 143 isolates of Colletotrichum spp. show the following haplotype distribution within the species identified in phylogenetic analyses: Colletotrichum sp. 74 isolates belong to haplotypes H1 (46), H2 (19) and H4(9); C. fructicola: 2 isolates, all in H5; C. asianum 58 isolates, H3 (35) and H7 (23); C. tropicale 7 isolates, H8 (6) and H6 (1); Colletotrichum karstii 2 isolates, all in H9.

Morphological and cultural characterization

The Colletotrichum isolates grouped into seven morphotypes, based on colony characteristics (Fig. 3). Twelve percent of all isolates belonged to morphotype 1, which comprised white mycelia lacked conidial masses. Morphotype 2 (29 %) comprised isolates which had pale greenish grey (33””f), in reverse buff (19”f) colonies. Eleven percent of all isolates belonged to morphotype 3 with rosy vinaceous colonies (5”d), in reverse rosy buff (13”f). Isolates belonging to morphotype 4 (6 %) had cinnamon (15”b), in reverse cinnamon (15”i) colonies. Isolates belonging to morphotype 5 (8.3 %) had sky grey colonies (45””d), which in reverse were sky grey (53””d). The morphotype 6 (4.1 %) had pale mouse grey (15””’d) colonies which were pale mouse grey (15””’b) in reverse. Isolates belonging to morphotype 7 (4.1 %) which had orange (13b) colonies which were saffron (13d) in reverse. Isolates identified as C. asianum are represented in the morphotypes 1, 2, 3, 4, 5 and 6. The C. fructicola isolates were all included in morphotype 1. C. tropicale includes the morphotypes 1, 2 and 3. Isolates of Colletotrichum sp. comprised morphotypes 1, 2, 3, 4 and 5. The isolates of C. karstii belonged to morphotype 7. The species of Colletotrichum found in this study show differences in conidial size and growth rates. The previously described species show conidial dimensions and growth rate compatible with what is described in the literature (Table 3).

Fig. 3
figure 3

Morphotypes (1 to 7) of isolates of Colletotrichum from mango fruits. Colony characteristics: Plates in A aerial view, reverse view in B

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Table 3 Summary of morphological data of Colletotrichum isolates
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Taxonomy

Colletotrichum dianesei N. B. Lima, M. P. S. Câmara & S. J. Michereff sp. nov. (Fig. 4a–f)

Fig. 4
figure 4

Colletotrichum dianesei (from holotype.). Upper (a) and reverse (b) sides of cultures on PDA 7 days after inoculation; c brown to dark brown conidiomata and dark brown setae; d conidiogenous cells; e appressoria; f conidia. Bars: 10 μm

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MycoBank: MB803223.

Etymology: named in honor of professor José Carmine Dianese, University of Brasília (Brazil), for his contribution to Brazilian mycology.

Description: colonies on PDA at first white and becoming pale brownish to pinkish, reverse pale yellowish to pinkish, max. of 47.8 mm diam. in 5 days at 28 °C, growth rate 7.60–9.55 mm day−1 (\( \overline{x}=8.60\pm 0.95 \), n = 6) (Fig. 4a, b). Aerial mycelium greyish white, dense, cottony, absent conidial masses. Sclerotia absent. Acervuli brown to dark brown present in culture. Setae present but rare (Fig. 4c, d). Conidia common in mycelium, one-celled, smooth-walled, hyaline, cylindrical with obtuse to slightly rounded ends, sometimes oblong, 10.5–14.5 × 4–5.5 μm (\( \overline{x} = 12.0\pm 0.95\times 4.5\pm 0.40 \), n = 50) (Fig. 4f). Appressoria in slide cultures, brown, ovoid, sometimes clavate, 6.0–10.0 × 4.5–7.0 μm (\( \overline{x} = 7.5\pm 0.9\times 5.5\pm 0.5 \), n = 50) (Fig. 4e).

Teleomorph: not produced in culture.

Holotype: BRAZIL–Pernambuco, Petrolina, Farm Mapel (40° 34′ 23″, 9° 18′ 40″), on Mangifera indica fruits, 11 Apr 2010, Coll. N. Lima (holotype living culture CMM 4083; isotype in MFLU 1300058);

Known distribution: Pernambuco, Brazil.

Additional specimens examined: Additional specimens examined: Brazil, Pernambuco, Petrolina, Farm Mapel (40°34′ 23″, 9°18′40″), on Mangifera indica fruit, 11 May 2010, coll. N. B. Lima (paratype living cuture CMM4081, ex-paratype living culture MFLU 1300056). Brazil, Pernambuco, Petrolina, Farm Mapel (40°34′ 23″, 9°18′40″), on Mangifera indica fruit,11 May 2010, coll. N. B. Lima (paratype living cuture CMM4082, ex-paratype living culture MFLU 1300057). Brazil, Rio Grande do Norte, Ipanguaçu, Farm São João (36°53′03″, 5°31′29″), on Mangifera indica fruit, 9 Jun 2010, coll. N. B. Lima (paratype living cuture CMM4097, ex-paratype living culture MFLU 1300062). Brazil, Rio Grande do Norte, Ipanguaçu, Farm São João (36°53′03″, 5°31′29″), on Mangifera indica fruit, 9 Jun 2010, coll. N. B. Lima (paratype living cuture CMM4096, ex-paratype living culture MFLU 1300061). Brazil, Pernambuco, Petrolina, Farm copa fruit (40°34′00″, 9°23′08″), on Mangifera indica fruit, 23 May 2010, coll. N. B. Lima (paratype living cuture CMM4089, ex-paratype living culture MFLU 1300060). Brazil, Pernambuco, Petrolina, Farm copa fruit (40°34′00″, 9°23′08″), on Mangifera indica fruit, 23 May 2010, coll. N. B. Lima (paratype living cuture CMM4088, ex-paratype living culture MFLU 1300059).

Pathogenicity and virulence in fruits

All isolates of Colletotrichum were pathogenic to mango fruits. Ripe fruits affected by anthracnose develop sunken, prominent, dark brown to black decay. C . asianum were most virulent. There were no significant (P≤0.05) differences in virulence among the other species (Fig. 5).

Fig. 5
figure 5

Mean lesion lengths (mm) caused by Colletotrichum species associated with mango anthracnose in northeastern Brazil, 10 days after inoculation with conidia suspension (106 conidia/ml) onto wounded fruits of Tommy Atkins cultivar. Bars above columns are the standard error of the mean. Columns with same letter do not differ significantly according to Fisher’s LSD test (P≤0.05)

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Discussion

This study represents the first attempt to characterize Colletotrichum species associated with anthracnose of mango fruits in Brazil using a phylogenetic approach. Although C. gloeosporioides has previously been shown to be the causal agent of tropical fruit rots, the most striking discovery of this study is that none of the 47 strains isolated from mango fruits with anthracnose symptoms belong to the species C. gloeosporioides. Phoulivong et al. (2010) showed that C. gloeosporioides was actually not a common pathogen in the tropics and it was not the cause of anthracnose in mango in Laos and Thailand.

Phylogenetic analysis showed that most of the strains included in this study belong to the ‘gloeosporioides’complex. Five species of Colletotrichum were found associated with anthracnose of mango fruits, C asianum, C. fructicola, C. tropicale, C. dianesei and C. karstii, while C. asianum and C. karstii have been previously recorded from mango (Damm et al. 2012; Weir et al. 2012).

Colletotrichum dianesei was the most frequently isolated species with 51.1 % all the isolates, indicating it is the most frequent Colletotrichum species associated to mango in northeastern of the Brazil. Phylogenetic analysis reveals high suppot for the C. dianesei clade, which is closely related to C. siamense (Fig. 1). These two species are similar in conidial shape but differ in conidial size, C. dianesei having longer conidia with a mean length 12.06 μm, when compared with C. siamense (10.18 μm). Colletotrichum dianesei also differs from C. siamense in growth rate, with 8.60 mm day−1 and 9.30 mm day−1, respectively.

Colletotrichum asianum was the second most prevalent species with 27.7 % of all isolates. This species was originally described by Prihastuti et al. (2009) in Coffea arabica from Thailand. This is the first report this species associated with the mango fruits in Brazil. However, it is already known from Mangifera indica in Australia, Colombia, Japan, Panama and the Philippines (Weir et al. 2012).

Colletotrichum tropicale was described by Rojas et al. (2010) from Theobroma cacao in Panamá. This study represents the first report this species associated with the mango fruits worldwide. Rojas et al. (2010) noted that C. tropicale has been isolated from a wide range of hosts in forests in tropical America, from rotting fruit as well as leaf endophytes.

Colletotrichum fructicola was originally reported causing coffee berries in Thailand (Prihastuti et al. 2009). This species was also found as a leaf endophyte in several plants in Central America (originally described as C. ignotum (Rojas et al. 2010)). Colletotrichum fructicola biologically and geographically diverse. The species is presently known from C. arabica (Thailand), Pyrus pyrifolia (Japan), Limonium (Israel), Malus domestica and Fragaria × ananassa (USA), Persea americana (Australia), Ficus (Germany), Malus domestica (Brazil), Dioscorea (Nigeria), and Theobroma and Tetragastris (Panama) (Weir et al. 2012), and Vitis (China) (Peng et al. 2013). This is the first report of this species causing anthracnose in mango worldwide.

Colletotrichum karstii was recently reported from mango from Australia (Damm et al. 2012). It occurs on many host plants and is the most common and geographically diverse species in the C. boninense complex. This species was reported in China from Vanda sp. leaf and on several other orchids (Yang et al. 2011) and from Citrus leaves (Peng et al. 2012), as well as in infections from Phalaenopsis orchid petals in United States (Jadrane et al. 2012). Some isolates from Passiflora edulis in Brazil that were initially identified as C. boninense (Tozze et al. 2010), but later revealed to be C. karstii by GPDH sequences (Damm et al. 2012).

Pathogenicity testing using isolates from the five species of Colletotrichum showed that all species were pathogenic to mango fruits. Symptoms development may vary considerably with factors such as variety and condition of the fruit, humidity and temperature, and the concentration of inoculum (Simmonds 1965; Freeman et al. 1998). This result may not accurately reflect the true virulence potential of these species. Additional research should be conducted to determine the virulence potential of Colletotrichum species according to natural infections rather than artificial inoculations.

The Colletotrichum isolates from mango analyzed in this study showed high variability based on GPDH gene and the morphological characteristics. Five species were identified, with the majority of the species had more than one haplotype and a high number of morphotypes. The greater the genetic diversity of a population, greater evolutionary potential and hence the more likely it to adapt to changing environmental conditions (McDonald and Linde 2002). That is, the greater the diversity, the greater the chance there is an individual that is adapted to certain restrictive condition covering the population. Accordingly, such information is relevant because it can assist in the implementation of disease control measures more effectively.