Genetic Diversity of Colletotrichum spp. an Endophytic Fungi in a Medicinal Plant, Brazilian Pepper Tree

In this study, we reported thirty-nine endophytic fungi identified as Colletotrichum spp. associated with Brazilian pepper tree or aroeira (Schinus terebinthifolius Raddi. Anacardiaceae) in Paraná state, Brazil. These endophytes were identified by morphological and molecular methods, using PCR taxon-specific with CaInt/ITS4, CgInt/ITS4, and Col1/ITS4 primers, which amplify specific bands in C. acutatum, C. gloeosporioides lato sensu, and Colletotrichum boninensis, respectively, and by DNA sequence analysis of the nrDNA internal transcribed spacer region (ITS1, 5.8S, ITS2). We also assayed the presence of dsRNA particles in Colletotrichum spp. isolates. Combining both morphological characters and molecular data, we identified the species C. gloeosporioides, C. boninense, and C. simmondsii. However, we found a high genetic variability intraspecific in C. gloeosporioides which suggests the existence of several other species. Bands of double-stranded RNA (dsRNA) were detected in three of thirty-nine isolates. Identity of these bands was confirmed by RNAse, DNAse, and S1 nuclease treatments for the isolates LGMF633, LGMF726, and LGMF729. This is the first study reporting these particles of dsRNA in C. gloeosporioides.

Colletotrichum has been isolated from numerous plant species especially as symptomatic pathogens but can be found as asymptomatic endophytes. The genus has wide geographic distribution, being more important in the tropics. Studies involving the complex C. gloeosporioides and C. boninense revealed high genetic variability and molecular diversity [21,[29][30][31]. There is significant interest in developing a fast, simple, and efficient method to identify species of Colletotrichum. Several authors have described new species and morphological characteristics associated to species of Colletotrichum [32][33][34][35][36]. Afanador-Kafuri et al. [32] developed specific primers to C. boninense [34]. The species C. acutatum was also organized and divided into three species, C. acutatum, C. fioriniae comb. et stat. nov., and C. simmondsii sp. nov. [36].
Several authors have investigated the influence of viral particles on fungi [37]. Fungal virus genomes are commonly 2 ISRN Microbiology composed of dsRNA that can modulate plant-fungal symbioses [38]. The associations between fungal viruses and their hosts are similar to those involved in plant-endophyte interactions [37]. Changes in morphological characteristics and increased production of conidia have been reported as associated with the presence of dsRNA in Beauveria bassiana [39], Metarhizium anisopliae [40,41], and Nectria radicicola [34].
In the present study, we isolated endophytic fungi from leaves of medicinal tree called aroeira (Schinus terebinthifolius Raddi). These endophytes were identified by morphological and molecular methods. We also assayed the presence of dsRNA particles in Colletotrichum spp. isolates.

Fungal Isolates.
Isolates were obtained from leaves of plants of Brazilian pepper tree (S. terebinthifolius Raddi), located in the campus of the University of Paraná, Paraná, Brazil. The isolates were obtained as described by Petrini [42] and identified by macroscopic and microscopic reproductive structures after growth on PDA medium. The cultures are permanently stored in the fungal collection of the Laboratory of Microorganisms (LabGeM-UFPR), Paraná, Brazil.

Molecular Characterization
3.1. DNA Extraction. Colletotrichum isolates were grown on PDA medium for 3 days at 28 • C. The mycelium was harvested, lyophilized for 24 h, and ground with a mortar and pestle under liquid nitrogen. Genomic DNA was obtained according to methods described by Raeder and Broda [43], modified by Glienke-Blanco et al. [44].
According to the method described by Pileggi et al. [47], PCR reactions were performed in a total volume of 25 μL, containing 1X buffer solution, 1.5 mM MgCl 2 , 0.2 mM of each dNTP (Invitrogen, CA, USA), 0.5 μM primer, 1.5 Unit of Taq DNA polymerase (Invitrogen, CA, USA), and 20 ng of genomic DNA. Amplifications were carried using the following conditions: an initial denaturation at 95 • C for 5 min, followed by 40 cycles of 30 s at 95 • C, 30 s at 65 • C, and 1.5 min at 72 • C, and a final extension at 72 • C for 3 min.
To identify C. acutatum and C. gloeosporioides complex, the PCR reactions were performed as previously described. Amplifications were carried out in a gradient thermocycler with an initial denaturation period of 5 min at 95 • C, followed by one cycle of 30 sec at 94 • C, 45 seconds at 62 • C, 90 seconds at 72 • C, one cycle of 30 seconds at 94 • C, 45 seconds at 60 • C, 90 seconds at 72 • C, followed by 33 cycles of 30 seconds at 94 • C, 45 seconds at 58 • C e 90 seconds at 72 • C, and a final extension period of 3 minutes at 72 • C.

DNA Analysis and
Sequencing. The primers V9G [48] and ITS4 [45] were used to amplify the internal transcribed spacer region (ITS) of the nuclear ribosomal RNA operon, including the 3 end of the 18S rRNA, the first internal transcribed spacer region, the 5.8S rRNA gene; the second internal transcribed spacer region and the 5 end of the 28S rRNA gene. PCR was performed in total reaction volume of 50 μL, which was composed of 1 × PCR Buffer Amplified rDNA fragments were cleaned with 50 μL 20% PEG and resuspended in 15 μL of ultrapure water. To confirm the presence of DNA in the sample, 1 μL was applied on a 1.4% agarose gel. rDNA Internal Transcribed Spacer (ITS) was sequenced with primers ITS4 and ITS1 [45]. PCR was performed in 10 μL volumes of a reaction mixture containing sterile distilled water, 0.5 μL PCR buffer (10x, Applied Biosystems), 0.5 μL of primer (50 pmol), 0.5 μL of Big Dye (Applied Biosystems), and 1 μL PCR products. Thirty five cycles were performed: 96 • C for 10 s (denaturation), 50 • C for 5 s (annealing), 60 • C for 4 min (extension), and 60 s initial and terminal delay. Sequencing was performed on an ABI 3130 automatic sequencer (Perkin-Elmer, Massachusetts, USA).

Sequence Assembly and Alignment.
Sequences were edited using BioEdit 7.0 [40]. ITS sequences were aligned on the basis of similarity by means of the sequence editor CLUSTAL-W 1.7 [49]. Sequence analysis was performed using the sequence alignment software BLASTn run against the NCBI database (National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/)).

dsRNA Analysis.
After genomic DNA extraction was performed electrophoresis on 0.7% agarose gel to observe the occurrence of bands of dsRNA. For confirmation, the total DNA of the isolates was submitted separately to the treatment of enzymatic digestion with RNAse, DNAse, and S1 Nuclease. Three digestions were performed as described by Azevedo et al. [1].
LGMF666 showed amplification using primer Col1/ITS4, that amplifies a specific band for C. boninense and LGMF738 amplifies a specific band with CaInt/ITS4 primer, and was identified as C. acutatum complex (Table 1).

DNA Analysis and Sequencing.
Twenty five isolates were sequenced for the ITS1-5.8S-ITS2 of rDNA, generating fragments between 600 and 700 bp. Phylogenetic analysis grouped the Colletotrichum isolates into three clades (Figure 1). The first clade included the C. acutatum complex species, with 100% bootstrap support. The isolate LGMF738 clustered with C. simmondsii (GU183331) holotype strain. All Colletotrichum isolates that clustered in clade II included isolates of the C. gloeosporioides from GenBank (AB439815; AB273196; EU734587; EU552111; EU687190) with 84% bootstrap support. The third clade clustered the isolate LGMF625 with C. boninense species (EU822802 and AB051401).  LGMF625

Discussion
The taxonomy of Colletotrichum is confused, both for the anamorphic species and its teleomorph Glomerella. The combined use of molecular diagnostic tools along with traditional morphological techniques is at present an appropriate approach for studying Colletotrichum species complexes [6,47].
Afanador-Kafuri et al. [32] proposed the use of two pairs of primers for the identification of Colletotrichum species, one for C. gloeosporioides (CgInt) and another for Colletotrichum sp (Col1). Moriwaki et al. [30] proposed the classification of isolates originally identified as C. gloeosporioides as belonging to a new species, they called C. boninense. Pileggi et al. [47] suggested that the primers pair developed by Afanador-Kafuri et al. [32] for Colletotrichum sp amplify isolates of the new species C. boninense proposed by Moriwaki et al. [30]. Moriwaki et al. [30] showed that the ITS1 region of C. boninense was 190 bp, whereas, for C. gloeosporioides, this region was 171 bp. Consequently, the difference between C. boninense and C. gloeosporioides should reflect interspecific relationships and should be further investigated.
In this paper, we proposed PCR identification speciesspecific anticipated results, enabling identification of approximately 70% of isolates without the need for extensive morphological analysis. ITS sequence analysis confirmed species-specific results and resolved the identification of C. boninense (LGMF625) and C. gloeosporioides complex. However, the PCR species-specific mistakenly identified the isolate LGMF738 as C. acutatum when in fact it belongs to the new species C. simmondsii ( Table 1).
The analyzed Brazilian pepper trees were colonized by three different species of Colletotrichum and showed high genetic diversity; including the species C. gloeosporioides sense lato, C. boninense, and C. simmondsii. The ecological roles of endophytes are diverse and varied. Colletotrichum gloeosporioides complex is a worldwide plant pathogen that infects many plant species. These isolates will need more examination to ensure the correct identification. Zou et al. [27] reported one endophytic isolate of C. gloeosporioides from stem of Artemisia mongolica that produced the colletotric acid, with antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Sarcina lutea, and Helminthosporium sativum [27].
Many studies discriminate Colletotrichum complex using ribosomal ITS sequence data; however, due to the limited number of informative sites been identified, other regions of the Genome, such as the β-tubulin gene, have been identified suitable for the phylogenetic reconstruction [56]. Hyde et al. [57] suggest epitypification and use of multilocus phylogeny to delimit species and gain a better understanding of the genus. Our data corroborated the existence of more than one species in the C. gloeosporioides complex. Also, our data corroborated the reassessment of Colletotrichum acutatum complex and the new species C. simmondsii introduced by Shivas and Tan [58]. It was the first report of C. simmondsii as an endophyte from Schinus terebinthifolius. The host range and host specificity of C. simmondsii are not clear [36].
This paper is the first study describing the existence of dsRNA particles in C. gloeosporioides isolates. The presence of dsRNA in entomopathogenic fungi is described in a long time [41,[59][60][61][62][63]. Dalzoto et al. [39] described the horizontal transfer and hypovirulence associated with dsRNA in the fungus B. bassiana. The authors suggest the increased production of conidia in strains without dsRNA when compared with the strains positive to dsRNA particles.
Double-stranded RNA viruses have been described for a long time in a wide variety of filamentous fungi and yeasts [9, 37, 65-67]. Marquez et al. [38] suggested the associations between fungal viruses and their hosts are similar to plantendophyte associations. In this study, Marquez et al. [38] found no differences in colony morphology among isolates with dsRNA and those free dsRNA. Also, the authors did not find any association between the presences of dsRNA and genetically different groups. In the Colletotrichum genus, it is not yet known the influence that these particles can have on fungi morphology or physiology. So, we suggest the investigation by scanning electron microscopy and also by the study of these strains after cure (elimination) of dsRNA.