Eur J Plant Pathol DOI 10.1007/s10658-012-0065-7 Morphological, pathological and genetic diversity of Colletotrichum species responsible for anthracnose in papaya (Carica papaya L) Claudia Torres-Calzada & Raul Tapia-Tussell & Inocencio Higuera-Ciapara & Daisy Perez-Brito Accepted: 16 August 2012 # KNPV 2012 Abstract Recently, anthracnose has become a major problem in papaya production and postharvest stages. The occurrence of both Colletotrichum gloeosporioides and Colletotrichum capsici has been demonstrated in this crop. The differential response of these pathogens to fungicides has highlighted the need to use rapid and accurate techniques to identify them. Thus, the objective of this study was to reveal the genetic diversity of Colletotrichum isolates in Mexican papaya fields. C. gloeosporioides-and C. capsicispecific primers were successfully used to detect the pathogens from different papaya parts. A combination of morphological characters, molecular techniques and pathogenicity tests were used to characterize 37 isolates from different localities of five papaya-producing states. Analyses of the 5.8-ITS region and arbitrarily primed-PCR revealed intraspecific groups; most of the isolates within these groups have the same geographical location and morphological characteristics. Knowledge of the genetic diversity of Colletotrichum spp. in Mexican papaya fields will facilitate the C. Torres-Calzada : R. Tapia-Tussell : D. Perez-Brito (*) Laboratorio GeMBio, Centro de Investigación Científica de Yucatán A.C., Calle 43 # 130, Col. Chuburná de Hidalgo, Mérida, Yucatán 97200, Mexico e-mail: daisypb@cicy.mx I. Higuera-Ciapara Centro de Investigación Científica de Yucatán A.C., Calle 43 # 130, Col. Chuburná de Hidalgo, Mérida, Yucatán 97200, Mexico identification of the pathogen population in this crop in order to select the appropriate fungicide to control anthracnose, as well as to improve genetic resistance breeding programs. Keywords AP-PCR . Characterization . 5.8S-ITS region Abbreviations AP-PCR Arbitrarily primed-polymerase chain reaction d. a. i. Days after inoculation ITS Internal transcribed spacers Introduction Anthracnose, caused by Colletotrichum species, is an economically important disease in tropical and subtropical areas worldwide, affecting a wide host range including vegetables, legumes, cereals and fruits (Bailey and Jeger 1992). During the last 5 years in Mexico, the prevalence of anthracnose in papaya production fields and postharvest has increased with subsequent losses of over 50 %, even after repeated treatment with fungicides. It has been shown this disease in papaya is caused by C. capsici and C. gloeosporioides in Mexico (TapiaTussell et al. 2008), USA (Tarnowski and Ploetz 2010) and Trinidad and Tobago (Rampersad 2011). The importance of species differentiation is critical for control Eur J Plant Pathol purposes, because Colletotrichum spp. often differ in their sensitivity to fungicides, resulting in inefficient control (Freeman et al. 1998). A reliable identification method therefore, can facilitate the development of appropriate disease management strategies. Also studies on the variability of the pathogen populations are needed to direct breeding efforts towards long-term resistance to anthracnose as has been seen in the development of Phaseolus vulgaris resistant cultivars against Colletotrichum lindemuthianum (Mahuku and Riascus 2004). Traditionally, Colletotrichum species have been identified by a range of cultural and morphological characteristics, such as conidial morphology, presence or absence of setae, fungicide sensitivity, colony colour and growth rate (Adaskaveg and Hartin 1997). Although valuable, these criteria alone are not always adequate as morphological characteristics may vary under different environmental conditions (Cannon et al. 2000). Molecular techniques, combined with morphological studies have proven to be effective for characterization of Colletotrichum species (Sreenivasaprasad and Talhinhas 2005; Van Hemelrijck et al. 2010). Sequence analyses of the ribosomal DNA (rDNA) (Photita et al. 2005), PCR with species-specific primers (Mills et al. 1992), fingerprinting with UP-PCR (Schiller et al. 2006), AP-PCR (Talhinhas et al. 2005) and RAPD (Sangdee et al. 2011) have been used extensively to determine genetic diversity within the genus. To our knowledge, molecular analyses to assess the genetic diversity among the Colletotrichum isolates responsible for anthracnose in papaya have not been performed. Thus, the objective of this work was to characterize the Colletotrichum species from Mexican papaya fields, using a range of molecular techniques including PCR with species-specific primers, ITS sequences and AP-PCR analyses, as well as morphological and pathological assays. Materials and methods Sampling and isolation of Colletotrichum spp. Papaya fruit, leaves and flowers with characteristic symptoms of anthracnose were collected at different papaya production sites in Mexico (Table 1). Diseased plant tissues taken from the advanced margin of lesions were cut into small pieces (5×5 mm) and disinfested by immersing them in 3 % NaOCl solution, followed by rinsing with sterile distilled water three times. They were then transferred onto Potato Dextrose Agar (PDA) medium and incubated at room temperature (25 °C) for 7 days. Pure cultures were obtained by single spore isolation and were maintained on PDA dishes at 25 °C for 7 days, before observation of cultural and morphological characteristics. Morphological and cultural characterization Each isolate was plated onto PDA at room temperature. Three 5-mm plugs were aseptically punched from actively sporulating areas near the growing edge of a 5day-old culture of these isolates. Each plug was placed onto PDA dishes and incubated under the same conditions as starter cultures. After 7 days, colony size, shape, margin and colour were recorded. Colony diameter of every culture was recorded daily for 7 days. Growth rate was calculated as the 7-day average of mean daily growth (mm per day). Three cultures of each isolate were investigated and experiments were conducted twice. For examination of conidial morphology, all isolates were subcultered as mentioned above. Cultures were washed with sterile water and drops of the suspension were placed on microscope slides and mixed with lactophenol/cotton blue to stain the conidia. Length and width were measured for 30 conidia per isolate. Conidial shape (cylindrical or falcate) was also recorded. Genomic DNA extraction and identification with species-specific primers For genomic DNA extraction, 10 pieces of agar culture (1×1 cm) obtained from the 7-day-old colonies grown on Richard’s V8 (RV8) medium were transferred into 250-ml Erlenmeyer flasks containing 50 ml of Nutrient Broth (NB). After incubation at 28 °C on an orbital shaker (100 rpm) for 7 days, the mycelia were collected by filtration, frozen at −80 °C for 2 h and lyophilized (LABCONCO 77530) until use. The total genomic DNA was extracted according to the method described by Tapia-Tussell et al. (2006) and diluted to a final concentration of 50 ng/μl. Eur J Plant Pathol Table 1 Colletotrichum isolates used in this study Species Isolate Tissue Location PCR amplification CgInt/ITS4 CcapF/CcapR C. gloeosporioides CGP1 Fruit Morelos, Q. Roo + − C. gloeosporioides CGP2 Fruit Quizás, Q. Roo + − C. gloeosporioides CGP3 Fruit Quizás, Q. Roo + − C. gloeosporioides CGP4 Fruit Quizás, Q. Roo + − C. gloeosporioides CGP5 Fruit Quizás, Q. Roo + − C. gloeosporioides CGP6 Fruit Quizás, Q. Roo + − C. gloeosporioides CGP7 Fruit Quizás, Q. Roo + − C. gloeosporioides CGP8 Fruit Quizás, Q. Roo + − C. gloeosporioides CGP9 Fruit Soledad de doblado, Veracruz + − C. gloeosporioides CGP10 Fruit Soledad de doblado, Veracruz + − C. gloeosporioides CGP11 Fruit Soledad de doblado, Veracruz + − C. gloeosporioides CGP12 Fruit Soledad de doblado, Veracruz + − C. gloeosporioides CGP13 Fruit Soledad de doblado, Veracruz + − C. gloeosporioides CGP14 Leaf Conkal, Yucatan + − C. gloeosporioides CGP15 Leaf Conkal, Yucatan + − C. gloeosporioides CGP16 Leaf Conkal, Yucatan + − C. gloeosporioides CGP17 Flower Conkal, Yucatan + − C. gloeosporioides CGP18 Flower Conkal, Yucatan + − C. gloeosporioides CGP19 Fruit Quizas, Q. Roo + − C. gloeosporioides CGP20 Fruit Quizas, Q. Roo + − C. gloeosporioides CGP21 Fruit Quizas, Q. Roo + − C. gloeosporioides CGP22 Fruit Quizas, Q. Roo + − C. capsici Ccg1 Fruit Tapachula, Chiapas − + C. capsici Ccg2 Fruit Tapachula, Chiapas − + C. capsici Ccg6 Fruit Morelos, Q. Roo − + C. capsici Ccg7 Fruit Morelos, Q. Roo − + C. capsici Ccg15 Leaf Valladolid, Yucatan − + C. capsici CCP1 Fruit Champoton, Campeche − + C. capsici CCP4 Fruit Champoton, Campeche − + C. capsici CCP6 Fruit Morelos, Q. Roo − + C. capsici CCP10 Fruit Quizás, Q. Roo − + C. capsici CCP11 Fruit Quizás, Q. Roo − + C. capsici CCP12 Fruit Quizás, Q. Roo − + C. capsici CCP14 Fruit Quizás, Q. Roo − + C. capsici CCP15 Fruit Quizás, Q. Roo − + C. capsici CCP16 Fruit Quizás, Q. Roo − + C. capsici CCP17 Fruit Quizás, Q. Roo − + Molecular identification was carried out using C. gloeosporioides-specific primers CgInt/ITS4 (Mills et al. 1992) and C. capsici-specific primers CcapF/ CcapR (Torres-Calzada et al. 2011a). PCR reaction (25 μl final volume) contained 25 ng of DNA, 1× PCR buffer (10×: 200 mM Tris–HCl, 500 mM KCl, pH 8.4; Invitrogen), 0.20 mM of each dNTP (Invitrogen), 1.5 mM MgCl2, 1 μM primers and 1 U Eur J Plant Pathol Taq polymerase (Invitrogen). DNA amplification was performed in a GeneAmp 9700 DNA Thermal Cycler (Perkin-Elmer), and consisted of an initial denaturing step at 95° for 5 min, followed by 25 cycles of 30 s at 94 °C, 2 min at 62 °C and 2 min at 72 °C, and a final extension step of 5 min at 72 °C. PCR products were separated by electrophoresis in 1.5 % (w/v) agarose gels and visualized by ethidium bromide staining. 5.8S-ITS sequence analysis The internal transcribed spacer regions, including the 5.8S rDNA, were amplified using universal primers ITS1 and ITS4 (White et al. 1990). The PCR reaction was carried out in 25 μl consisting of 25 ng of DNA, 1× PCR buffer (Invitrogen), 0.20 mM of each dNTP (Invitrogen), 1.5 mM MgCl2, 1 μM primers and 1 U Taq polymerase (Invitrogen). DNA amplification was performed in a GeneAmp 9700 DNA Thermal Cycler (Perkin-Elmer), and consisted of an initial denaturing step at 95° 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 editing were carried out with the BioEdit Sequence Alignment program (Altschul et al. 1990). Sequences were then compared against C. gloeosporioides and C. capsici sequences downloaded from the GenBank Database. Data were analyzed using the Molecular Evolutionary Genetic Analysis (MEGA) software version 5.0 (Tamura et al. 2011), distances were calculated using the JukesCantor model (assumes equal probability of independent change at all sites), and a tree was produced using the UPGMA, Neighbour Joining and Maximum Likelihood methods. A bootstrap analysis using 1,000 re-samples of the data was done. The ITS5.8S sequence of Colletotrichum anthrisci was obtained from the GenBank database and used as an outgroup. AP-PCR (arbitrarily- primed PCR) analysis Five 15-bp primers derived from the repeated sequences (GTG)5, (GACAC)3, (CAG)5, (GAC)5 and (TCC) 5 were used for AP-PCR analysis (Nguyen et al. 2009). PCR reaction was carried out in 25 μl reaction volumes containing 50 ng of DNA, 1× PCR buffer (Invitrogen), 0.25 mM of each dNTP (Invitrogen), 2 mM MgCl2, 0.8 μM primers and 1 U Taq polymerase (Invitrogen). DNA amplification was performed in a GeneAmp 9700 DNA Thermal Cycler (Perkin-Elmer), and consisted of an initial denaturing step at 95° for 5 min, followed by 40 cycles including: denaturing at 95 °C for 40 s, annealing for 1 min at either 42 °C for (GACAC)3 and (TCC)5, or 50 °C for (GTG)5 and (GAC)5, or 60 °C for (CAG)5, extension for 1 min at 72 °C; and a final extension step of 5 min at 72 °C. The amplified PCR products were loaded onto a 1.5 % agarose gel and the bands were detected by ethidium bromide staining. The presence or absence of bands produced by AP-PCR was recorded and a binary matrix was generated for each marker. A similarity matrix was calculated using the Dice coefficient and employed later in a cluster analysis performed by the Unweighted Pair-Group Method with Arithmetic averaging, using PAST software (Hammer et al. 2001). Branch support of the tree was assessed by bootstrapping (Felsestein 1985) using 1,000 replications. Pathogenicity tests Pathogenicity tests were performed with a representative set of isolates, from all morphological groups and locations, using papaya fruit (C. papaya var. Maradol). Fruit were disinfested by immersing them in 1 % NaOCl solution for 1 min, washed twice with sterile distilled water and dried at room temperature. An aqueous conidial suspension (1 × 106 spores ml−1) was prepared from 7-day-old cultures of each isolate and then placed on the fruit by the wound/drop method (Kanchana-Udomkan et al. 2004). This method involved pin-pricking the surface of the fruit to a 1mm depth and then placing 20 μl of conidial suspension over the wound. Three fruits were tested per isolate and experiments were conducted twice. The inoculated fruit, along with appropriate controls (fruit inoculated with sterile distilled water) were incubated at room temperature (25 °C) in humid chamber. Symptoms were recorded 5 days after inoculation (d.a.i.) and re-isolation, according to Koch’s postulates, was made from all resulting lesions. Eur J Plant Pathol Statistical analyses Identification with species-specific primers Statistical analyses for conidial morphology, growth rate and pathogenicity tests were performed using the R statistical package version 2.13.1 (Ihaka and Gentleman 1996). Data were analyzed using ANOVA in order to determine the significance of differences in conidia dimensions, growth rates and lesion diameters post-inoculation with C. capsici and C. gloeosporioides. Means were compared using Tukey’s multiple range test. All 37 Colletotrichum spp. isolates collected from papaya, plus the C. capsici reference isolate (C. capsici ATCC 48574), were amplified with the species-specific primers for C. gloeosporioides and C. capsici. A 450-bp DNA fragment was amplified with the C. gloeosporioides- specific primers CgInt and ITS4 in 22 of the 37 isolates obtained. The rest, 15 of 37 isolates, and the reference C. capsici ATCC 48574 isolate, amplified a 390-bp DNA fragment when the C. capsici-specific primers CcapF and CcapR were used (Fig. 2). Results Morphological and cultural characterization Colletotrichum isolates were obtained mainly from lesions on papaya fruit, but some isolates were obtained from leaves, petioles and flowers. In total, thirty seven were obtained from seven different orchards located in five papaya producing states in Mexico (Fig. 1). Preliminary identification was based on the morphological description of Colletotrichum species (Shenoy et al. 2007; Sutton 1992). Of all isolates collected, 15 fitted the description of C. capsici and 22 fitted the description of C. gloeosporioides. Differences in colony characteristics among the isolates resulted in formation of morphological groups. C. gloeosporioides isolates formed six distinct groups and C. capsici isolates formed another three different groups. Cultural characteristics Isolates of the C. gloeosporioides groups 1, 2, 3 and 4 produced white colonies. In some of them, it was possible to observe orange conidial masses in the Fig. 1 Map of Mexico, where 37 isolates of Colletotrichum spp. were collected from papaya plants with anthracnose symptoms Eur J Plant Pathol Fig. 2 Amplification products obtained using species-specific primers, a PCR amplification with the primer pair CgInt/ITS4 for detection of Colletotrichum. gloeosporioides and b PCR amplification with the primer pair CcapF/CcapR for detection of C. capsici. Lane M, DNA marker (1 kb DNA ladder); lanes 1–7, representative C. gloeosporioides isolates; lanes 8–12, representative C. capsici isolates; lane 13, C. capsici ATCC 48574; lane 14, negative control centre (group 1 and 2) or distributed in concentric rings throughout the colony (group 3). Colonies produced by C. gloeosporioides isolates from group 5 and 6 were olive to black coloured, but the presence of dark grey-coloured conidia were observed only in group 6. Most of the C. capsici isolates (67.5 %, group 9) had pale grey to black coloured colonies with beigecoloured conidial masses forming concentric rings around the dish. Colonies from C. capsici group 7 and 8 were both light salmon, but the distribution of conidial masses was different for each group (Table 2, Fig. 3). isolates (P 00.001), especially those belonging to group 4 (Table 3). Growth rate There was no significant difference in growth rate among isolates of C. gloeosporioides. However, isolates from group 7 of C. capsici grew faster than those in groups 8 and 9 (P00.003). The C. gloeosporioides isolates grew significantly faster than the C. capsici Conidial morphology Two types of conidia were observed: cylindrical and falcate. C. capsici isolates had significantly longer conidia than those of C. gloeosporioides. C. capsici isolates produced one-celled, hyaline, falcate conidia with acute apex. The average length and width of the conidia were 22.8–23.8 μm and 3–3.02 μm, respectively. The C. gloeosporioides conidia were all cylindrical, with both ends rounded. The average length and width of the conidia were 13.56–14.24 μm and 4– 4.02 μm, respectively (Table 3). 5.8S-ITS sequence analysis The ITS region, including the 5.8S gene of all isolates was successfully amplified and sequenced. The data Table 2 Morphological features of the Colletotrichum species causing anthracnose in papaya Colletotrichum species Group % isolates Colony appearance Shape 5.4 Margin Circular Entire Colour C. gloeosporioides 1 C. gloeosporioides 2 8.1 Circular Curled White flocculose, with orange conidial masses in centre C. gloeosporioides 3 10.8 Circular Curled White to pale grey, with orange conidial masses produced in concentric rings C. gloeosporioides 4 5.4 Circular Curled White flocculose, with no visible conidial mass C. gloeosporioides 5 24.3 Circular Entire Olive to black, with no visible conidial mass C. gloeosporioides 6 5.4 Circular Curled Dense, olive to black, with dark grey-coloured conidial masses in centre C. capsici 7 5.4 Circular Entire Light salmon to pale grey, with conidial masses in centre C. capsici 8 8.1 Circular Undulate White to light salmon, with conidial masses produced in concentric rings C. capsici 9 27.0 Irregular Entire White to orange, with orange conidial masses in centre Pale grey to black, with conidial masses produced in concentric rings Eur J Plant Pathol Fig. 3 Morphotypes of Colletotrichum gloeosporioides and C. capsici isolated from papaya, a upper side of the colony and b reverse side of the colony set contained 450 characters of which 53 were parsimony informative. The BLAST similarity search confirmed the results obtained by the species-specific PCR analyses. Eleven reference sequences from other reports were downloaded from the NCBI database and used in this study: HQ896483, HQ845103, HQ264179, HM562711 and FJ972609 for C. gloeosporioides; HQ271458, GQ369594, HQ271468, DQ453990 and EF683602 for C. capsici; and GU227845 for C. anthrisci. Phylogenetic analyses performed by UPGMA, Neighbour Joining and Maximum Likelihood methods produced similar topologies; one of the trees is shown in Fig. 4. Phylograms separated the Colletotrichum isolates into two distinct groups named A and B. Group A (100 % bootstrap) includes all C. gloeosporioides isolates and group B (100 % bootstrap) includes all C. capsici isolates. Group A was divided into four subgroups designated A1, A2, A3 and A4. Subgroup A1 included 59 % of the C. gloeosporioides isolates, along with the reference sequences HQ896483, HQ264179 and HM562711. A2 included the CBS reference isolate C. gloeosporioides FJ972609, and isolates CGP14, CGP15 and CGP16, showing 99 % identity. Group B was divided into subgroups B1 and B2. Most of the isolates (57.14 %) were included in subgroup B1. B2 included six isolates which were 99 % identical to the reference isolates HQ271458, GQ369594, HQ271468, DQ453990 and the C. capsici sequence from the epitype strain EF683602. In some cases, isolates were separated according to geographical origin, for example group A3 contains isolates Table 3 Conidial characteristics of the morphological groups of Colletotrichum species Species Group Conidial characteristics Shape Length(μm) Width(μm) Growth ratea (mmday−1) Lesionb diameter (mm) C. gloeosporioides 1 Cylindrical, with rounded ends 13.98±0.04bc 4.01±0.02abc 11.02±0.69abc 23.89±0.13bc C. gloeosporioides 2 Cylindrical, with rounded ends 13.97±0.05b 4.02±0.02ab 10.76±0.68ab 24.17±0.31b C. gloeosporioides 3 Cylindrical, with rounded ends 14.24±0.49b 4.0±0.0b 11.15±0.71ab 23.43±0.49b C. gloeosporioides 4 Cylindrical, with rounded ends 14.02±0.01b 4.0±0.0b 11.57±0.42a 22.18±0.28b C. gloeosporioides 5 Cylindrical, with rounded ends 13.77±0.51b 4.0±0.0b 10.90±0.57ab 23.76±0.67b C. gloeosporioides 6 Cylindrical, with rounded ends 13.56±0.62b 4.0±0.0b 10.93±0.31ab 24.34±0.36b C. capsici 7 Falcate, with acute apex 23.76±0.20a 3.0±0.0d 10.42±0.49b 19.31±0.21a C. capsici 8 Falcate, with acute apex 22.83±0.07a 3.02±0.017c 8.17±0.07c 18.78±0.45a C. capsici 9 Falcate, with acute apex 23.84±0.21a 3.0±0.0d 9.13±0.56c 19.09±0.37a a Growth rate measured as lesion diameter in mm per day b Lesion diameter recorded 5 d.a.i. c Mean values in the same column and followed by the same letter are not significantly different (P≤0.05) according to Tukey test Eur J Plant Pathol Fig. 4 Maximum Likelihood (ML) tree based on the ITS data from collected isolates and published sequences. The tree was rooted with Colletotrichum anthrisci and Tamura-Nei model was used. Bootstrap test was done (1,000 replicates) and values above 50 % are shown on internal branches only from Quintana Roo state. Also, groups A2 and A4 are formed exclusively for isolates from Yucatan and Veracruz states respectively. different primers. Gels showing diversity among representative isolates using the primer (CAG)5 are presented (Fig. 5). The average number of polymorphic bands per primer was 11 and ranged in size from 300 to 5,000 bp. Information on banding patterns obtained from all the primers was used to determine genetic distance between isolates and to construct a dendrogram. AP-PCR analysis AP-PCR analysis was done to characterize 31 representative Colletotrichum isolates using six Eur J Plant Pathol Fig. 5 Band patterns of arbitrarily primed polymerase chain reaction amplified genomic DNA of representative a Colletotrichum gloeosporioides and b C. capsici isolates using primer (CAG)5. Lane M, DNA marker (1 kb DNA ladder); lane C, negative control According to this dendrogram, the Colletotrichum spp. we studied could be divided into 11 main groups (Fig. 6). Groups A1 to A6 had all the C. gloeosporioides isolates, while groups B1 to B5 contained the C. capsici isolates. The number of isolates within a cluster ranged from two to five. However, isolate Ccg2 formed a separate group. The clustering of the C. gloeosporioides isolates based on AP-PCR data showed a relationship with geographical distribution of isolates in wellsupported clusters. However, the clustering of the C. capsici isolates was not always associated with the geographical localities from which the isolates had been obtained as only isolates in groups B1 and B2 clustered according to their region, whilst groups B3 and B4 contained isolates from three different geographic regions in the same cluster. Pathogenicity tests Symptoms characteristics of anthracnose were observed at the inoculation site 5 d.a.i. All C. gloeosporioides and C. capsici isolates were capable of causing infection, but there were significant differences between the species in mean lesion diameter (P00.001) (Table 3). Two different types of lesions could be distinguished on the inoculated fruit, corresponding to each of the species tested (Fig. 7). Lesions produced by C. gloeosporioides were water soaked, sunken and contained orange conidial masses. Lesions caused by C. capsici were brown to black, Eur J Plant Pathol Fig. 6 Unweighted Pair-Group Method with Arithmetic average dendrogram showing diversity and relationships among Colletotrichum isolates from papaya based on Arbitrarily Primed-PCR analyses using a combined dataset from primers (GAC)5, (GTG)5, (TCC)5, (CAG)5 and (GACAC)3. One thousand bootstrap test and Dice coefficient distance matrices were used with grey spore masses and abundant acervuli arranged concentrically. known as an aggressive pathogen in pepper (Shenoy et al. 2007) and has recently been associated with anthracnose in papaya (Tapia-Tussell et al. 2008) and physic nut (Torres-Calzada et al. 2011b). Consequently, there has been considerable interest in the development and utilization of PCR-based tools that allow the rapid and accurate diagnosis of these species. In our study, C. gloeosporioides isolates could be distinguished from C. capsici isolates by using speciesspecific primers. Previous reports indicated that C. capsici-specific primers were successfully used to detect the pathogen from different papaya parts such as fruit, petioles, leaves and flowers (Torres-Calzada et al. 2011a). Similarly, C. gloeosporioides-specific primers have been used for the detection of this species in other tropical crops like tamarillo, passiflora and mango (Afanador-Kafuri et al. 2003). All these studies Discussion A combination of morphological characters, molecular techniques and pathogenicity tests confirmed that C. gloeosporioides and C. capsici are the causative agents for anthracnose disease of papaya fruit in Mexico. C. gloeosporioides and C. capsici are both pathogens of many economically important hosts. C. gloeosporioides is a common pathogen on a variety of tropical crops such as mango, avocado and papaya (Silva-Rojas and Avila-Quezada 2011; Tapia-Tussell et al. 2008). On the other hand, C. capsici is best Eur J Plant Pathol Fig. 7 Pathogenicity test showing anthracnose symptoms 5 days after inoculation. a–c Colletotrichum gloeosporioides; d–f C. capsici; a, d artificially infected fruits; b conidial masses on fruit surface; e acervuli with setae; c, f conidia demonstrate the reliability of these primers for fast and accurate identification of pathogens, especially in cases where morphological identification is complicated (Whitelaw-Weckert et al. 2007). The cultural characteristics we observed separated C. gloeosporioides into six different morphotypes. The isolates used in this study, belonging to groups 2, 3 and 6, were similar in appearance to those described by Photita et al. (2005) in reference to the groups they designated as 1, 2, and 3. Also, the C. gloeosporioides groups 4 and 6 fit the description of C. gloeosporioides group 4 and 2 characterized by Rampersad (2011) and Than et al. (2008), respectively. Cultural characteristics separated C. capsici isolates into three different morphotypes. The C. capsici group 3 described in this paper is consistent with the morphological characterization made by Shenoy et al. (2007). However, the appearances of morphotypes 1 and 2 have not been previously reported. Within the Colletotrichum genus, analyses of ribosomal DNA have been extensively used for species delimitation (Mills et al. 1992). In the present study, ITS sequence analysis was reliable for interspecific separation among Colletotrichum spp. Moreover, this comparison was useful to elucidate the differences between the isolates even below the species level, separating C. gloeosporioides into groups A1 to A4 and C. capsici into groups B1 and B2. Interestingly, in some cases, geographical distribution was related to phylogeny, as shown in groups A2, A3 and A4, where the isolates within the groups belong to the same location. Johnston and Jones (1997) reported the presence of different groups based on rDNA sequence analyses among the Colletotrichum spp. isolates from fruit-rot in New Zealand. Their results discriminate among isolates of different morphological and cultural groups. We also assessed the diversity of the Colletotrichum spp. populations using AP-PCR molecular markers. The clustering in the AP-PCR dendrogram for both analyzed species was associated with the geographic localities from which the isolates were obtained in all cases, except clusters B3 and B4, which contained isolates from different geographic regions. It is interesting to note that isolates within clusters A5, A6 and B1 also belong to the same morphological group. These findings indicate a link between genotype, geographical distribution, morphological Eur J Plant Pathol characteristics and phylogeny. Genetic diversity of C. gloeosporioides and C. capsici has been described previously for other hosts (Afanador-Kafuri et al. 2003; Than et al. 2008). Similarly to previous reports, higher variability was observed within the C. gloeosporioides populations where the genetic heterogeneity may be explained by the presence of a perfect stage (Freeman et al. 1998). Pathogenicity tests with the Colletotrichum species isolated, showed that all were able to infect and cause symptoms in wounded papaya fruit, proving that both species were causal agents of anthracnose infection on papaya. These results provide evidence that, in anthracnose pathosystems, the same host is often infected by different Colletotrichum species as has been observed in crops such as avocado (Silva-Rojas and Avila-Quezada 2011) and pepper (Than et al. 2008). In this study, it was possible to isolate both C. gloeosporioides and C. capsici from fruit, flowers and leaves, which concurs with previous reports indicating that some Colletotrichum species are commonly found infecting not only fruit, but other parts of the plant (Howard et al. 1992). Further studies using different inoculation methods should be carried out to assess the cross-infection potential of these two species from papaya. To our knowledge, this is the first study conducted to determine the genetic diversity of Colletotrichum species causing anthracnose in papaya. The high diversity observed in both species might be due to the different climate conditions found within our country. Also, the ability of both species to infect different hosts suggests the possibility of cross-infection among cultivars. In our region, papaya orchards are often located adjacent to other horticultural crops such as Habanero pepper (Capsicum chinense), facilitating dispersion of the pathogen from one crop to another and therefore representing a high-risk situation for disease control in the field. Determining the genetic diversity present facilitates the identification of pathogen populations in crops in order to select appropriate control measures, as different species or even subgroups vary in their sensitivity to fungicides (Van Hemelrijck et al. 2010), thereby allowing producers to reduce the economic losses in crop production caused by this disease. Also this knowledge will be useful for breeding programs for genetic resistance to anthracnose in papaya, considering that the obtaining of an anthracnose resistant papaya variety is the best control measure. Acknowledgments This research was supported by the Quintana Roo Produce Foundation through Grant 23-2009-1712. 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