potential sources of new natural products have been explored in medicine , agriculture , and industry . endophytic fungi have been recognized as useful sources of bioactive secondary metabolites , especially those isolated from medicinal plants [ 2 , 3 ] . various important characteristics are currently attributed , such as the increase in resistance to stressful conditions ; alteration in physiological properties ; production of phytohormones , toxins , medicinal substances , immunosuppressants , antitumor agents , and compounds of biotechnological interest such as enzymes [ 1 , 413 ] . brazilian pepper tree also called aroeira ( schinus terebinthifolius raddi - anacardiaceae ) is native to argentina , brazil , and paraguay . in brazil , the bark leaves and fruits have been used in popular medicine due to their medicinal properties [ 1518 ] . actions anti - inflammatory and antiseptic for treatment of wounds , urinary , and respiratory infections are listed as medicinal properties popularly known . studies showed antimicrobial activity [ 15 , 18 , 2024 ] , antifungal activity [ 25 , 26 ] , as antioxidant [ 15 , 27 ] , and antitumor [ 18 , 28 ] . despite its importance , there are a few records about the endophytic community in this plant . colletotrichum has been isolated from numerous plant species especially as symptomatic pathogens but can be found as asymptomatic endophytes . studies involving the complex c. gloeosporioides and c. boninense revealed high genetic variability and molecular diversity [ 21 , 2931 ] . 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 [ 3236 ] . the species c. acutatum was also organized and divided into three species , c. acutatum , c. fioriniae comb . fungal virus genomes are commonly composed of dsrna that can modulate plant - fungal symbioses . the associations between fungal viruses and their hosts are similar to those involved in plant - endophyte interactions . changes in morphological characteristics and increased production of conidia have been reported as associated with the presence of dsrna in beauveria bassiana , metarhizium anisopliae [ 40 , 41 ] , and nectria radicicola . in the present study , we isolated endophytic fungi from leaves of medicinal tree called aroeira ( schinus terebinthifolius raddi ) . isolates were obtained from leaves of plants of brazilian pepper tree ( s. terebinthifolius raddi ) , located in the campus of the university of paran , paran , brazil . the isolates were obtained as described by petrini   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 ) , paran , brazil . 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 , modified by glienke - blanco et al . . species - specific amplifications were performed using the primer its4   with specific primers for c. gloeosporioides complex ( cgint : 5-ggcctcccgcctccgggcgg-3 ) , c. boninense ( col1 : 5-gccgtcccctgaaaag ) , or c. acutatum complex ( caint2 : 5-ggggaagcctctcgcgg-3 ) . according to the method described by pileggi et al . , pcr reactions were performed in a total volume of 25  l , containing 1x buffer solution , 1.5  mm mgcl2 , 0.2  mm of each dntp ( invitrogen , ca , usa ) , 0.5  m 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 95c for 5  min , followed by 40 cycles of 30  s at 95c , 30  s at 65c , and 1.5  min at 72c , and a final extension at 72c for 3  min . to identify c. acutatum and c. gloeosporioides complex , amplifications were carried out in a gradient thermocycler with an initial denaturation period of 5  min at 95c , followed by one cycle of 30  sec at 94c , 45 seconds at 62c , 90 seconds at 72c , one cycle of 30 seconds at 94c , 45 seconds at 60c , 90 seconds at 72c , followed by 33 cycles of 30 seconds at 94c , 45 seconds at 58c e 90 seconds at 72c , and a final extension period of 3 minutes at 72c . genomic dna of positive control of c. gloeosporioides ( col11 ) , c. boninense ( col7 ) , is obtained from pileggi et al . . genomic dnas of c. acutatum strains fdc89a08 and fdc31a08 were obtained from fundecitrus , sp . the primers v9 g   and its4   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  l , which was composed of 1  pcr  buffer ( applied biosystems , foster city , usa ) , 2  mm mgcl2 , 40  m dntps , 0.08  m of each forward and reverse primer , 0.5  u of taq dna polymerase ( roche diagnostics , indianapolis , usa ) , and 110  ng of genomic dna . thirty cycles were performed : 94c for 30  s , 56c for 1  min , 72c for 1  min , and 2  min initial and terminal delay . the second condition had a total reaction volume of 12.5  l , which was composed of 1  pcr  buffer ( bioline gmbh , luckenwalde , germany ) , 5.6% dmso ( v / v ) , 2  mm mgcl2 , 20  m dntps , 0.2  m of each forward and reverse primer , 0.25  u of biotaq taq dna polymerase ( bioline gmbh , luckenwalde , germany ) , and 110  ng of genomic dna . the pcr cycle conditions were 5  min of 94c , followed by 40 cycles of 94c for 30  s , 52c for 30  s , 72c for 30  s , and a final elongation step at 72c for 7  min . amplified rdna fragments were cleaned with 50  l 20% peg and resuspended in 15  l of ultrapure water . to confirm the presence of dna in the sample , 1  l was applied on a 1.4% agarose gel . rdna internal transcribed spacer ( its ) was sequenced with primers its4 and its1 . pcr was performed in 10  l volumes of a reaction mixture containing sterile distilled water , 0.5  l pcr buffer ( 10x , applied biosystems ) , 0.5  l of primer ( 50  pmol ) , 0.5  l of big dye ( applied biosystems ) , and 1  l pcr products . thirty five cycles were performed : 96c for 10  s ( denaturation ) , 50c for 5  s ( annealing ) , 60c for 4  min ( extension ) , and 60  s initial and terminal delay . sequencing was performed on an abi 3130 automatic sequencer ( perkin - elmer , massachusetts , usa ) . its sequences were aligned on the basis of similarity by means of the sequence editor clustal - w 1.7 . 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/ ) ) . the algorithm settings was the default , with 1.000 bootstraps ( 50 runs of 20 repetitions ) . bayesian tree search was done with parallel mrbayes version 3.1.2 [ 51 , 52 ] , using 20.000.000 generations and 4 independents runs . the model , sym+i , was selected with jmodeltest v0.1.1   using bayesian information criterion ( bic ) . the traces were analysed with tracer v1.5   and awty   to evaluate the stationary phase , setting the burn - in to 2.000.000 generations . to merge bootstrap replicates and posterior probabilities and summarize the consensus tree , sumtrees   from dendropy 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 . the isolates were investigated in pcr with specific primers for c. acutatum , c. gloeosporioides , and c. boninense ( table 1 ) . isolates lgmf625 , 666 , 667 , and 738 did not amplify the expected band with cgint / its4 primers . 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 ) . twenty five isolates were sequenced for the its1 - 5.8s - its2 of rdna , generating fragments between 600 and 700  bp . 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 ) . after electrophoresis of genomic dna was observed bands of approximately 3000  bp indicating the presence of dsrna in six isolates of c. gloeosporioides complex lgmf633 , 638 , 689 , 726 , 729 , and lgmf736 ( table 1 ) . 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 ] . 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 ) . proposed the classification of isolates originally identified as c. gloeosporioides as belonging to a new species , they called c. boninense . pileggi et al .   suggested that the primers pair developed by afanador - kafuri et al . for colletotrichum sp amplify isolates of the new species c. boninense proposed by moriwaki et al . . .   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 species - specific 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 . .   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 . 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 -tubulin gene , have been identified suitable for the phylogenetic reconstruction . .   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 . 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 , 5963 ] . the authors suggest the increased production of conidia in strains without dsrna when compared with the strains positive to dsrna particles . morphological changes of colonies associated with the presence of the dsrna particles were described in chalara elegans , metarhizium anisopliae [ 40 , 41 ] , diaporthe ambigua , and nectria radicicola . double - stranded rna viruses have been described for a long time in a wide variety of filamentous fungi and yeasts [ 9 , 37 , 6567 ] . marquez et al .   suggested the associations between fungal viruses and their hosts are similar to plant - endophyte associations . in this study , marquez et al .   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 .
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 paran 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 .