Morphocultural and molecular characterization of papaya tree Colletotrichum spp .

Papaya cultivation has great economic importance in tropical and subtropical countries, and Brazil is one of the largest producers of papaya (Carica papaya L.) in the world. However, productivity is hampered by plant health problems, particularly the pathogen Colletotrichum spp., which causes anthracnose and great damages on postharvest handling. This study characterized the morphocultural and genetic diversity of 21 isolates of papaya Colletotrichum spp. from different Brazilian states. The species were identified using the taxon-specific primers for Colletotrichum gloeosporioides and Colletotrichum acutatum. Eleven ISSR molecular markers were used to investigate the genetic diversity of the isolates. Moreover, the mycelial growth rate, pathogenicity, average diameter and colony color parameters were used in the morphocultural characterization. Of the 21 isolates, 19 were identified as C. gloeosporioides and two as C. acutatum. The ISSR markers showed great genetic diversity between the C. gloeosporioides and C. acutatum isolates, especially those from different locations. The morphocultural aspects displayed high polymorphism, and Linhares-1 and Linhares-2 isolated stood out for having unique characteristics when compared to other isolates. The pathogenicity test was positive for all isolates, but with different severity degree. C. gloeosporioides and C. acutatum displayed high genetic diversity among the isolates from different locations, and great morphocultural variability among isolates of papaya Colletotrichum.


INTRODUCTION
Brazil is the third largest fruit producer in the world and ranks second in papaya production (Carica papaya L.), accounting for approximately 12.5% of world production.
Over 1.5 million tons were produced in 2012 (FAO 2015).In recent years, however, the industry has been facing complex problems of a different nature, particularly *Corresponding author.E-mail: juliana.uem@outlook.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License regarding plant health.In this context, anthracnose, a disease caused by fungi of the Colletotrichum genus, stands out.
Anthracnose results in pre-and post-harvest losses.In general, pathogens associated with complex papaya anthracnose symptoms are classified as Colletotrichum gloeosporioides.However, recent studies have shown that this species is not always the only one involved in the disease (Prihastuti et al., 2009;Phoulivong et al., 2010).There are several species of the Colletotrichum genus that cause numerous diseases in various hosts, whose symptoms are not always visible until the fruit begins the maturation process (Prusky, 1996).The hosts include more than 470 genus of plants, where the fungi coexist as pathogens or endophytes (Cannon et al., 2008;Lu et al., 2004).Therefore, it is essential to identify the prevailing species since the epidemiology of fungal species and the control methods vary (Torres-Calzada et al., 2013).In addition, the precise determination of the Colletotrichum species is critical to the quarantine programs, for plant genetic improvement, and even to control these pathogens (Freeman et al., 1998;Cai et al., 2009).
There are many fungal species considered cryptic, forming a complex, including C. gloeosporioides and C. Dematium, among others (Shivas and Cai, 2012).According to Crous and Groenewald (2005), this complex can be extended to virtually every species of plant pathogenic fungi.The cryptic species are important due to the diversity of symptoms, host range, and geographic distribution.
Several works on the Colletotrichum-Carica papaya pathosystem have been published in recent years, including epidemiological aspects (Vásquez-López et al., 2012), genetic diversity, phylogeny, population structures (Andrade et al., 2007;Phoulivong et al., 2010;Rampersad, 2011), control (De la Cruz, et al. 2014;Barrera-Necha et al., 2008;Al-Eryani-Raqeeb et al., 2009).However, given the importance of the problem, and despite the great contribution of information created, there is a need for additional information regarding taxonomic analysis, to help define control strategies and other purposes.
Molecular methods based on DNA sequences are indispensable especially for cryptic species (Shivas and Cai, 2012).In general, studies of this nature are becoming popular for Colletotrichum spp.since the methods based on morphological and cultural tests have not always been satisfactory (Freeman et al., 1998).The alternative inter simple sequence repeat (ISSR) technique characterizes complex genomes, allowing the detection of polymorphisms in regions flanked by microsatellite DNA without isolating and sequencing specific DNA fragments (Cançado et al., 2012).
The ISSR marker is multilocus and, therefore, does not require prior knowledge of the DNA to be evaluated.It is a highly reproducible technique, low cost, and easy to use (Sserumaga et al., 2013).The ISSR markers are very effective in understanding the phylogenetic relationships of fungi in genetic diversity studies due to their high polymorphism (Schwarzenbach et al., 2007).When studying the several pathosystems, it is important to investigate the existence of genetic structures among the isolates of a particular pathogen from different producing regions, as well as those from specific niches, and their possible genetic divergence levels.The study on the species complex is important because it contributes to understanding its population dynamics.Genetic structure and gene flow influence the generation and distribution of new genotypes that affect the evolutionary and adaptive potential of the pathogen under changing selection pressure, and contribute to population viability and survival (Meng and Chen, 2001;Rampersad, 2013).
It is assumed that isolates from papaya that have never been treated with a fungicide may have specific characteristics compared to those from intensive production areas.Genetic data can contribute to quantifying genetic diversity and, also, lead to results that can contribute and guide the breeding programs.The molecular information complements the knowledge about the morphocultural aspects of the isolates, assesses, validates and facilitates comparisons to species that have already been described (Gaiero et al., 2011).To date, the literature on the genetic diversity of Colletotrichum from papayas in Brazil is somewhat generic and scarce.Studies in this field have been published by Peres et al. (2002) and Andrade (2007).This study characterized the morphocultural and genetic diversity of 21 Colletotrichum spp.isolates of papayas from different Brazilian states.

Origin, cultivation and preservation of Colletotrichum spp.
A total of 21 isolates of Colletotrichum were collected from two types of papaya, 'Formosa' and 'Solo' (Sunrise Solo, Improved Sunrise Solo cv.72/12 'or Tainung 1 or 2) in Itápolis/SP, Jaboticabal/SP, Maringá/PR, Umuarama/PR, Linhares/ES and Freitas Teixeira/BA, in 2014 (Table 1).The fruits were collected, stored in coolers and transported to the Phytopathology Laboratory of FCAV/UNESP, in Jaboticabal, where the pathogen was isolated.Most of the fruit was collected from producing areas, where all agricultural practices indicated for the culture are common, including the use of pesticides.However, the fruit collected in Jaboticabal originated from family farms where commercial and agronomic practices, including the use of pesticides, were not used.The harvested fruits were maintained in the laboratory at room temperature under 12/12 h photoperiod, and at the onset of the symptoms, the pathogen was isolated.Symptomatic tissue fragments of approximately 5 mm 2 were removed, disinfected, rinsed in sterile water, dried on filter paper and placed in Petri dishes containing potato dextrose agar (PDA) medium.Subsequently, the petri dishes were incubated in BOD incubators at 25°C and 12 h photoperiod.After seven days, the typical Colletotrichum spp.colonies were selected, followed by subculturing, and subsequent identification based on the morphophysiological aspects of the culture (Sutton, 1992).From the typical and pure colonies of the pathogen, monosporic cultures were obtained and stored in mineral oil.The Colletotrichum spp.isolates were used in subsequent studies.

Molecular identification of Colletotrichum spp. isolates
For DNA extraction, the discs with the isolates' colonies were transferred to 110 ml glass bottles containing PD (potato-dextrose) liquid culture medium and kept for ten days.After this, the mycelium was sieved out from the culture medium, washed with distilled water, drained and dried on a Petri dish, and kept at room temperature for approximately 12 h.The dried mycelium was then macerated to form a dry powder in liquid nitrogen, which was transferred to 2.0 ml Eppendorf tubes.
DNA extractions were based on the Kuramae-Izioka protocol (1997).The quantity and quality of extracted DNA were assessed by measuring the absorbance of each sample using a NanoDrop 100 spectrophotometer (Uniscience).The DNA samples extracted from Colletotrichum spp.were subjected to polymerase chain reaction (PCR) reaction with eleven ISSR primers (Table 2) and with two specific primers designed and developed by Mills et al. (1992), for amplifying the bands of about 500 bp for CaInt2 and 450 bp for CgInt, specific for C. acutatum and C. gloeosporioides, respectively, which were used in conjunction with the universal primer ITS4 to amplify the ITS region (White et al., 1990).Two isolates, one from C. acutatum, and one from C. gloeosporioides were used as amplification standards.They originated from tissues containing typical anthracnose symptoms, previously identified by sequencing the ITS2-ITS1-5.8Sregion, using the primer pair ITS1/ITS4 (White et al., 1990).The Table 3. Grades attributed to edge and reverse coloring of colonies while determining the presence of sectors as much as possible.PCR reactions were performed using 1X buffer (50 mM KCl, 200 mM Tris-HCl, pH 8.4); dNTP's (dATP, dTTP, dGTP, dCTP, 2.5 mM each) 0.2 mM, 2.0 U Taq polymerase, 2 mM MgCl2, 5 pmol primer, 80 ng DNA and sterile pure water q.s.p. 20 μl.The amplification reactions with primers were performed in a Nexus thermal cycler (Eppendorf), using one cycle at 95°C for 3 min, 35 cycles of 94°C for 40 s, temperature-specific primer for amplification for 1 minute and 72°C for 1 min, and ending with 1 cycle at 72°C for 10 min.The PCR product was revealed by electrophoresis in TEB 1X buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) using 1.5% agarose gel containing ethidium bromide (0.5 μg/ml) and visualized under the UV light of the Gel Doc XR (Bio-Rad) photographic equipment.The molecular size standard used was the 100 bp "DNA Ladder Plus" marker (Fermentas).The primers used were Invitrogen Life Technologies.The choice of primers was based on work carried out with species of the Colletotrichum genus or that presented high polymorphism (Gupta et al., 1994;Ratanacherdchai et al., 2010;Rampersad, 2013).

Morphocultural characterization of Colletotrichum sp.
All isolates used in molecular identification were previously grouped based on their origin.The appearance and color of the colonies were assessed.To this end, colonies' discs of 5 mm diameter were extracted from the margins of 7 day cultures, grown on PDA medium and transferred to new dishes containing the same medium.These cultures were then incubated at 25°C under a continuous fluorescent light.After ten days of incubation, period in which the colonies uniformly reached the edges of the plates while color stabilized, edge and reverse were evaluated, and the possible presence of sectors, as well.Three replications were used for each isolate.Each sample unit was represented by a Petri dish containing their isolates, the color and the presence of sectors were graded qualitatively (Table 3).
For the pathogenicity test, healthy papaya cv.Formosa fruits were subjected to the disinfection method according to Sanchez (1990).The fruits were superficially wounded with a sterile needle, and a drop of a suspension containing 1-2 x 10⁶ conidia.mL of each isolate (7-day cultures on PDA), was placed over the wound using a 20 µl pipette.The fruits were placed in plastic bowls with polystyrene and water film at the bottom, covered with plastic wrap and kept in a cold chamber at 25°C and a 12 h photoperiod.The isolates were evaluated at 2, 4 and 6 days after inoculation (Table 3) (Andrade et al., 2007 modified).The experiment was conducted in a completely randomized design with four replicates per isolate.Each repetition consisted of a fruit inoculated at one point.As a control, fruits were inoculated with sterile water.
Colony diameters were measured daily perpendicular to each other, up to when the first colony of an isolate reached the edge of the plate.From these data, the mycelial growth rate (MGR) was determined according to Nechet (1999), using the formula employed by Oliveira (1991).

Data analysis
The amplification products, visualized in the gel, produced by each primer were used for preparing a genetic similarity array: present (1) or absent (0).The binary matrices were used to obtain estimates of genetic similarities, using the XLSTAT software (Addinsoft®, 2014 version) and the Jaccard coefficient.The Unweighted Pair Group Method with Arithmetic Average (UPGMA) was used to group the genotypes.
The results of IVCM and diameter of the colonies after 7 days of culture were analyzed statistically first as individual characteristics.The statistical design was completely randomized with 21 isolates and three replicates per isolate.Each sample was represented by a petri dish, where the Colletotrichum isolates were cultivated.Means were compared using the Tukey test (P≤ 0.05).Multivariate exploratory analysis, cluster analysis by the hierarchical method and principal component analysis, which allowed group evaluation of all variables, were also performed as a complement.
The hierarchical clustering technique links the samples by their associations, producing a dendrogram where similar samples are grouped together according to the chosen variables (Moita Neto and Moita, 1998).The similarity between the centroids of each isolate was measured using the Euclidean distance (a measure of dissimilarity) for a set of seven variables while the method of Ward was adopted for the grouping strategy.The result of the analysis to help characterize the groups is presented as a dendrogram.
The principal component analysis allows summarizing the greatest amount of original information contained in p variables (edge and reverse coloring of the colonies, presence or absence of sectors, colony diameter, and mycelial growth rate, MIGS) in orthogonal latent variables called major components, which are linear combinations of the original variables created with the eigenvalues of the data covariance matrix (Hair, 2005).The Kaiser criterion is used to designate the main components.The eigenvalue preserves the relevant information when it is greater than one.

RESULTS AND DISCUSSION
Only two of the 21 isolates subjected to PCR with the CaInt2/ITS4 primer pair molecular identification (T from F BA1 and Linhares ES1) were amplified for the 450 bp specific band of C. acutatum.The remaining isolates  4).
In recent years, ISSR primers have become an important tool in molecular research of the genetic diversity of Colletotrichum (McKay, 2009;Rampersad, 2013;Ratanacherdchai, 2010) and other fungal genera due to the high reproducibility compared to other markers based on non-specific PCR, such as RAPD.
In this study, two species of Colletotrichum spp.associated with the symptoms of anthracnose in the analyzed fruits have been identified: C. gloeosporioides and C. acutatum similar to the results in the literature.These results demonstrate the feasibility of using ISSR molecular markers in the taxonomy of Colletotrichum spp.Species associated with papaya.However, it is acknowledged that follow-up studies with Colletotrichum isolates from more locations and in greater numbers may help to detect additional new species, which have not yet been found in studies conducted under Brazilian conditions.
Generally, it is accepted in various parts of the world that the symptoms of anthracnose in papaya result from C. gloeosporioides infections (strictu sensu).However, the results of recent studies related to the taxonomy of the causative agents associated with the anthracnosetype symptoms, in several species of plants, including tropical fruits, should bring deep modifications in this scenario.Among these studies, there are analyses related to the diversity of fungal species (Phoulivong et al., 2010;Torres-Calzada et al., 2013), which obviously are going to contribute significantly to better understanding the pathogen-host relationship, including the development and validation of new and efficient control strategies of their causative agents.According to Andrade et al. (2007), to develop appropriate control strategies it is essential to thoroughly understand the various epidemiological relationships.
The 11 ISSR primers used allowed the coding of 103 loci, ranging from 7 (P10 and AF80822) to 14 (P14).The obtained dendrogram shows four distinct groups with common traits within the groups, the origin of the isolates.Group I consists of isolates from Paraná (Maringá PR1, Maringá PR2, Umuarama PR1 and Umuarama PR2); Group II, from Espírito Santo (Linhares ES1, Linhares ES2 and Linhares ES3), and Group III, from Bahia (Teixeira de Freitas BA1 and Teixeira de Freitas BA2).Last, Group IV consists of the isolates from papaya fruits collected in São Paulo (Figure 1).This study displayed a mean genetic similarity of 0.289, which shows a great genetic diversity among the isolates studied.Group I, the Umuarama PR1 and PR2, Colletotrichum spp.isolates from Paraná displayed the greatest genetic similarity of this study (0.971).The lowest genetic similarities (0.135 to 0.2) were found between the isolates from Linhares ES and Jaboticabal SP.
Agronomically, the culture managements between these two areas, Jaboticabal SP and Linhares ES, are completely different.In Linhares, Espirito Santo, papaya is cultured under intensive management, with various applications of pesticides to control insects, mites, and fungi.Unlike Jaboticabal, SP, where the plants were cultured in backyards as extractive exploration, without any technical and agro-economic concerns.Possibly, the ISSR markers were also efficient in detecting the biological nuances resulting from the effects associated with responses from these two ecosystems.The excessive use of systemic pesticides are factors that increase the strength of selection exerted on the populations of pathogens, favoring new virulence genes and polymorphism in the structure of these isolates (Araya, 2003).
The Linhares ES1 and TdeF BA1 access belong to the species C. acutatum and this particular trait/feature may have influenced the low similarity (less than 0.29), compared to the isolates from other locations.Rampersad (2013) used only 5 ISSR primers to evaluate C. gloeosporioides genetic structure from papaya fruit, and obtained two distinct groups, one from the South and the other from the North regions of Trinidad and Tobago.In this study, the Colletotrichum grouping showed an evident relationship with the geographical distribution.The genotype separation according to geographic distribution is not uncommon for ISSR, and has been done in Mexico (Torres-Calzada et al., 2013), Trinidad and Tobago (Rampersad, 2013) and Malaysia (Mahmodi et al., 2014).
Results of studies using either a limited number of isolates or from the same geographical site are usually inadequate to detect possible genetic diversity, which is better represented by a numerically and geographically better set of isolates.Generalized conclusions derived from a group of isolates of the same location, and without 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1a  Means followed by the same letter do not differ.MGR: CV = 5.45 and DMS = 0.264 and DC: CV = 7.10 and = 1.66 DMS.
the backing of a molecular study, should be avoided.The colors of Colletotrichum colonies varied from gray to salmon/white/grayish (Table 5).The edge color ranged from white to gray while the reverse, from white to salmon/white.Sectors were observed in some isolates.Analysis of variance of both the IVCM data and diameter of the colonies after seven days of culture showed a significant difference in the behavior of the isolates (Tukey at 5%).The isolates T de F BA2, Umuarama PR2, Maringa PR1, Maringa PR2 and Linhares ES1 displayed the lowest MGR and differed significantly from the isolates from Jaboticabal SP, Itápolis SP, Linhares ES2, Linhares ES3 and TdeF BA1 (Table 4).On the other hand, the isolates from Itápolis SP8, SP7 and SP3; Jaboticabal SP1, SP3, and SP4 displayed the highest values for this variable.
The isolates Maringa PR1 and PR2; TdeF BA2; and, Linhares ES1 displayed the lowest value for the diameter of the colony parameter while the largest was recorded for Jaboticabal SP4 (Table 4).
The hierarchical cluster analysis method applied to the variables, coloration of the colonies, board and reverse, presence or absence of sectors, mycelial growth rate and diameter of colonies after seven culture days showed a great variability among the isolates, that were clustered in three different groups: Group I (GI), group II (GII) and group III (GIII) (Figure 2).GI group consisted of the isolates from Itápolis (SP1, SP2, SP3, SP7, and SP8) and Jaboticabal (SP1, SP2, SP3, and SP4).A high degree of similarity was observed among them, given the existence of common features, especially for the Jaboticabal SP3 and Itápolis SP3 isolates, whose Euclidean distances were close to zero.The isolates of this group displayed white colored colonies and edges, lack of sectors, and the largest MGR and diameter of the colony.In the molecular analysis, these genotypes also shared the same grouping.
GII consisted of the isolates Itápolis (SP4, SP5 and SP6), Umuarama PR2 and Linhares ES2, which were characterized by the presence of sectors and the isolates Itápolis SP4 and SP5, which displayed gray colonies.The similarity in this group was low, with high Euclidean distances, as shown in Figure 2.
The isolates of GIII displayed predominantly white colonies, white edge, and no sectors.The exception was the Linhares ES1 isolate, which presented salmon/white colony and gray edge.T de F BA1 and BA2 isolates had gray reverse colonies while Maringa PR1 and PR2 were white.Linhares ES1 and ES3 isolates had salmon/ white/green reverse while Umuarama PR1 isolate, salmon/white.Only two of the 21 studied isolates were Two eigenvalues were greater than unity, which generated two main components, preserving 62.20% of the original variability (38.86% in CP1 and 23.34% in CP2).Thus, the initial set of six dependent variables was characterized by two new independent latent variables, located in two-dimensional figures (Figure 3) (ordering of isolates by the main components).
The graphical representation of the main components allowed to characterize the variables that discriminated the most when forming the Groups I, II and III.The mycelial growth rate (MGR), diameter of colonies after seven days of culture (DC) weighed the most in Group I, displaying the highest values of MGR and DC while in Group III, MGR and DC displayed the lowest values.The presence of sectors separated the isolates of Group II from the others.All isolates that displayed sectors are in Group II, but this group was not only discriminated by this feature.Isolate Linhares ES2 showed salmon/black/ green/white reverse color (C_R); furthermore, this is the only group in which all isolates have pathogenicity 4. The characteristic colony (C_C) and edge (C_E) colors also helped to separate the isolates of Group III, which displayed predominantly gray colony and white edge, only Linhares ES1 had special characteristics, salmon/ white colony and gray edge, and thus is away from the center of mass.It is known that this isolate belongs to C. acutatum species, and could, therefore, be responsible for its peculiar characteristics compared to C. gloeosporioides.The pathogenicity classification did not correlate with any of the characteristics of the colonies.The most pathogenic isolates were found in various geographic regions, all isolates from Paraná and Bahia displayed pathogenicity 4, depressed lesions with sporulation, and the only region that did not have any isolate with pathogenicity 4 was Jaboticabal, in São Paulo.
In terms of morphocultural aspects, there is, in general, high polymorphism among the species of fungi, especially between those belonging to the Colletotrichum genus.This behavior is, usually, due to environmental influences, particularly the aspects related to the types of culture media, ambient temperature and luminosity (Maia et al., 2011;Nechet and Abreu, 2002).Some species of fungi display cultures characteristics very different from the original, even when using sub-colonies that originated from the same colony.On the other hand, in the case of Colletotrichum, the morphology is greatly similar, resulting in complex taxon revisions (Weir et al., 2012).C. acutatum.Ferraz (1977), classified the isolates of the Colletotrichum genus into groups, according to their cultural characteristics, but in the present study there was a great molecular and morphocultural variability in individuals of the same species and, therefore, the classification in groups considering the cultural characteristics of the isolates was not possible.

CONCLUSION
(i) Isolates from 'Formosa' and `Solo' papaya from four Brazilian states were analyzed and classified as belonging to the C. gloeosporioides and C. acutatum species; (ii) There was great genetic diversity among C. gloeosporioides and C. acutatum isolates from different locations, and in contrast, greater similarity among isolates from the same region;

Figure 1 .
Figure 1.Different Colletotrichum spp.represented by UPGMAa and genetic similarity by Jaccard coefficient.

Figure 2 .
Figure 2. Dendrogram resulting from the hierarchical cluster analysis showing the groups formed according to the variables: color of colonies, edge and reverse, presence or absence of sectors, mycelial growth rate, diameter of colonies after seven days cultivation.

Figure 3 .
Figure 3. Distribution according to the results of the morphocultural analysis of isolates of Colletotrichum spp., in the principal components 1 and 2.

Table 1 .
Colletotrichum spp.isolates, the origin of the papaya fruits, number of isolates and variety.

Table 2 .
List of primers used, their respective sequences, amplification temperatures and number of fragments observed in Colletotrichum sp.isolates from papaya.

Table 4 .
List of Colletotrichum spp.isolates according to the origin of the papaya fruit, isolates reference, analyzed variety, and species.

Table 5 .
Mean mycelial growth rate (MGR), diameter of the colonies (DC) and notes corresponding to the color of the colonies (C_C), the edge (C_E) and reverse (C_R), and the absence or presence sectors (S) and the pathogenicity of Colletotrichum spp.isolates in BDA medium, at 25°C.