ABSTRACT

Studies involving endophytic fungi isolated from endemic plants and their antibacterial potential are largely unknown in the Pampa biome. In this study, we identified endophytic fungi isolated from Kelissa brasiliensis (Iridaceae), an endemic species of the Brazilian Pampa, and assessed their antibacterial activity. Endophytic fungi were first grouped based on macro-and micro-morphology, and one representative of each morphospecies was analyzed using sequences from the internal transcribed spacer (ITS) rDNA region. We then tested the fungal extracts against laboratory isolates of Staphylococcus aureus and Escherichia coli for antibacterial activity. A total of 30 endophytes were isolated from the tissues of K. brasiliensis, with the majority from the leaves. Endophytes were then grouped into seven morphospecies based on their morphological features and one representative from each was selected for phylogenetic analysis. The inference from the ITS rDNA sequences identified the endophytes of the seven selected morphospecies as belonging to six taxonomic groups: Colletotrichum (two), Diaporthe (one), Epicoccum (one), Fusarium (one), and Pestalotiopsis (one). The endophyte extracts revealed better results against E. coli than S. aureus, although the extracts from Colletotrichum and Pestalotiopsis sp. were statistically similar to the control antibiotic. Our study is a basis for endophytic fungi studies in Pampa.

Keywords:
“bibi-pintadinha”; Brazilian biome; Colletotrichum; endophytic fungi; Pestalotiopsis

Introduction

Kelissa is a monospecific genus endemic to the Pampa biome, and Kelissa brasiliensis (Iridaceae), popularly known in Brazil as “bibi-pintadinha,” is an herbaceous bulbous species with a flowering period in the spring (September to November) and seed dispersal in December. This species is found in rocky environments in the southeastern depression, and in the center of the state of Rio Grande do Sul (Barroso 2006Barroso CM. 2006. Propagação de espécies nativas com potencial ornamental: Kelissa brasiliensis (Baker) Ravenna e Sinningia lineata (Hjelmq.) Chautems. MSc Thesis, Universidade Federal do Rio Grande do Sul, Porto Alegre.; Aguiar et al. 2009Aguiar LW, Martau L, Strehl T. 2009. Coleções de plantas vivas do Jardim Botânico da Fundação Zoobotânica do Rio Grande do Sul, Porto Alegre, Brasil: Begoniaceae. Iheringia, Série Botânica 64: 101-107.), and is considered vulnerable because of habitat reduction in its natural areas, mainly through agriculture (Barroso 2006Barroso CM. 2006. Propagação de espécies nativas com potencial ornamental: Kelissa brasiliensis (Baker) Ravenna e Sinningia lineata (Hjelmq.) Chautems. MSc Thesis, Universidade Federal do Rio Grande do Sul, Porto Alegre.).

Kelissa brasiliensis is an endemic species that contributes to the environment of the Pampa biome, which contains approximately 3,000 plants (Pillar et al. 2009Pillar VDP, Müller SC, Castilhos ZMS, Jacques AVA. 2009. Campos Sulinos - conservação e uso sustentável da biodiversidade. Brasília, Ministério do Meio Ambiente.). However, due to the expansion of intense agricultural activities in the native fields, these species are threatened (De Oliveira et al. 2017De Oliveira TE, Freitas DS, Gianezini M, et al. 2017. Agricultural land use change in the Brazilian Pampa Biome: The reduction of natural grasslands. Land Use Policy 63: 394-400.). Pampa comprises more than 45 % of the Iridaceae species in Brazil (86 species in 14 genera of the total taxa described for the country), yet no studies involving the mycobiota associated with these species have been noted (Gil et al. 2015Gil A, Eggers L, Lovo J, Chukr N. 2015. Iridaceae in Lista de Espécies da Flora do Brasil. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB136. 20 Nov. 2020.
http://floradobrasil.jbrj.gov.br/jabot/f... ; Pastori et al. 2020Pastori T, Eggers L, Chauveau O. 2020. Kelissa in Lista de Espécies da Flora do Brasil. http://floradobrasil.jbrj.gov.br/reflora/floradobrasil/FB36344. 1 Nov. 2018.
http://floradobrasil.jbrj.gov.br/reflora... ). The reference to K. brasiliensis has only been identified in studies of plant propagation, chromosomal numbers, and floral lists (Barroso 2006Barroso CM. 2006. Propagação de espécies nativas com potencial ornamental: Kelissa brasiliensis (Baker) Ravenna e Sinningia lineata (Hjelmq.) Chautems. MSc Thesis, Universidade Federal do Rio Grande do Sul, Porto Alegre.).

Many plants have been studied as hosts of endophytic fungi in all other biomes in Brazil, given their excellent environmental service and biotechnological potential (Bezerra et al. 2019Bezerra JDP, Silva LF, Souza-Motta CM. 2019. The explosion of Brazilian endophytic fungal diversity: taxonomy and biotechnological potentials. In: Satyanarayana T, Deshmukh S, Deshpande M. (eds.) Advancing Frontiers in Mycology & Mycotechnology. Singapore, Springer. p. 405-433.). Endophytic fungi are microorganisms that live in plant tissues without causing adverse effects to the plant. These fungi have been described in relation to hundreds of plants, and several studies have demonstrated their ability to produce a wide range of secondary metabolites with antibacterial, anticancer, and antifungal properties (Mishra et al. 2014Mishra Y, Singh A, Batra A, Sharma MM. 2014. Understanding the biodiversity and biological applications of endophytic fungi: a review. Journal of Microbial & Biochemical Technology S8: 004. doi: 10.4172/1948-5948.S8-004
https://doi.org/10.4172/1948-5948.S8-004... ; Rana et al. 2019Rana KL, Kour D, Sheikh I, et al. 2019. Biodiversity of endophytic fungi from diverse niches and their biotechnological applications. In Advances in endophytic fungal research. Cham, Springer. p. 105-144.). Saffron (Crocus sativus) is a unique species in the family Iridaceae with reports of endophytic fungi and biotechnological potential (Zheng et al. 2012Zheng CJ, Li L, Zou JP, Han T, Qin LP. 2012. Identification of a quinazoline alkaloid produced by Penicillium vinaceum, an endophytic fungus from Crocus sativus. Pharmaceutical Biology 50: 129-133.; Raj et al. 2013Raj P, Khan SS, Modak M, Lone ZA, Rather SA, Yaqoob M. 2013. Biodiversity of endophytic fungi in saffron (Crocus sativus) and antimicrobial activity of their crude extract. Indo American Journal of Pharmaceutical Research 3: 3702-3713.; Wani et al. 2016Wani ZA, Mirza DN, Arora P, Riyaz-Ul-Hassan S. 2016. Molecular phylogeny, diversity, community structure, and plant growth promoting properties of fungal endophytes associated with the corms of saffron plant: An insight into the microbiome of Crocus sativus Linn. Fungal Biology 120: 1509-1524.; Chamkhi et al. 2018Chamkhi I, Sbabou L, Aurag J. 2018. Endophytic fungi isolated from Crocus sativus L.(saffron) as a source of bioactive secondary metabolites. Pharmacognosy Journal 10: 1143-1148.). In Brazil, the fungal endophyte community of Iridaceae species is still unknown.

Plants that live in poorly explored habitats similar to the Pampa biome have been used as sources for the discovery of new species of endophytic fungi, thereby providing increased data on the richness, diversity, and biological activity of this mycobiome (Hokama et al. 2016Hokama Y, Savi DC, Assad B, et al. 2016. Endophytic fungi isolated from Vochysia divergens in the pantanal, mato grosso do sul: Diversity, phylogeny and biocontrol of Phyllosticta citricarpa. In: Hughes E. (ed.) Endophytic Fungi: Diversity, Characterization and Biocontrol. New York, Nova Science Publishers. p. 1-27.). Endemic species and plants at risk of extinction are among the most important hosts for revealing endophytes and their biotechnological potential (Omeje et al. 2017Omeje EO, Ahomafor JE, Onyekaba TU, et al. 2017. Endophytic Fungi as Alternative and Reliable Sources for Potent Anticancer Agents. In Natural Products and Cancer Drug Discovery. London, InTech Open Press.). Thus, the identification of endophytic fungi from the Iridaceae species, K. brasiliensis, which is endemic to the Pampa biome, presents a promising option for uncovering fungi with the potential to produce antibacterial agents for further biotechnological applications.

This study aimed to report the endophytic fungi richness of K. brasiliensis, an endemic plant of the Pampa Biome, and evaluate the antibacterial activity of these endophytes against clinical isolates.

Materials and methods

Study area and plant collection

Kelissa brasiliensis individuals were collected from a private area of the Pampa biome with native vegetation near the Reserve of Sanga da Bica (30°20′03″ S, 54°19′18″ W), in São Gabriel, Rio Grande do Sul, Brazil. Countryside vegetation and a temperate climate are predominant in this region, with an average temperature of 18 °C and annual precipitation ranging from 1,250 to 1,600 mm. The aerial parts of 12 healthy K. brasiliensis plants up to 7 cm tall and having one flower were randomly collected at three locations (30°20′50″ S, 54°19′10″ W; 30°20′48″ S, 54°19′10″ W; and 30°20′47″ S, 54°19′09″ W) (Fig. 1), along a pre-existing trail on the edge of the forest during the spring of 2017. Four specimens of K. brasiliensis were randomly collected at each sampling point. The plants were stored individually in sterilized plastic bags, transported to the laboratory at ± 24 °C, and processed on the same day as collected.

Isolation of endophytic fungi

Under aseptic conditions, the plants were divided into three parts (capsules, stems, and leaves). The central part of each fragment was cut into three subfragments measuring approximately 1 cm, which were subjected to superficial disinfestations (de Andrade et al. 2018de Andrade GAK, Cañón ERP, Alves RP, et al. 2018. First Record of Juncaceicola as Endophytic Fungi Associated with Deschampsia antarctica Desv. Diversity 10: 107. doi: 10.3390/d10040107
https://doi.org/10.3390/d10040107... ). Briefly, the plant tissues were washed in 70 % alcohol for 1 min, followed by 2 % sodium hypochlorite for 3 min, and finally in distilled and autoclaved water for 2 min. The disinfected sub-fragments were inoculated in Petri dishes containing potato dextrose agar (PDA) culture medium supplemented with chloramphenicol (100 µg mL^-1) to restrict bacterial growth. The efficiency of the disinfestation protocol was tested by inoculating 1 mL of the washing water in Petri dishes containing PDA medium. Plates were incubated at 20 °C under a photoperiod (16 h light and 8 h dark) for up to 20 days. When the fungal endophytes were first observed from the seventh day, they were isolated in Petri dishes containing PDA and incubated under the same conditions described above.

Fungal endophyte identification

Fungal endophyte isolates were identified and grouped into morphological species according to the macro- (size of colonies after seven days growing in PDA culture medium, color, shape, and texture) and micro-morphological characteristics of their somatic and reproductive structures (e.g.,Booth 1971Booth C. 1971. The genus Fusarium. London, Kew, Commonwealth Mycological Institute Press.; Barnett & Hunter 1999Barnett HL, Hunter BB. 1999. Illustrated genera of imperfect fungi. Minnesota, American Phytopathological Society Press.; Guo et al. 2000Guo LD, Hyde KD, Liew ECY. 2000. Identification of endophytic fungi from Livistona chinensis based on morphology and rDNA sequences. The New Phytologist 147: 617-630.). After grouping the initial morphological species, seven isolates were selected for molecular analysis.

Molecular identification of fungal isolates was performed using rDNA sequences of the internal transcribed spacer (ITS) region. Fungal isolates were inoculated on PDA and the DNA was extracted on day 7^th day using the PureLink^tm Plant Kit. The ITS rDNA region was amplified using primers ITS1 and ITS4 (White et al. 1990White TJ, Bruns TD, Lee SB. 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics, In: Gelfand DH, Sninsky JJ, White TJ. (eds.) PCR Protocols: A Guide to Methods and Applications. Innis, MA, Academic Press. p. 315-322.), as described by Andrade et al. (2018)de Andrade GAK, Cañón ERP, Alves RP, et al. 2018. First Record of Juncaceicola as Endophytic Fungi Associated with Deschampsia antarctica Desv. Diversity 10: 107. doi: 10.3390/d10040107
https://doi.org/10.3390/d10040107... (95 °C for 5 min, 40 cycles of 94 °C for 60 s, 50 °C for 60 s, and 72 °C for 60 s, with a final extension at 72 °C for 5 min). The resulting PCR amplifications were purified using the Wizard® SV Gel kit and PCR Clean-Up System, following the manufacturer’s protocol. Sequencing was conducted using an ABI Prism 3500 Genetic Analyzer (ACTGene Company).

Sequences from the endophytes were analyzed and edited manually using BioEdit v.7.2.5 (Hall 1999Hall TA. 1999. BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT. Nucleic acids Symposium Series 41: 95-98.), and the consensus sequence was determined using the SeqMan software by DNASTAR (Burland 2000Burland TG. 2000. DNASTAR’s Lasergene sequence analysis software. Bioinformatics Methods and Protocols 132: 71-91.). The BLASTn tool of the NCBI GenBank database was used to search for similar sequences to those of the seven endophytic fungi, and the resulting selections (Tab. S1 in supplementary material) were used to construct a matrix that was aligned using MEGA v. 7 (Kumar et al. 2016Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.). Phylogenetic analysis based on maximum likelihood (ML) was conducted with the RAxML-HPC BlackBox v.8.2.8 (Kozlov et al. 2019Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. 2019. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 35: 4453-4455.) using the CIPRES Science Gateway (Miller et al. 2010Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Gateway Computing Environments Workshop (GCE). New Orleans, IEEE Conference Press. p. 1-8.). The best nucleotide model, GTR + I + G, with 1,000 bootstrap replicates were used in this analysis. ML bootstrap (ML-BS) values equal to or higher than 70 % are shown near the nodes. Sequences generated during this study were submitted to GenBank under the accession numbers MK778368-MK778374, and the ITS alignment was deposited in TreeBASE (study S24809). After identification, the isolated morphotypes were stored in distilled water (Castellani 1967Castellani A. 1967. Maintenance and cultivation of common pathogenic fungi of man in sterile distilled water. Further Researches. Journal of Tropical Medicine and Hygiene 70: 181-184.) and deposited in the Herbário Bruno Edgar Irgang at the Universidade Federal do Pampa (codes: HBEI 0009-HBEI 0015).

Frequency and abundance of endophytic fungi

The frequency of plant tissue colonization (FC %) was estimated as suggested by Rajagopal & Suryanarayanan (2000Rajagopal K, Suryanarayanan TS. 2000. Isolation of endophytic fungi from leaves of neem (Azadirachta indica A. Juss.). Current Science 78: 1375-1378.) using the formula FC % = Ni/Nf × 100, where Ni = the number of endophytic isolates and Nf = the total number of fragments inoculated. To calculate the percentage abundance (PA %) of each genus, we applied the methodology used by Rosa et al. (2010Rosa LH, Almeida VML, Santiago IF, Rosa CA. 2010. Endophytic fungi community associated with the dicotyledonous plant Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae) in Antarctica. FEMS Microbiology Ecology 73: 178-189.). Thus, we considered the occurrence of each genus (Og) and the occurrence of all genera (Otg) (PA % = Og × 100/Otg). These values were used to determine the prevalence of different genera in the fungal endophyte community of K. brasiliensis.

The absolute (f) and relative (fr) frequencies were calculated according to the method described by Larran et al. (2002Larran S, Perelló A, Simón MR, Moreno V. 2002. Isolation and analysis of endophytic microorganisms in weat (Triticum aestivum L.) leaves. World Journal of Microbiology and Biotechnology 18: 683-686.). The absolute frequency (f) was calculated according to the total number of fungi isolated from K. brasiliensis, and the relative frequency (fr) from the number of isolates of each morphotype divided by the total number of isolates.

Fungal fermentation and antibacterial activity

Fermentation and antibacterial activity tests were performed as described by Teixeira et al. (2011Teixeira MFS, Silva TA, Carneiro ALB, Palheta RA, Atayde HM. 2011. Fungos da Amazônia: uma riqueza inexplorada (aplicações biotecnológicas). Manaus, Editora da Universidade Federal do Amazonas - Edua.). The seven selected endophytes were cultured on PDA for 14 days at 25 °C, after which the 9 mm diameter culture disks were transferred to 500 mL Erlenmeyer flasks containing 200 mL Czapek-Dox liquid medium (0.5 g KCl, 1 g KH₂PO₄, 2 g NaNO₃, 30 g sucrose, 0.01 g FeSO₄.H₂O, and 0.5 g MgSO₄.7 H₂O per 1,000 mL distilled water) previously sterilized at 120 °C for 20 min. Five discs of each culture were inoculated per flask and maintained on shaker tables at 180 rpm for 7 d at 25 °C. The cultures were then subjected to two successive vacuum filtrations using Whatman #1 paper and filtered using 0.22 μm Millipore membranes. The final extract was stored at 4 °C until antibacterial assays were performed.

Antibacterial activity was evaluated using quantitative biological assays in triplicate with strains of Escherichia coli (TOP10 Invitrogen®) and a wild strain of Staphylococcus aureus. The S. aureus strain was isolated from milk products and identified using selective media, biochemical tests, microscopy, and gram staining. The strains can be accessed at the voucher UNI15 in the Microbiology Research Laboratory of the Federal University of Pampa, São Gabriel Campus, Brazil, for future studies and reproducibility tests.

The cup-plate diffusion technique (Rose & Miller 1939Rose SB, Miller RE. 1939. Studies with the agar cup-plate method: I. A standardized agar cup-plate technique. Journal of Bacteriology 38: 525-537.) was used to verify the potential of the endophyte extract against the two test microorganisms. The amoxicillin + clavulanate (AMC) (40 mg/mL) antibiotic was used as a positive control and the fermentation medium without fungus was used as a negative control. The tested microorganisms were pre-inoculated in Luria-Bertani (LB) liquid medium (1 g tryptone, 0.5 g yeast extract, and 1 g NaCl to 1,000 mL of distilled water) and incubated at 37 °C for 16 h in the dark. Bacteria were seeded (100 μL) on the surface of Muller Hinton medium (2.0 g meat extract, 17.5 g cassava acids, 1.5 g starch, and 15 g agar to 1,000 mL distilled water). Three 9 mm cup-plates were equidistantly drilled in each Petri dish, and 100 μL of the fungal extract was added at each point. Plates with extracts were incubated for 20 min in a laminar flow cabinet until the culture medium fully absorbed the extract. The plates were incubated at 37 °C for 24 h in the dark. The test was performed in triplicate and the antibacterial activity was evaluated by the formation and measurement of the inhibition halo. The results were considered using the mean values of the three replicates per plate.

The size of the inhibition halo values did not follow a normal distribution (Wickham 2016Wickham H. 2016. ggplot2: Elegant Graphics for Data Analysis. Houston, Texas, Springer.) and were statistically evaluated using Wilcoxon's nonparametric test (Bauer 1972Bauer DF. 1972. Constructing confidence sets using rank statistics. Journal of the American Statistical Association 67: 687-690. ; Hollander & Wolfe 1973Hollander M, Wolfe DA. 1973. Nonparametric Statistical Methods. New York, John Wiley and Sons. ) in the R software (R Development Core Team 2019R Development Core Team. 2019. R: A language and environment for statistical computing. Vienna, Austria, R Foundation for Statistical Computing. http://www.R-project.org. 10 Aug. 2019.
http://www.R-project.org... ). Values were compared using the p-value, where p < 0.05 indicated that the treatments differed statistically from the control.

Results

Endophytic fungi of Kelissa brasiliensis

In total, 30 endophytic fungi were isolated from 108 plant fragments, with a colonization frequency rate (FC) of 27.7 % (Tab. 1). Fifteen fungi were isolated from leaves (FC = 41.66 %), followed by ten from capsules (FC = 27.77 %) and five from stems (FC = 13.88 %) (Tab. 1). Thirty endophytes isolated were grouped into seven morphospecies according to their morphological characteristics (isolates F6KB, F8KB, F13KB, F14KB, F16KB, F17KB, and F18KB) and subsequently identified at the genus level through ITS rDNA region sequencing.

Phylogenetic analysis (Fig. 2) using the sequences obtained from the endophytes and those from the GenBank database showed that the endophytic fungi belonged to five families of the Sordariomycetes and Dothideomycetes (Ascomycota) classes represented by five genera. Three of the isolates (F6KB, F16KB, and F17KB) were phylogenetically grouped with sequences of Colletotrichum (Glomerellaceae), while F16KB and F17KB were grouped as the same species because of the formation of a separate cluster. The remaining isolates were grouped as follows: F18KB with Pestalotiopsis (Amphisphaeriaceae), F13KB with Fusarium (Nectriaceae), F14KB with Diaporthe (Diaporthaceae), and F08KB with Epicoccum (Didymelaceae). All genera clustered with significant support values (bootstrap value >95 %). Using the results from morphological grouping species along with phylogenetic analysis, Colletotrichum was the most representative genus (15 isolates, PA = 50 %), followed by Pestalotiopsis (PA = 33.33 %, ten isolates), and Diaporthe (PA = 10 %, three isolates). Epicoccum and Fusarium were represented by one isolate each.

Antibacterial activity of endophytic fungi

All extracts tested showed activity against E. coli and S. aureus with inhibition halos varying from 11-32.4 mm (Tab. 2). Escherichia coli exhibited the highest sensitivity to endophytic fungi extracts (halos of 18-27.3 mm), and the extract from Colletotrichum sp. 2 F17KB presented the greatest activity in the test (27.3 mm) when compared to the control (27.4 mm). The extracts were less efficient against S. aureus (halos of 11.1-32.4 mm) when compared to the control (38.2 mm). The most efficient extracts were obtained from Colletotrichum sp. 2 F17KB (32.4 mm), Fusarium sp. F13KB (31.1 mm), and Pestalotiopsis sp. F18KB (30.1 mm).

The extracts from Colletotrichum sp. 2 F17KB and Pestalotiopsis sp. F18KB showed the highest inhibition values against E. coli and S. aureus (Tab. 2), whereas that of Epicoccum sp. F8KB presented the lowest (18.6 and 11 mm) compared to the other tested extracts.

Statistical analysis indicated the highest sensitivity of E. coli to endophytic extracts (p> 0.05) when compared with the positive control (amoxicillin + clavulanate) (Tab. 2). In contrast, although some extracts had high inhibition values against S. aureus, they showed statistical differences from the positive control (p <0.05) (Fig. 3).

Discussion

The Pampa is the only Brazilian biome not mentioned in a survey of studies on endophytic fungi (Bezerra et al. 2019Bezerra JDP, Silva LF, Souza-Motta CM. 2019. The explosion of Brazilian endophytic fungal diversity: taxonomy and biotechnological potentials. In: Satyanarayana T, Deshmukh S, Deshpande M. (eds.) Advancing Frontiers in Mycology & Mycotechnology. Singapore, Springer. p. 405-433.; Savi et al. 2019Savi DC, Aluizio R, Glienke C. 2019. Brazilian plants: An unexplored source of endophytes as producers of active metabolites. Planta Medica 85: 619-636.). This is the first known study of endophytic fungi from plants of the Pampa biome, revealing the fungal endophyte community associated with K. brasiliensis and its antibacterial activity.

The richness of endophytic fungi associated with K. brasiliensis is similar to that observed in other two studies of Iridaceae species (Raj et al. 2013Raj P, Khan SS, Modak M, Lone ZA, Rather SA, Yaqoob M. 2013. Biodiversity of endophytic fungi in saffron (Crocus sativus) and antimicrobial activity of their crude extract. Indo American Journal of Pharmaceutical Research 3: 3702-3713.; Wani et al. 2016Wani ZA, Mirza DN, Arora P, Riyaz-Ul-Hassan S. 2016. Molecular phylogeny, diversity, community structure, and plant growth promoting properties of fungal endophytes associated with the corms of saffron plant: An insight into the microbiome of Crocus sativus Linn. Fungal Biology 120: 1509-1524.). We isolated 30 fungal endophytes from 108 fragments and found a colonization rate of 27.7 %. Studies with saffron (Crocus sativus, Iridaceae) showed similar colonization rates of endophytic fungi of 21 % (Raj et al. 2013Raj P, Khan SS, Modak M, Lone ZA, Rather SA, Yaqoob M. 2013. Biodiversity of endophytic fungi in saffron (Crocus sativus) and antimicrobial activity of their crude extract. Indo American Journal of Pharmaceutical Research 3: 3702-3713.) and 29.7 % (Wani et al. 2016Wani ZA, Mirza DN, Arora P, Riyaz-Ul-Hassan S. 2016. Molecular phylogeny, diversity, community structure, and plant growth promoting properties of fungal endophytes associated with the corms of saffron plant: An insight into the microbiome of Crocus sativus Linn. Fungal Biology 120: 1509-1524.). The colonization rate in our study was also similar to the isolation rates of several other Brazilian plants, such as two species of medicinal herbs, Mentha piperita (40.8 %) and M. canadensis (14.6 %) (Herrmann et al. 2019Herrmann LW, Poitevin CG, Schuindt LC, Almeida AB, Pimentel IC. 2019. Diversity of fungal endophytes in leaves of Mentha piperita and Mentha canadensis. International Journal of Botany Studies 4: 44-49.); a shrub species of the family Apocynaceae, Calotropis procera (32.1 %) (Nascimento et al. 2015Nascimento TL, Oki Y, Lima DMM, Almeida-Cortez JS, Fernandes GW, Souza-Motta CM. 2015. Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity. Fungal Ecology 14: 79-86.); and the medicinal plant Myracroduon urundeuva (10.43 % in the Caatinga forest and 39.58 % in an upland forest) (Pádua et al. 2019Pádua APSLD, Freire KTLDS, Oliveira TGLD, et al. 2019. Fungal endophyte diversity in the leaves of the medicinal plant Myracrodruon urundeuva in a Brazilian dry tropical forest and their capacity to produce L-asparaginase. Acta Botanica Brasilica 33: 39-49.).

In our study, leaves showed the highest colonization rate (41.66 %), followed by capsules (27.77 %), and stem (13.88 %). In studies with Iridaceae plants, the most significant number of endophytic isolates were found in corms (Raj et al. 2013Raj P, Khan SS, Modak M, Lone ZA, Rather SA, Yaqoob M. 2013. Biodiversity of endophytic fungi in saffron (Crocus sativus) and antimicrobial activity of their crude extract. Indo American Journal of Pharmaceutical Research 3: 3702-3713.; Wani et al. 2016Wani ZA, Mirza DN, Arora P, Riyaz-Ul-Hassan S. 2016. Molecular phylogeny, diversity, community structure, and plant growth promoting properties of fungal endophytes associated with the corms of saffron plant: An insight into the microbiome of Crocus sativus Linn. Fungal Biology 120: 1509-1524.) although leaves were not used for endophyte isolation. Similar to our study, leaves were the most colonized tissues (colonization rate of 50.41 %) of Lippia sidoides, a wild medicinal bush found in northeastern Brazil, whereas stems revealed a lesser colonization rate (35.40 %) (Siqueira et al. 2011Siqueira VM, Conti R, Araújo JM, Souza-Motta CM. 2011. Endophytic fungi from the medicinal plant Lippia sidoides Cham. and their antimicrobial activity. Symbiosis 53: 89-95.). In another study with Acmella ciliate, a small Asteraceae species, Ortiz-Ojeda et al. (2020Ortiz-Ojeda CP, Andrade SL, Procópio REL. 2020. Antifungal activity of endophytic microorganisms isolated from Acmella ciliate (Asteraceae). Genetic and Molecular Research 19: GMR18570. https://repositorio.utp.edu.pe/bitstream/handle/20.500.12867/3054/Cinthya%20Paola%20Ortiz%20Ojeda_Articulo_Genetics%20and%20Molecular%20Research_en_2020.pdf?sequence=1&isAllowed=y.
https://repositorio.utp.edu.pe/bitstream... ) reported that the leaves were the most colonized plant tissue (40 isolates), followed by the stems (16 isolates).

The 30 endophytes isolated were represented by seven morphospecies and six taxonomic groups, with the Colletotrichum (15 isolates) genus being the most abundant (PA 50 %). In contrast, saffron (Iridaceae) showed the greatest abundance with the genus Rhizoctonia (27.7 %, 13 isolates) among the six genera observed, followed by Fusarium (25.5 %, 12 isolates) (Raj et al. 2013Raj P, Khan SS, Modak M, Lone ZA, Rather SA, Yaqoob M. 2013. Biodiversity of endophytic fungi in saffron (Crocus sativus) and antimicrobial activity of their crude extract. Indo American Journal of Pharmaceutical Research 3: 3702-3713.). In a similar study with saffron, Wani et al. (2016Wani ZA, Mirza DN, Arora P, Riyaz-Ul-Hassan S. 2016. Molecular phylogeny, diversity, community structure, and plant growth promoting properties of fungal endophytes associated with the corms of saffron plant: An insight into the microbiome of Crocus sativus Linn. Fungal Biology 120: 1509-1524.) reported 294 isolates belonging to 19 genera, with Phialophora (15 %, 44 isolates) and Cadophora (12.9 %, 18 isolates) as the most common. Our results, along with these unique studies of fungal endophytes from Iridaceae species, show that the fungal communities may vary among plant tissues, species of the same family, and biomes.

The endophytic fungal genera found in our study have previously been listed as endophytes in different parts of plants in other biomes (Vieira et al. 2014Vieira WA, Michereff SJ, de Morais MA, Hyde KD, Câmara MP. 2014. Endophytic species of Colletotrichum associated with mango in northeastern Brazil. Fungal Diversity 67: 181-202.; Hamzah et al. 2018Hamzah TNT, Lee SY, Hidayat A, Terhem R, Faridah-Hanum I, Mohamed R. 2018. Diversity and characterization of endophytic fungi isolated from the tropical mangrove species, Rhizophora mucronata, and identification of potential antagonists against the soil-borne fungus, Fusarium solani. Frontiers in Microbiology 9: 1707. doi: 10.3389/fmicb.2018.01707
https://doi.org/10.3389/fmicb.2018.01707... ; Larran et al. 2018Larran S, Siurana MPS, Caselles JR, Simón MR, Perelló A. 2018. Fusarium sudanense, endophytic fungus causing typical symptoms of seedling blight and seed rot on wheat. Journal of King Saud University-Science.; Pádua et al. 2019Pádua APSLD, Freire KTLDS, Oliveira TGLD, et al. 2019. Fungal endophyte diversity in the leaves of the medicinal plant Myracrodruon urundeuva in a Brazilian dry tropical forest and their capacity to produce L-asparaginase. Acta Botanica Brasilica 33: 39-49.). Colletotrichum species are one of the most common endophytes, and are the most abundant in the study of the endophytic community associated with Carapichea ipecacuanha (Rubiaceae), a medicinal plant in Brazil, Colombia, and Central America (Ferreira et al. 2020Ferreira MC, de Assis JCS, Rosa LH. 2020. Diversity of endophytic fungi associated with Carapichea ipecacuanha from a native fragment of the Atlantic Rain Forest. South African Journal of Botany 134: 225-229.), as well as in Begonia fishcheri and B. olsoniae (Begoniaceae) in the Brazilian Atlantic rainforest (Correia et al. 2018Correia AM, Lira SP, Assis MA, Rodrigues A. 2018. Fungal endophyte communities in Begonia species from the Brazilian Atlantic rainforest. Current Microbiology 75: 441-449.).

Endophytic Pestalotiopsis isolates were found only in capsules of K. brasiliensis, whereas isolates of Colletotrichum were obtained only from the fragments of leaves and stems, confirming specificity. According to Petrini et al. (1992Petrini O, Sieber TN, Toti L, Viret O. 1992. Ecology, metabolite production and substrate utilization in endophytic fungi. Natural Toxins 1: 185-196.), different organs and plant tissues comprise specific microenvironments in which the endophytic communities may have a certain distribution in the tissues (e.g., Ganley & Newcomb 2006Ganley RJ, Newcombe G. 2006. Fungal endophytes in seeds and needles of Pinus monticola. Mycological Research 110: 318-327.; Xing et al. 2010Xing X, Guo S, Fu J. 2010. Biodiversity and distribution of endophytic fungi associated with Panax quinquefolium L. cultivated in a forest reserve. Symbiosis 51: 161-166.; Bezerra et al. 2015Bezerra JDP, Nascimento CCF, Barbosa RN, et al. 2015. Endophytic fungi from medicinal plant Bauhinia forficata: diversity and biotechnological potential. Brazilian Journal of Microbiology 46: 49-57.).

In Brazil, Pestalotiopsis species have been registered in some geographical regions as saprophytes, phytopathogens, and endophytes (Kruschewsky et al. 2014Kruschewsky MC, Luz EDMN, Bezerra JL. 2014. O gênero Pestalotiopsis Ascomycota, ‘Coelomycetes’ no Brasil. Agrotrópica (Brasil) 26: 89-98.). For example, Pestalotiopsis oryzae has not yet been reported in Brazil, but it has been isolated from Telopea sp. (Proteaceae), Paris polyphilla (Mellathiaceae), Dysosma versipellis (Berberidaceae), and rice (Oryza sativa) (Poaceae), which together with soybeans, are the largest monocultures in the Pampa region (Sentelhas et al. 2015Sentelhas P, Battisti R, Câmara G, Farias J, Hampf A, Nendel C. 2015. The soybean yield gap in Brazil - magnitude, causes and possible solutions for sustainable production. The Journal of Agricultural Science 153: 1394-1411. ). The genus Epicoccum, is a cosmopolitan plant pathogen mainly found on grasses in tropical regions (De Oliveira et al. 2018De Oliveira RC, Queiroz LC, Correa B. 2018. Epicoccum sorghinum in food: occurrence, genetic aspects and tenuazonic acid production. Current Opinion in Food Science 23: 44-48.). Some species, including Paspalum guenoarum (Poaceae), have previously been reported in the Pampa territory of Brazil (Gasparetto et al. 2017Gasparetto BF, Franke LB, Andrade CCL, et al. 2017. First Report of Bipolaris micropus, Curvularia geniculata, Epicoccum sorghinum, and Fusarium incarnatum on Paspalum guenoarum seeds in Rio Grande do Sul, Brazil. Plant Disease 101: 1679-1679.; Liu et al. 2018Liu PQ, Wei MY, Zhu L, et al. 2018. First report of leaf spot on taro caused by Epicoccum sorghinum in China. Plant Disease 102: 682-682.; Zeng et al. 2018Zeng H, Lu Q, Li R. 2018. First report of leaf spot of lily caused by Epicoccum sorghinum in China. Plant Disease 102: 2648-2648.).

All extracts of the endophytic fungi tested had antibacterial potential against S. aureus and E. coli, six of which showed high inhibition values. The potential of endophytic fungi may be related to the habitat of the host, and new hosts may be a promising source of antibacterial metabolites (Strobel & Daisy 2003Strobel G, Daisy B. 2003. Bioprospecting for microbial endophytes and their natural products. Microbiology and Molecular Biology Reviews 67: 491-502.; Schulz & Boyle 2005Schulz B, Boyle C. 2005. The endophytic continuum. Mycological Research 109: 661-686.). Raj et al. (2013Raj P, Khan SS, Modak M, Lone ZA, Rather SA, Yaqoob M. 2013. Biodiversity of endophytic fungi in saffron (Crocus sativus) and antimicrobial activity of their crude extract. Indo American Journal of Pharmaceutical Research 3: 3702-3713.) observed similar results with the strong potential of saffron endophytes to produce bioactive compounds against E. coli. In a similar study, the potential of secondary metabolites produced by endophytic fungi from saffron showed antibacterial activity, highlighting the Rhizopus ethyl acetate extract, which could inhibit all the bacteria tested (Chamkhi et al. 2018Chamkhi I, Sbabou L, Aurag J. 2018. Endophytic fungi isolated from Crocus sativus L.(saffron) as a source of bioactive secondary metabolites. Pharmacognosy Journal 10: 1143-1148.). In a survey of endophytic fungi from six plant species of the Cyperaceae family in Sri Lanka, Ratnaweera et al. (2018Ratnaweera PB, Walgama RC, Jayasundera KU, et al. 2018. Antibacterial activities of endophytic fungi isolated from six Sri Lankan plants of the family Cyperaceae. Bangladesh Journal of Pharmacology 13: 264-272.) observed that 91.6 % of the endophytes had metabolites with antibacterial properties, of which 33 % were able to inhibit the growth of E. coli.

Studies of endophytic Colletotrichum, Fusarium, Epicoccum, and Pestalotiopsis isolates have revealed antibacterial action against S. aureus and E. coli (Xing et al. 2011Xing YM, Chen J, Cui JL, Chen XM, Guo SX. 2011. Antimicrobial activity and biodiversity of endophytic fungi in Dendrobium devonianum and Dendrobium thyrsiflorum from Vietman. Current Microbiology 62: 1218-1224.; Radić & Štrukelj 2012Radić N, Štrukelj B. 2012. Endophytic fungi-The treasure chest of antibacterial substances. Phytomedicine 19: 1270-1284.). In our study, the extracts of Colletotrichum sp. 2 F17KB and Pestalotiopsis sp. F18KB presented the highest growth inhibition values for both microorganisms tested. Endophytic Colletotrichum and Pestalotiopsis isolates had antibacterial activity against gram-positive and gram-negative bacteria, with extracts showing high inhibition values (95.4 to 100 mm) (Maria et al. 2005Maria GL, Sridhar KR, Raviraja NS. 2005. Antimicrobial and enzyme activity of mangrove endophytic fungi of southwest coast of India. Journal of Agricultural Technology 1: 67-80.; Ferreira et al. 2015Ferreira MC, Vieira MDA, Zani CL, et al. 2015. Molecular phylogeny, diversity, symbiosis and discover of bioactive compounds of endophytic fungi associated with the medicinal Amazonian plant Carapa guianensis Aublet (Meliaceae). Biochemical Systematics and Ecology 59: 36-44. ).

Similar to saprophytes and plant pathogens, endophytic microorganisms can exhibit varying degrees of host specificity, ranging from permanent associations with a single plant species to relationships involving a wide range of hosts (Petrini 1996Petrini O. 1996. Ecological and physiological aspects of host specificity in endophytic fungi. In: Redlin SC, Carris LM. (eds.) Endophytic Fungi in Grasses and Woody Plants. St Paul, Minnesota, APS Press. p. 87-100.). The host specificity exhibited by endophytic microbes can be complex, and although many strict associations seem to be formed in nature, this can be manipulated somewhat when developing novel endophyte-host associations. Cannon and Simmons (2002Cannon PF, Simmons CM. 2002. Diversity and host preference of leaf endophytic fungi in the Iwokrama Forest Reserve, Guyana. Mycologia 94: 210-20.) described frequent isolation of identical endophytic strains from plant tissues of several species, which suggests that the extent of host preference/specificity in leaf endophytes is small; therefore, molecular research is being conducted on two key genera (Pestalotiopsis and Colletotrichum), the same common groups found in our study. The analysis of these factors is beyond the scope of this study but the loss of diversity, especially of endemic species, can result in negative consequences such as the loss of potentially associated organisms (Mommer et al. 2018Mommer L, Cotton TEA, Raaijmakers JM, et al. 2018. Lost in diversity: the interactions between soil-borne fungi, biodiversity and plant productivity. New Phytologist 218: 542-553. ; Chen et al. 2019Chen C, Chen HYH, Chen X, Huang Z. 2019. Meta-analysis shows positive effects of plant diversity on microbial biomass and respiration. Nature Communications 10: 1-10.; Li et al. 2020Li J-L, Sun X, Zheng Y, Lü P-P, Wang Y-L, Guo L-D. 2020. Diversity and community of culturable endophytic fungi from stems and roots of desert halophytes in northwest China. MycoKeys 62: 75-95.). Plant diversity is an essential factor for microbial biomass, and both the diversity and richness of fungi and bacteria are positively affected by the plants found in a given location. When losses in plant diversity are found, microbial biomass immediately decreases (Chen et al. 2019Chen C, Chen HYH, Chen X, Huang Z. 2019. Meta-analysis shows positive effects of plant diversity on microbial biomass and respiration. Nature Communications 10: 1-10.).

Our findings in this first study show that Iridaceae endemic to the Pampa biome has endophytic fungi with antibacterial potential, thus highlighting the importance of further studies with these fungal isolates. Endophytic fungi were more abundant in the leaves of the plant, and Colletotrichum and Pestalotiopsis were the most abundant genera and presented the best inhibition values in antibacterial tests. This survey is fundamental for the study of endophytic fungi in the Pampa biome, providing important information for the development of future studies of endophytic fungal communities in this Brazilian environment.

Acknowledgements

Special thanks to the researchers Rutilene Roll and Andrés Cañedo of the Laboratório de Pesquisa em Microbiologia of UNIPAMPA for the strains of microorganisms used in the experiments of antimicrobial activity given to the first author. This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Finance Code 001) and the Conselho Nacional de Desenvolvimento Científico (grant number 446234/2015-0). All authors thank the Universidade Federal do Pampa for their support in the development of this work.

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