Skip to main content
Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Diversity of cultivable fungal endophytes in Paullinia cupana (Mart.) Ducke and bioactivity of their secondary metabolites

  • Fábio de Azevedo Silva ,

    Contributed equally to this work with: Fábio de Azevedo Silva, Marcos Antônio Soares

    Roles Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

    Affiliation Departamento de Botânica e Ecologia, Instituto de Biociências, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brasil

  • Rhavena Graziela Liotti,

    Roles Methodology, Writing – review & editing

    Affiliations Departamento de Botânica e Ecologia, Instituto de Biociências, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brasil, Instituto Federal de Mato Grosso, Cáceres, Mato Grosso, Brasil

  • Ana Paula de Araújo Boleti,

    Roles Methodology, Writing – review & editing

    Affiliation Escola de Ciências Biológicas e Ambientais, Universidade Federal de Grande Dourados, Dourados, Mato Grosso do Sul, Brasil

  • Érica de Melo Reis,

    Roles Methodology, Writing – review & editing

    Affiliation Faculdade de Medicina, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brasil

  • Marilene Borges Silva Passos,

    Roles Methodology, Writing – review & editing

    Affiliation Faculdade de Medicina, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brasil

  • Edson Lucas dos Santos,

    Roles Methodology, Writing – review & editing

    Affiliation Escola de Ciências Biológicas e Ambientais, Universidade Federal de Grande Dourados, Dourados, Mato Grosso do Sul, Brasil

  • Olivia Moreira Sampaio,

    Roles Methodology, Writing – review & editing

    Affiliation Departamento de Química, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brasil

  • Ana Helena Januário,

    Roles Methodology, Writing – review & editing

    Affiliation Centro de Pesquisa em Ciências Exatas e Tecnológicas, Universidade de Franca, Franca, São Paulo, Brasil

  • Carmen Lucia Bassi Branco,

    Roles Methodology, Writing – review & editing

    Affiliation Faculdade de Medicina, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brasil

  • Gilvan Ferreira da Silva,

    Roles Methodology, Writing – review & editing

    Affiliation Embrapa Amazônia Ocidental, Manaus, Amazonas, Brasil

  • Elisabeth Aparecida Furtado de Mendonça,

    Roles Project administration

    Affiliation Faculdade de Agronomia, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brasil

  • Marcos Antônio Soares

    Contributed equally to this work with: Fábio de Azevedo Silva, Marcos Antônio Soares

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – review & editing

    Affiliation Departamento de Botânica e Ecologia, Instituto de Biociências, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brasil


Paullinia cupana is associated with a diverse community of pathogenic and endophytic microorganisms. We isolated and identified endophytic fungal communities from the roots and seeds of P. cupana genotypes susceptible and tolerant to anthracnose that grow in two sites of the Brazilian Amazonia forest. We assessed the antibacterial, antitumor and genotoxic activity in vitro of compounds isolated from the strains Trichoderma asperellum (1BDA) and Diaporthe phaseolorum (8S). In concert, we identified eight fungal species not previously reported as endophytes; some fungal species capable of inhibiting pathogen growth; and the production of antibiotics and compounds with bacteriostatic activity against Pseudomonas aeruginosa in both susceptible and multiresistant host strains. The plant genotype, geographic location and specially the organ influenced the composition of P. cupana endophytic fungal community. Together, our findings identify important functional roles of endophytic species found within the microbiome of P. cupana. This hypothesis requires experimental validation to propose management of this microbiome with the objective of promoting plant growth and protection.


Paullinia cupana var. sorbilis (Mart.) Ducke is a Brazilian Amazonia plant species. Commonly known as guaraná, it is used as central nervous system stimulant due to its high concentration of caffeine [1]. The first P. cupana crops were grown by indigenous tribes, but the beverage industry has prompted the development of commercial-scale crops. Nowadays, guaraná seeds not only represent a global trend in the market of soft and energy drinks [2], but are also promising raw materials for the development of drugs and supplements with different properties, such as anti-fatigue [3,4], antioxidant [57], antimicrobial [810], antiproliferative [11], anxiolytic [12] and cardioprotective [13,14]. However, plant diseases like anthracnose and oversprouting, caused by Colletotrichum guaranicola F. C. Albuq and Fusarium decemcellulare Brick, respectively, have seriously damaged P. cupana crops and limited their expansion and productivity [1518]. There are promising alternatives for disease control–such as cultivation of resistant P. cupana genotypes [19,20]–or the biological control of phytopathogens by endophytic microrganisms [21,22].

Endophytes are microorganisms that inhabit the inner tissue of their hosts, at least during a phase of the endophyte’s life cycle, and perform various ecological relationships without showing visible symptoms of infection in the host [2325]. Endophytes are intensively studied for bioprospecting purposes, but their interaction with the host and associated ecological factors in tropical regions remain poorly investigated [2628]. Some studies have reported the endophytic fungal diversity of P. cupana leaves [18,29,30] and endophytic bacterial diversity of P. cupana leaves, seeds and roots [31,32]. However, many aspects of the P. cupana endophytic fungal community have not yet been assessed, such as: (i) whether abiotic factors, such as weather, geographic distance, plant tissue, ultraviolet radiation and culture system influence its endophytic fungal community’s structure and composition, as observed in other host plants [3338]; (ii) whether different P. cupana genotypes host different endophytic fungal communities, as occurs in other crops [39,40]; and (iii) the bioprospecting potential and functional traits of P. cupana endophytes [30,32,41].

Several functional traits such as synthesis of hydrolytic enzymes and indole acetic acid are related to the protection against phytopathogens, plant growth promotion and resistance to environmental stress, which are relevant for their multiple applications in agriculture [4244]. The same is true for natural products synthesized by endophytes, which have important applications in human health [4550]. There is a strong demand for new and more effective antibiotics and anticancer drugs, which can be synthesized by endophytes [5154].

Considering that different factors can influence the structure of endophytic communities, the present study aims to identify cultivable endophytic fungal species in P. cupana roots and seeds and to examine how the plant organ, genotype and geographic location influence the endophytic fungal community structure and production of functional traits for plant growth promotion and induction of tolerance to pathogens. In addition, we have isolated and identified the major special metabolites from two endophytic fungal species (Trichoderma asperellum and Diaporthe phaseolorum) and examined their antibacterial, antitumor and genotoxic activity in vitro.

Materials and methods

Isolation and molecular identification of endophytic fungi from P. cupana roots and seeds

This study was carried out with genotypes obtained by vegetative propagation (clonal cultivar) to ensure the analysis of the same genetic material in plants grown in two different places. The seeds and roots of five adult healthy plants of P. cupana from each clonal cultivars CMU 871 (tolerant to anthracnose and oversprouting) and CMU 300 (tolerant to anthracnose and susceptible to oversprouting) [16,55] were collected in November 2014 in the Embrapa (Brazilian Agricultural Research Company) experimental farms located in the cities of Manaus (2° 53' 29.14''S and 59° 58' 39.90''W, 99 m high) and Maués (3° 22' 54'' S and 57° 42' 55'' W, 18 m high), State of Amazonas, Brazil. The plant material was collected at the end of the dry season, when the accumulated rainfall and average temperature were respectively 196.0 mm and 28.46°C in Manaus, and 272.3 mm and 28.17°C in Maués [56,57].

The plant material was stored and transported under refrigeration until processing. Roots and seeds were washed in tap water and neutral detergent. Surface disinfection was performed in a laminar flow hood by soaking in 70% ethanol for 30 s and 2.5% NaClO for 8 min (roots) or 20 min (seeds), and rinsing five times with sterile distilled water [58]. The efficacy of surface disinfection was verified by inoculating the last rinse water on tryptone soy agar (TSA) plates. Disinfected seeds and roots were inoculated on potato dextrose agar (PDA) and TSA culture medium (Kasvi, Curitiba, PR, Brazil) supplemented with streptomycin, chloramphenicol and tetracycline (200 mg/L), and incubated for 15 days. The fungal strains were grouped morphologically [59], and the grouping was confirmed by analyzing microscopic traits in microculture slides [60] and comparing the obtained results with taxonomic keys [61,62]. The isolates were stored under refrigeration in PDA medium and in tubes with sterilized rice. DNA of one strain from each morphological group was extracted using the Genomic DNA Isolation Kit (Norgen Biotek Corporation, Thorold, ON, Canada), according to the manufacturer’s protocol. The internal transcribed spacer regions (ITS) were amplified using the ITS1 and ITS4 primers [63,64]. The polymerase chain reaction products were purified using ExoSAP-IT (USB Corp., Cleveland, OH, USA) and sequenced by the Sanger method, using the Big Dye Terminator kit (Applied Biosystems, Foster City CA, USA). The obtained sequences were analyzed with BioEdit Sequence Alignment Editor (version 7.2.5) and compared with sequences deposited in the NCBI GenBank database (National Center for Biotechnology Information).

Community structure analysis

The endophytic colonization rate [65] and the relative frequency of fungal species [28] were evaluated. The species diversity, richness and dominance were estimated using the non-parametric Shannon-Weaver index [66], Chao 1 index [67] and Simpson index [68], respectively. The degree of similarity among different communities was determined using the Jaccard index [69], while the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cluster analysis grouped the endophytes into distinct clusters. To examine whether a given endophytic community plays predominant roles, we analyzed their ecological roles. The species were classified into the categories phytopathogen, saprophyte, mycotroph, entomopathogen and coprophilous, according to the criteria established by literature reports and the Agricultural Research Service of the United States Department of Agriculture (USDA-ARS) database [70].

Non-metric multidimensional scaling (NMDS) based on Jaccard's coefficient was used to visually detect the preference of the species in relation to each host plant geographic location, genotype and organ. The distinctiveness of fungal assemblages was tested with one-way analysis of similarity (ANOSIM) considering the incorporation of 999 permutations. The indicator value method (IndVal) [71] was used to identify the most characteristic species of each community, and the ones more prevalent in a single community. Permutation test was performed to confirm whether the indicator value was statistically significant (p < 0.05).

A network was built to analyze the existence of interspecific interactions. Correlation between species were considered positive when the Spearman correlation coefficient (ρ) was > 0.6, and statistically significant when p < 0.05. The rate of connectivity between nodes was determined by analyzing betweenness centrality, while the Louvain algorithm was used to detect modularity [72].

Root associations, endophytic potential and functional traits

P. cupana roots were washed, clarified with KOH (10%, v/v), and stained with trypan blue in lactoglycerol (0.05%, v/v) for visualization of fungal-root associations [73]. Fifteen root fragments from each individual were analyzed in the Nikon Eclipse E200 microscope coupled with a camera (Bel Photonics), in order to identify the presence of dark septate endophytic fungi (DSE) and endomycorrhizal structures, such as arbuscles and vesicles. The identity of the structures was confirmed by comparison with literature reports [74].

Endophytic fungi with dark mycelium and septate hyphae isolated from P. cupana were selected to examine their infection capacity and identify typical structures of dark septate endophytes. P. cupana seeds were not used due to the difficulty in promoting their germination [75], and alternatively, seeds of the Sorghum cultivar BRS 373 were used (Sorghum sp.). Sorghum sp seeds were disinfected [76] and incubated in minimum mineral medium (MM) [77] at room temperature (approximately 27°C) under natural light. After 5 days, the rootlets of seedlings were inoculated with mycelium fragments. Non-inoculated seedlings were used as controls. The roots were cleared, stained, and analyzed after 15 days of interaction [73]. Stained roots were preserved in polyvinyl lactoglycerol and observed under light microscope [78].

The production of cellulase [79], esterase [80], amylase, phosphatase and protease [81] was analyzed in vitro in all the isolated endophytic fungal species, using essentially qualitative tests. The siderophore production was determined in cromoazurol S medium [82]. Discs of endophytic mycelium (⌀1 mm) were used as inoculum and the formation of halos was determined after 24 h incubation. The production of indoleacetic acid (IAA) was determined by a colorimetric method, using BD broth supplemented with tryptophan (0.73 μmol/ml) as the negative control [83]. The IAA concentration was determined using a standard curve prepared with commercial IAA.

Antibacterial activity

A lineage of each endophytic fungal species was grown in PDA medium for the production of ethyl acetate (EtOAc) extracts, as reported by Rosa [84]. The qualitative antibacterial activity of the endophytes’ EtOAc extracts (20 mg/mL) was assessed as reported by Ichikawa [85], against the following strains: susceptible Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 6538), and multiresistant E. coli (1A), P. aeruginosa (2A) and S. aureus (3A). The extracts that suppressed bacterial growth the most strongly were selected for determination of the minimal inhibitory concentration (MIC)–the lowest sample concentration at which bacteria do not grow [86]–using serial dilutions ranging from 5,000 μg/mL to 39 μg/mL. Tetracycline (0.05 mg/mL) and LB broth were used as the positive and negative controls, respectively. The minimum concentration of death (MCD) was determined using Mueller-Hinton agar medium [87].

Purification and structural elucidation of special metabolites

The species Trichoderma asperellum (1BDA) and Diaporthe phaseolorum (8S) were selected for the chemical analysis because they exhibited significant functional traits. Crude EtOAc extracts from D. phaseolorum (8S) (235 mg) and T. asperellum (1BDA) (240 mg) were prepared as reported by Rosa [84] and further purified through reversed-phase column chromatography using C-18 resin (230–400 mesh, Merck) as stationary phase and CH3OH:H2O (3:7, 7:3 and 10:0 v/v) as eluent.

High-performance liquid chromatography (HPLC) was performed in the Varian ProStar 215 chromatograph and data were acquired using the software Galaxie Chromatography Data System (Varian). The CH3OH fraction from T. asperellum (1BDA) was subjected to semi-preparative HPLC (Phenomenex column, 10.0 × 250 mm, 5 μm, C18, equipped with a precolumn) under elution with CH3OH/H2O/HCO2H (7:2,9:0.1) in isocratic condition to afford 1-hydroxy-8-methoxyanthraquinone (sample 17A, 1.5 mg) as a light-yellow solid.

The CH3OH:H2O fractions from D. phaseolorum (8S) were submitted to column chromatography employing silica gel 60 (40–63 mesh, Merck). The fraction CH3OH:H2O (7:3) was eluted with EtOAc:CH3OH (1:1 v/v), hexane and CH3OH to provide the di-(2-ethylhexyl)phthalate (DEHP, sample 070, 2.1 mg) as a dark yellow solid. The fraction CH3OH:H2O (3:7) was sequentially eluted with hexane:EtOAc (6:4) and CHCl3 to furnish 3-hydroxypropionic acid (3HPA, sample 3A, 2.7 mg) as a colorless solid.

Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Ascend 500 spectrometer operating at 500 MHz for 1H nuclei and 125 MHz for 13C nuclei. Chemical shifts were quoted in parts per million (ppm), referenced to the appropriate residual solvent peak. Two-dimensional spectroscopy (COSY, HSQC and HMBC) were used for structural determination. Gas chromatography coupled to mass spectrometry (GC-MS) were performed on a Shimadzu GC17A chromatograph coupled to a GCMSQP5050A detector. After structural determination, the purified samples were tested for antibacterial activity using the method reported in the previous section. MIC was determined with serial dilutions ranging from 30 μg/mL to 0.2 μg/mL.

Antitumor activity

The elucidated purified molecules (DEHP and 3HPA) were tested for antitumor activity. The Chinese hamster ovary cells (CHO) and Mus musculus skin melanoma B16F10 cell lines were kindly provided by Dr. Edgar Julian Paredes Gamero from Federal University of São Paulo (UNIFESP, São Paulo, SP, Brazil) and cultured in the Laboratory of Cell Culture and Molecular Biology at the Federal University of Grande Dourados (Dourados, MS, Brazil). CHO and B16F10 cells were cultured in DMEM + F10 and RPMI-1640 medium, respectively, containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, in a humidified incubator at 37°C and under 5% CO2.

The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay provides a sensitive measurement of the cell metabolic status, in particular the mitochondrial function, which reflects early cellular redox changes [88]. CHO and B16F10 cells (6x103 and 1x103cells/well, respectively) were grown in 96-well tissue culture plates and treated with the isolated compounds (3.12, 6.25, 12.5, 25, 50 and 100 μg/mL) for 24, 48 and 72 h. The cells were washed with 0.1 mL of 0.1 M phosphate buffered saline (PBS), pH 7.4, at 37°C, and further incubated with 100 μL of MTT solution (1 mg/mL in culture medium) for 3 h, at 37°C. The assay medium was removed and the dark-blue formazan crystals formed in intact cells were dissolved in DMSO. The absorbance was recorded at 630 nm in a microplate reader. Three independent experiments were performed in triplicate. Doxorubicin was used as positive control; it inhibits CHO and B16F10 cell growth by 50% (IC50) at the concentration of 1 and 0.1 μg/mL, respectively. The results were expressed as the percentage of MTT reduction relative to the control group (untreated cells).


The alkaline comet assay [89] was used to assess the genotoxicity of compound 3A isolated from D. phaseolorum (8S) EtOAc extract. This is a sensitive method capable of detecting DNA strand breaks, alkali-labile sites and DNA-DNA/DNA-protein cross-linking [90]. A prerequisite to perform this assay is cell viability >70% because cytotoxic concentrations of a given sample may cause DNA damage that does not reflect the genotoxic effect. Thus, first we used the MTT assay to address whether compound 3A was cytotoxic towards V79 cells. Briefly, after a 24-h treatment with this sample (12.5, 25.0, 50.0 or 100.0 μg/mL) at 36.5°C, the assay medium was removed and the cells were incubated with 100 μL of MTT (0.5 mg/mL) for 2 h at 36.5°C. The assay medium was replaced by 100 μL of DMSO and, after five minutes, the samples were transferred to another plate to record their absorbance at 540 nm, using a microplate reader (Multiskan EX, Thermo Electron Corporation). Untreated cells and 10.0 μM doxorubicin (Sigma-Aldrich, St. Louis, MO, USA) represented the negative and positive controls, respectively. The results were expressed as the percentage of MTT reduction relative to the negative control group (100% survival).

The comet assay was performed using the method reported by Speit [91], with minor modifications. The V79 cells were treated with sample 3A (25.0, 50.0, or 100.0 μg/mL) for 3 h, at 36.5°C, protected from light to prevent additional DNA damage. Cells treated with medium and 200 μM H2O2 were used as the negative and positive controls, respectively. Aliquots of 20 μL of cell suspension were mixed with 100 μL of low-melting point agarose (0.5% in PBS) and layered onto a microscope slide previously thin-layered with 1.5% normal-melting point agarose. The slides were immersed in freshly prepared lysing solution (100 mM EDTA, 10 mM Tris, 2.5 M NaCl, 1% Triton X-100, and 10% DMSO pH 10 –all from Sigma-Aldrich, St. Louis, MO, USA) for at least 24 h, at 4°C. Then, the slides were immersed in alkaline buffer (0.2 M EDTA, 10 M NaOH, pH >13, both from Sigma-Aldrich, St. Louis, MO, USA) for 25 min, at 4°C, and submitted to electrophoresis at 0.92 V/cm and 300 mA, for further 25 min. Next, the slides were immersed in neutralization buffer (0.4 M Tris-HCl, pH 7.5, Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and fixed in 100% ethanol for 5 min.

The slides were air-dried, stained with 0.3X Sybr Gold Nucleic Acid Gel Stain (Invitrogen, Carlsbad, CA), and analysed in an Ecliple Ci fluorescence microscope (Nikon, Japan) equipped with a 20X objective. Images of at least 100 nucleoids were obtained using the Lucia Comet Assay software (version 7.30, Laboratory Imaging, Czech Republic). The % DNA in the comet tail was considered as the parameter of genotoxicity. Three independent experiments were assayed in triplicate. Once the negative control of one experiment was excluded due to technical problems, we carried out an additional experiment without replicates. The data from each experiment are presented as the mean of median of 100 or 200 nucleoids.

Statistical analysis

The Past software [92] was used to determine diversity and equitability indexes and perform cluster analysis. The package vegan in the R software version 3.3.2 [93] was used to carry out the analyses of variance, similarity, permutation and correlation. Indicator species test was performed using package labdsv ( within the R environment. Network was constructed and visualized using the software Gephi version 0.9.1 [94]. Venn diagrams were generated with DrawVenn web tool ( Data from the antitumor activity assay were analyzed by the Dunnett's multiple comparison test, using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). Data from the comet assay (both cytotoxicity and genotoxicity) presented normal distribution and were statisticaly compared using one-way ANOVA and Bonferroni’s post-hoc test, at a significance level of 5%, with the aid of the GraphPad Prism software.

Results and discussion

Diversity of endophytic fungi in P. cupana roots and seeds

We isolated and identified 256 strains and 34 species of endophytic fungi from P. cupana roots and seeds. The phylum Ascomycota, the class Sordariomycetes, and the order Diaporthales had the highest relative frequency (Table 1). The aforementioned taxa often colonize tropical plant communities [28,95].

Table 1. Endophytic fungal taxa isolated with the highest relative frequency (%) from Paullinia cupana, stratified according to the plant organ, genotype and geographic location.

The genus Xylogone and the species Xylogone ganodermophthora had the highest total relative frequency in root endophytic communities (Table 1). The genus Xylogone was described only once as endophyte of Taxus chinensis [96], and X. ganodermophthora was described as mycopathogen [97] and as an antifungal species against watermelon pathogens [98]. Here we report for the first time that the species X. ganodermophtora is an endophyte of P. cupana. Phomopsis asparagi, the species with the second highest total relative frequency (Table 2), was reported as endophyte [99] and phytopathogen in asparagus crops [100].

Table 2. Relative frequency (%) of endophytic fungal species isolated from different microbial communities in Paullinia cupana.

Diaporthe phaseolorum and P. asparagi were the species with the highest relative frequency in seed endophytic communities (Table 2). Only the former was present in all the endophytic communities of P. cupana seeds (Table 2). The genus Phomopsis and the species D. phaseolorum are predominantly endophytic in many tropical species [101104].

Only the genera Diaporthe, Fusarium, Glomerella and Phomopsis were identified in endophytic communities of both the roots and seeds of P. cupana (Table 2). The genera Diaporthe, Pestalotiopsis and Phomopsis were previously identified in endophytic communities of phyllospheres [29]. Thus, we hypothesize that endophytic fungi that colonize P. cupana have organ-specificity, as reported for other tropical and temperate host plants [105107].

Most of the endophytic species occurred at a total relative frequency lower than 10%. The species Arxiella dolichandrae, Mariannaea camptospora, Mycena robusta, Paraphaeosphaeria arecacearum, Parapleurotheciopsis inaequiseptata, Peyronellaea pinodella and Pochonia boninensis occur in tropical regions [108114], while X. ganodermophthora occurs only in temperate regions [115]. Nevertheless, their occurrence in endophytic communities has not been reported yet, indicating that P. cupana is a repository of fungal species in the Brazilian Amazonia forest. As Peyronellaea pinodella [116] and Arxiella dolichandrae [117] have already been reported as phytopathogens in other hosts, we believe that they are endophytes or latent pathogens in P. cupana because the plant-endophyte relationship can vary from mutualism to parasitism [42,118]; however, this hypothesis requires experimental validation.

Regarding the ecological role of the isolated species (S1 Table), all the endophytic fungal communities isolated from P. cupana had at least one species that is reported as endophyte, phytopathogen, saprophyte, mycoparasite, entomopathogen or coprophile. Some fungal species have been reported as both endophytic and phytopathogenic. It is known that endophytic communities present latent phytopathogens [119] and several phytopathogens can behave as endophytes depending on the local biotic and abiotic conditions [120,121]. In addition, the genetic diversity of some endophytic fungi may enable them to act as phytopathogens or saprophytes [122], and some phytopathogens may not be able to develop the disease due to host defense mechanisms [123,124].

Several species reported as saprophytes, coprophiles and entomopathogens were isolated from P. cupana (S1 Table). Coprophilous fungi are transmitted to plant tissues by herbivores [125], while saprophytic fungi mainly derive from soil and rhizosphere [126,127]. Entomopathogenic fungi may have an endophytic phase of life [128] and some endophytic fungal genera have entomopathogenic species [129]. Endophytes with entomopathogenic potential are frequently tested for their ability to inhibit agricultural pests [127,130,131]. These reports indicate that P. cupana may host endophytic fungal species with potential for pest control; this hypothesis requires further experimental validation.

Mycoparasitic species may be abundant in endophytic communities [132134], such as Colletotrichum gloeosporioides [135], Fusarium oxysporum [136], Humicola fuscoatra [137], Talaromyces pinophilus [138], Trichoderma asperellum [139], Trichoderma harzianum [140], and Xylogone ganodermophthora [115]. Several species of mycoparasitic fungi behave as endophytes, such as C. gloeosporioides [141], H. fuscoatra [142], T. asperellum [143], and T. harzianum [144].

Analysis of the indicator value revealed that few species preferred a specific community. Regarding the geographic location, P. asparagi significantly preferred Maués (IndVal = 0.314, P = 0.01). Regarding the P. cupana organs, X. ganodermophthora significantly preferred root communities (IndVal = 0.475, P = 0.001), while D. phaseolorum significantly preferred seed communities (IndVal = 0.15, P = 0.001). H. fuscoatra was significantly associated with the root endophytic communities of the susceptible P. cupana genotype grown in Manaus (IndVal = 0.2, P = 0.012). No species was significantly associated with a specific genotype (susceptible or tolerant).

The P. cupana genotype influenced the endophytic fungal community diversity (Table 3). Considering the communities from seeds collected in both geographic locations (Manaus and Maués), those isolated from the susceptible genotype exhibited higher richness, Shannon index and evenness when compared with those isolated from the tolerant genotype. Regarding the root endophytic communities from Manaus, those isolated from the susceptible P. cupana genotype had higher Shannon index, evenness and colonization rate than those isolated from the tolerant genotype. The endophytic communities isolated from the roots and seeds of the susceptible P. cupana genotype have higher richness and Shannon index than those isolated from the same organs of the tolerant genotype. The richness of microbial communities in the tolerant genotype was greater than that of the susceptible genotype only in the root endophytic community of plants grown in Manaus. The root endophytic communities isolated from the susceptible genotype grown in Maués had higher richness, Shannon index and dominance but lower evenness and colonization rate than the root endophytic communities from the tolerant genotype grown in the same location.

Table 3. Alpha diversity and colonization rate (%) of endophytic fungal communities isolated from P. cupana.

The root endophytic communities of the tolerant and susceptible genotypes grown in Manaus shared 11 species (Fig 1A), while those grown in Maués shared 6 species (Fig 1B). Communities isolated from the same organ shared a higher number of species in the susceptible (Fig 1C) and tolerant (Fig 1D) genotypes. Only the species F. oxysporum, G. acutata and P. asparagi occurred in both organs, explaining why communities of different organs shared few species. The root endophytic communities of plants grown in Manaus had the largest number of unique species (Fig 1C and 1D).

Fig 1. Venn diagrams representing the distribution of endophytic fungal species in the communities studied.

(A) Susceptible and tolerant genotypes from Manaus, (B) Susceptible and tolerant genotypes from Maués, (C) Roots and seeds from susceptible genotypes, (D) Roots and seeds from tolerant genotypes. Mu = Manaus; Me = Maués; Su = Susceptible genotype (CMU 300); To = Tolerant genotype (CMU 871); Se = seed; Ro = Root.

As reported in tobacco, tomato and potato cultivars [39,40,145], we found that different endophytic communities colonized the susceptible and tolerant genotypes of P. cupana, and that the susceptible genotypes exhibited higher species diversity–the last finding may be related to their vulnerability to microbial infections, which colonize tissues faster [146,147]. Moreover, roots can produce exudates that promote colonization of certain groups of microorganisms [148,149]. Exudates produced by different genotypes can influence the microbial community in rhizosphere and consequently affect the composition and richness of endophytic communities [150]. Additional studies are required to determine whether susceptible and tolerant P. cupana genotypes differ with respect to the composition of exudates.

The geographic location where P. cupana was grown also influenced the endophytic fungal community’s diversity (Table 3). Compared with the same genotype grown in Maués, the endophytic fungal communities isolated: (i) from seeds of the tolerant genotype grown in Manaus had higher colonization rate, dominance and richness; (ii) from seeds of the susceptible genotype grown in Manaus had lower colonization rate, evenness, richness and Shannon index; (iii) from roots of the tolerant genotype grown in Manaus had higher dominance, richness and Shannon index; (iv) from roots of the susceptible genotype grown in Manaus had higher colonization rate, evenness, richness and Shannon index.

The observed and estimated species richness (Chao index) in the endophytic fungal communities identified in Maués were similar to each other; in Manaus, the estimated richness was higher than that observed for most endophytic communities (Table 3). Therefore, the geographic location influences the endophytic fungal diversity in P. cupana. Manaus exhibited the highest species richness, more specifically in seeds of the tolerant genotype and roots of the susceptible genotype.

The level of particulate matter in the air of urban areas is high due to several anthropic activities, such as industrial and vehicular emissions, biomass burning and soil resuspension [151,152]. Particulate matter suspended in the air carries a high diversity of fungi and bacteria [153156] that can colonize the surface and integrate the epiphytic community of plants on which the particles deposit [157]. Several studies have reported epiphytic species as endophytes [28,158,159], indicating that a variety of species can colonize the inner plant tissues. Considering that Maués is located in a rural area within the Amazonia forest relatively distant from urban centers, and that Manaus is a large urban center, we hypothesized that the P. cupana samples grown in Manaus received more particulate matter from the air and thereby a greater amount of inoculum, which may explain their greater richness of endophytic fungal species. Urban and rural areas may present distinct endophytic communities even among hosts belonging to the same species [160162].

Other factors such as temperature, rainfall and soil composition can influence the diversity of endophytic species [163165]. These parameters differed between Manaus and Maués, which may have impacted on the endophytic diversity of each community. According to Koppen climate classification, Manaus climate is ranked as Am (monsoon), with average annual temperature of 26.7°C, average annual rainfall of 2.420 mm and soil characterized as yellow latosol. On the other hand, Maués climate is ranked as Af (humid equatorial), with average annual temperature of 25.5°C, mean annual rainfall of 2.101 mm and soil characterized as eutrophic gleysoil [166,167].

The plant organ is an important determining factor of the endophytic community’s composition in P. cupana, because the roots were colonized by communities with higher richness and diversity (S1 Fig). The isolation of seeds from the external environment for a long period [168] and the presence of antifungal compounds in seeds [9] may also lower endophytic species richness in this plant organ. P. cupana seeds are surrounded by a thick epicarp and partially surrounded by a thin membrane (the pith) [75], while the roots are in constant contact with the ground and can be infected by a great diversity of edaphic microorganisms.

Although the NMDS plot did not show separation between the endophytic fungal communities of distinct P. cupana genotypes and geographic locations, the communities of seeds and roots were clearly different and clustered as distinct groups (ANOSIM R2 = 0.27, P = 0.001, stress = 0.064) (S2 Fig). The root endophytic communities had a narrower distribution because they shared greater similarity in composition and abundance, indicating that the plant organ influences the community’s composition in P. cupana.

The Jaccard index of similarity (Fig 2) showed that (i) the communities from seeds and roots shared the lowest levels of similarity; (ii) the clonal type was crucial to distinguish the communities within each plant organ; and (iii) the location determined the highest rates of similarity between communities. The degree of similarity between endophytic communities may decrease as the geographical distance between same host species increases [28,34,35,165,169].

Fig 2. Analysis of the similarity degree between endophytic communities isolated from the seeds and roots of two P. cupana genotypes grown in Manaus and Maués.

Mu = Manaus; Me = Maués; Su = Susceptible phenotype (CMU 300); To = Tolerant phenotype (CMU 871); Se = Seeds; Ro = Roots.

Regarding the network analysis, the 34 nodes were connected by 107 edges, with average degree of 6.29 and clustering coefficient of 0.617 (S3 Fig). Ten species presented intermediate values of betweenness centrality, most of them were isolated from P. cupana roots and occured in both clonal types grown in both locations. X. ganodermophtora and P. asparagi were also identified in both clonal types from both locations and exhibited the highest betweenness centrality. X. ganodermophtora had the highest centrality degree, with 23 edges, binding to almost all species isolated from roots and acting as a local hub, while P. asparagi had the second highest centrality degree, with 20 edges mainly composed of species isolated from seeds. The network had two modules: the largest and the smallest ones were composed of endophytic fungal species isolated from P. cupana roots and seeds, respectively (S4 Fig). P. asparagi constituted an important node because it connected the seed and the root species modules. The formation of distinct modules for each organ, as well as the presence of different indicator species in each organ, stressed that different communities colonize the roots and seeds. Therefore, X. ganodermophtora and P. asparagi represented the central species of the endophytic fungal community of P. cupana and exerted the strongest influence on its structure, stability and dynamics.

In vitro endophytic ability

The presence of microsclerotia in the roots of all samples analyzed confirmed that P. cupana was associated with DSE (Fig 3). Associations with DSE fungi can become pathogenic, although most of them benefit the host by increasing nutrients absorption and promoting plant growth [170172].

Fig 3. Structure of mycorrhizal fungi and dark septate endophytic fungi detected within P. cupana roots (40x).

Arbuscle (AR), Coenocytic hyphae (HA), Coil hyphae (HC), Microsclerotium (M), Septate brown hyphae (HS), Vesicle (V).

P. cupana roots contained typical structures of mycorrhizal fungi. Arbuscules with the arum type morphology were detected in the roots of the susceptible genotype grown in Manaus; coil hyphae and brown septate hyphae were exclusively detected in tolerant host plants; and vesicles were observed only in the clonal cultivar grown in Maués. The literature reports the association between P. cupana and mycorrhizal fungi [173,174]. Colonization and sporulation are seasonal events strongly influenced by increased rainfall [173], favored by increased soil acidity and manganese concentration, and inhibited by high iron content [174].

The strains C. gloeosporioides (6TSA), P. asparagi (22TSA) P. pinodella (28S), and Sydowiella fenestrans (104BDA) were selected for the evaluation of endophytic capacity because they presented dark mycelium and septate hyphae. P. asparagi is frequently reported as phytopathogenic against asparagus crops [100], causing discoloration of the stem or trunk, oval lesions and brown spots on the roots [175,176]. Although the endophyte P. asparagi (22TSA) produced microsclerotia (Fig 4A), it also caused the same pathogenic symptoms reported for asparagus, probably due to the long cultivation period used in this study.

Fig 4. Microsclerotium in sorghum roots (40x).

(A) Phomopsis asparagi (22TSA) and (B) Peyronellaea pinodella (28S).

Sorghum roots colonized by P. pinodella (28S) contained microsclerotia (Fig 4B), but they did not show apparent symptoms of disease. Among the endophytic fungal species isolated from seeds, only P. pinodella (28S) exhibited dark mycelium and septate hyphae, the other fungal species colonized the seedling radicle without either developing dark septate structures or causing disease symptoms. There are no literature reports that the fungal species evaluated produce DSE structures; however, there are DSE species belonging to the genus Diaporthales [177], Phomopsis [178] and Pleosporales [179,180].

DSE are distributed in a wide variety of environments, such as sub-Antarctic regions, temperate and tropical forests, semi-arid regions, among others [181,182]. The currently available information on DSE mainly comes from studies carried out in arctic and temperate regions [183]. Several tropical plant families associate with DSE [181], but there are few reports on the association between DSE and species of the family Sapindaceae [184,185] and other species in the Amazonia region [186,187].

Analysis of functional traits

The EtOAc extract of the 27 endophytic fungal species isolated from P. cupana inhibited bacterial growth in the qualitative test (Table 4), and the EtOAc extract of 37 fungal strains suppressed the growth of at least one bacterial strain. Only D. phaseolorum (8S) inhibited the growth of P. aeruginosa susceptible and multiresistant strains. The antibacterial activity of several endophytic fungal species that we isolated was previously reported in the literature, such as C. gloeosporioides [188], D. melonis [189], D. phaseolorum [103], F. meliae [190], F. oxysporum [191], F. polyphialidicum [192], F. solani [193], G. acutata [194], G. zeae [195], H. fuscoatra [196], M. camptospora [197], P. janthinellum [198], P. microspora [199], T. asperellum [29] and T. harzianum [200]. To the best of our knowledge, this is the first report on the antibacterial activity of A. dolichandrae, D. hongkongensis, D. terebinthifolii, M. terrestris, M. robusta, N. mackinnonii, P. asparagi, P. arecacearum, P. boninensis, P. inaequiseptata, P. parvisporus and X. ganodermophthora. Only the antifungal activity of X. ganodermophthora was reported previously [201].

Table 4. Analysis of functional traits in endophytic fungi isolated from Paullinia cupana.

The EtOAc extracts of D. melonis (29TSA), D. phaseolorum (8S), D. terebinthifolii (24S), and T. harzianum (43BDA)–which suppressed the growth of the greatest number of bacterial strains in the qualitative test–were selected for determination of the minimal inhibitory concentration (Table 4). Analysis of the minimum concentration of death evidenced some microbial growth after treatment with all the extracts’ concentrations tested, indicating that they exerted bacteriostatic activity and that their minimum concentration of death was higher than 5.000 μg/mL (Table 4). The antimicrobial activity of D. melonis [189], D. phaseolorum [103] and T. harzianum [202204] has already been reported.

We identified 8 amylase-, 13 cellulase-, and 15 protease-producing endophytic fungal species in P. cupana (Table 4). Here we report for the first time the production of (i) amylase by D. melonis, M. robusta and M. terrestris; (ii) cellulase by A. dolichandrae, F. polyphialidicum, P. parvisporus, P. microspora, P. asparagi and X. ganodermophthora; (iii) and protease by A. dolichandrae, D. hongkongensis, D. melonis, D. phaseolorum, F. polyphialidicum, H. fuscoatra, M. terrestris, P. asparagi, P. boninensis, P. inaequiseptata, P. microspora and X. ganodermophthora. Some of the species that we identified are known for the production of (i) amylase, such as F. oxysporum, F. meliae, F. solani, G. zeae, P. janthinellum and T. harzianum [205209]; (ii) protease, such as F. meliae, F. oxysporum, F. solani, G. acutata, G. zeae, M. camptospora and P. janthinellum, T. harzianum [205,210214]; and (iii) cellulase, such as D. phaseolorum, F. meliae, F. oxysporum, F. solani, G. acutata, G. zeae, H. fuscoatra, M. terrestris and T. harzianum [205,215222].

We did not identify esterase-, phosphatase-, and siderophore-producing endophytic fungal species in P. cupana under the conditions assessed. Thirteen endophytic fungal species did not display any enzyme activity; however, enzyme release by some of them were already reported, such as the synthesis of (i) amylase and cellulase by C. gloeosporioides and P. macrospinosa [205,223]; (ii) amylase and protease by M. camptospora [214,224]; and (iii) amylase, cellulase and protease by T. asperellum [225227]. The species D. terebinthifolii, M. elegans, N. rigidiuscula, N. mackinnonii, P. arecacearum, P. macrospinosa, P. pinodella, P. lagerstroemiae and S. fenestrans–which did not present enzymatic activity in our work–, are not reported in the literature with any enzymatic activity.

Only the species F. oxysporum, M. terrestris and T. harzianum, which were isolated from tolerant and susceptible genotypes of P. cupana, exhibited amylolytic, cellulolytic and proteolytic activity. Although protease-, cellulase-, and amylase-producing endophytic fungal species colonized both P. cupana genotypes, the production of enzymes and antifungals in the roots may not be sufficient to prevent the development of anthracnose–this disease affects the aerial organs, mostly leaflets, petioles and young stems [18,30,228].

We identified 17 IAA-producing endophytic fungal species in P. cupana (Table 4), among which F. oxysporum, F. solani, T. harzianum and T. asperellum are known IAA producers in vitro [229231]. Although the other endophytic fungal species analyzed did not produce detectable levels of IAA, several species of Glomerella, Mycena and Pestalotiopsis were previously reported to synthesise IAA in vitro and promote plant growth [232234]. Endophytes can release IAA to promote plant growth in several crops such as rice, sugar cane [235] and coffee [236], and to favor germination [237]. Together, our findings demonstrate that endophytic fungi isolated from tolerant and susceptible genotypes of P. cupana also synthesize IAA in vitro, which could benefit the host.

Purification and structural elucidation of special metabolites

The CH3OH-H2O (3:7) fraction from D. phaseolorum (8S) afforded a colorless solid, named sample 3A. The 1H NMR spectrum showed two signals at 3.07 ppm (2H, t, J = 6.02 Hz) and 4.68 ppm (2H, t, J = 6.05 Hz); the 13C NMR spectrum showed three signals at 30.74, 69.29 and 174.51 ppm; and the 2D NMR spectrum showed two triplets coupled with each other (COSY). HMQC correlations demonstrated that protons with chemical shifts at 4.68 and 3.07 ppm were attached to the carbons that gave the signals at 69.29 and 30.74 ppm, respectively, while the HMBC correlation demonstrated that both proton signals gave long-range correlations to each other’s carbons as well as to the carbon at 174.51 ppm. The aforementioned spectral data are similar to those reported in the literature for 3-HPA [103,238] (Table 5). Therefore, sample 3A corresponds to 3-HPA (3-hydroxypropionic acid).

Table 5. NMR data of isolated compounds from P. cupana endophytic fungi.

The CH3OH subfraction–obtained from the CH3OH:H2O (7:3) fraction from D. phaseolorum (8S)–afforded a dark yellow solid sample named 070. Analysis of the 13C and 1H NMR spectra (Table 5) followed by the HSQC correlation map evidenced the correlation of the signals δH 7.52; 7.70; 4.20; 1.41; 1.28–1.33; 1.25; 0.88; and 0.90 with the signals δC 130.8; 128.8; 68.16; 23.74; 38.7–28.9–22.9; 29.69; 14.05 and 10.96 respectively. The HMBC data analysis evidenced the following fundamental correlations: δH 7.70 with δC 130.8 and 167.7, δH 7.52 with δC 128.8, δH 4.20 with δC 68, δH 1.33–1.28 with δC 22.9 and 23.74. Mass spectrum showed a molecular ion peak at m/z = 390. Together, the spectral data indicates that sample 070 corresponds to DEHP (di-(2-ethylhexyl) phthalate) [239].

The CH3OH extract fraction obtained from T. asperellum (1BDA) provided a solid light-yellow sample named 17A after a HPLC purification step. Analyses of 13C and 1H NMR spectra (Table 5) followed by the HSQC correlation map evidenced the correlation of the signals δH 7.95; 7.86; 7.72; 7.60; 7.33 and 4.06 with the signals δC 118.62; 135.72; 135.49; 118.22; 124.06 and 55.67, respectively. Mass spectrum showed a molecular ion peak at m/z = 254. Thus, sample 17A corresponds to 1-hydroxy-8-methoxyanthraquinone, corroborating literature reports [240].

DEHP is used as plasticizer in the plastics industry and is considered as a persistent environmental pollutant due to its extensive usage [241]. DEHP was isolated from endophytic microorganisms [242,243], but not from those belonging to the genus Diaporthe. Hence, this is the first report on the production of DEHP by species of the genus Diaporthe. As DEHP is present in laboratory equipment and accessories [244,245] and it is considered a common contaminant of analytical equipment [246,247], several authors have questioned whether DEHP is a natural product or an analytical contaminant [248250]. We believe that DEHP is a fungal product because it was exclusively detected in this sample.

3-HPA is commercially available and used in a range of industrial applications [251]. This compound can be synthesized through different metabolic pathways, from different precursors, and by both prokaryotes and eukaryotes [252], including endophytes [103,238,253]. There are few literature reports on 1-hydroxy-8-methoxyanthraquinone. It was produced through chemical synthesis [254] and isolated for the first time as a natural product from the phytopathogenic fungus Leptographium wageneri [240]. To the best of our knowledge, this is the first report on the isolation of 1-hydroxy-8-methoxyanthraquinone from an endophytic fungus and from a microorganism belonging to the genus Trichoderma.

Antibacterial, antitumor and genotoxic activity of the isolated compounds

Here we addressed whether the three special metabolites isolated from endophytic fungal species that colonize P. cupana display antibacterial, antitumor and genotoxic effecs. First, we used the minimal inhibitory concentration test to examine their antibacterial activity. 3-HPA and DEHP, at all the concentrations tested, exerted the strongest antimicrobial activity against P. aeruginosa susceptible and multiresistant strains (Table 6). The growth of bacterial colonies in culture medium indicated that 3-HPA and DEHP were bacteriostatic. The antibacterial and nematicidal activity of 3-HPA was previously reported [103,238], but the mechanisms underlying its bactericidal action remain unclear [252]. DEHP exerts antibiotic activity against resistant and susceptible pathogenic bacterial strains [255259]. In contrast, 1-hydroxy-8-methoxyanthraquinone did not suppress the growth of the bacterial strains tested and there are no literature reports on its antimicrobial activity.

Table 6. Minimal inhibitory concentration (μg/mL) of the special metabolites isolated from endophytic fungi of P. cupana.

Second, we assessed the antitumor effect of the three special metabolites against CHO and B16F10 cells in vitro. 3-HPA reduced the viability of both tumor cell lines in a concentration-dependent manner (S5 Fig). A 72-h treatment with 3-HPA at 100 μg/ml diminished CHO and B16F10 cell viability to 80% and 40%, respectively. 3-HPA is strongly cytotoxic towards prokaryotic cells [252], but there are no reports on its antitumor effect. Our data suggest that 3-HPA is toxic to both tumor and non-tumor cell lines and exerts stronger cytotoxic effect on the former.

There are several reports on the DEHP citotoxicity against tumour and non-tumor cell lines [257,258,260] and its risk to animal health by targeting several organs [261263]. DEHP was cytotoxic towards CHO and B16F10 cell lines: it decreased their cell viability to 70% and 50%, respectively, after a 72-h treatment at a concentration of 100 μg/ml (S6 Fig). On the other hand, 1-hydroxy-8-methoxyanthraquinone did not affect the viability of both cell lines investigated and there are no literature reports on its antitumor activity.

Third, we assessed the genotoxicity of 3-HPA in V79 cells. 3-HPA diminished the cell viability by less than 20%, even at concentrations as high as 100 μg/ml (S7 Fig); it excludes the possible interference of DNA damage caused by cytotoxic concentrations of the test-sample. 3-HPA did not induce DNA damage (genotoxicity), at least under the conditions assessed (S8 Fig). The cytotoxicity of 3-HPA is well-documented and can be mediated by DNA damage [264,265]. As we did not find information about its genotoxicity in the scientific literature, we recommend performing additional mutagenic tests to examine whether it is safe. 3-HPA was cytotoxic towards CHO cells but not towards V79 cells, corroborating literature reports that these cell lines have different sensibility to cytotoxic compounds, and that CHO cells can be several folds more sensible than V79 cells to toxic molecules [266].


We isolated and identified thirty-four endophytic fungal species in P. cupana: eight in seeds, twenty-three in roots and three in both organs. Eight species were not previously reported as endophytic, including X. ganodermophthora, which was the most abundant species in roots. These findings demonstrate the potential of the Amazonia forest as an environment with high diversity of endophytic fungi. The plant geographic location, clonal type, and organ are factors that influence the structure of the endophytic fungal community of P. cupana. The plant organ is the most important factor that causes differentiation in the community’s composition. We confirmed that mycorrhizae and DSE colonize susceptible and tolerant genotypes of P. cupana. P. asparagi (22TSA) and P. pinodella (28S) develop microsclerotia on roots under in vitro conditions. The endophytic fungal community of P. cupana harbors species bearing some plant growth-promoting traits, including the synthesis of enzymes, phytohormones and biologically active molecules. We isolated and identified 3-hydroxypropionic acid and di-(2-ethylhexyl)phthalate from Diaporthe phaseolorum (8S) and 1-hydroxy-8-methoxyanthraquinone from Trichoderma asperellum (1BDA). This study opens possibilities for further investigations, because some species have never been explored for the biological control of crop pests like insects, weeds or parasites, and for the production of chemically active constituents. Hence, they represent potential sources of new and valuable organic molecules.

Supporting information

S1 Table. Classification of the 34 identified endophytic fungal species in P. cupana seeds and roots into putative ecological roles.


S1 Fig. Distribution of endophytic fungal species of P. cupana within the communities studied.

Mu = Manaus; Me = Maués; Su = Susceptible phenotype (CMU 300); To = Tolerant phenotype (CMU 871); Se = Seeds; Ro = Roots.


S2 Fig. NMDS plot of the endophytic fungal community structure in P. cupana using the Jaccard coefficient.

Each point represents a single endophytic community. Permutation tests resulted a highly significant classification (P = 0.001). The lines separate communities from seeds and roots.


S3 Fig. Graphical representation of the network of all the culturable endophytic fungal species isolated from P. cupana roots and seeds.

The size of each node is proportional to its betweeness centrality. Blue, yellow, and red nodes indicate a high, intermediate, and low degree of betweenness centrality, respectively. Thick lines represent positive (Spearman’s ρ>0.6) and significant (P<0.05) correlations. Thin lines represent positive non-significant correlations (P>0.05).


S4 Fig. Modules within the network of culturable endophytic fungal species isolated from P. cupana roots and seeds.

Different colors represent the two modular communities identified. Green and blue modules represent the species isolated from seeds and roots, respectively (modularity index = 0.303, P < 0.01).


S5 Fig. Viability of Chinese hamster ovary (CHO) and Mus musculus skin melanoma (B16F10) cells treated with 3-hydroxypropionic acid (3-HPA) for 72 h.

*(CHO) and #(B16F10): P < 0.05 vs. control (untreated cells); Dunnett's multiple comparison test.


S6 Fig. Viability of Chinese hamster ovary (CHO) and Mus musculus skin melanoma (B16F10) cells treated with di-(2-ethylhexyl)phthalate (DEHP) for 72 h.

*(CHO) and #(B16F10): P < 0.05 vs. control (untreated cells); Dunnett's multiple comparison test.


S7 Fig. Viability (%) of V79 cells treated with 3-hydroxypropionic acid (3-HPA).

Negative control: untreated cells. Positive control: 10 μM doxorubicin. *P < 0.05 vs. negative control (Dunnett's multiple comparison test).


S8 Fig. DNA damage (% DNA tail) in V79 cells treated with 3-hydroxypropionic acid (3-HPA).

Negative control: untreated cells. Positive control: 200 μM hydrogen peroxide. * P < 0.05 vs. negative control (Dunnett's multiple comparison test).



  1. 1. Schimpl FC, Da Silva JF, Gonçalves JFDC, Mazzafera P. Guarana: Revisiting a highly caffeinated plant from the Amazon. Journal of Ethnopharmacology. 2013. pp. 14–31. pmid:23981847
  2. 2. Renfrew E. Trends in beverage markets. In: Ashurst PR, editor. Chemistry and Technology of Soft Drinks and Fruit. 3rd ed. Chichester, UK: John Wiley & Sons, Ltd; 2016. pp. 15–30.
  3. 3. Sette CV de M, Ribas de Alcântara BB, Schoueri JHM, Cruz FM, Cubero DIG, Pianowski LF, et al. Purified dry Paullinia cupana (PC-18) extract for chemotherapy-induced fatigue: results of two double-blind randomized clinical trials. Journal of Dietary Supplements. Taylor & Francis; 2017; 1–11. pmid:29190155
  4. 4. del Giglio A, del Giglio A. Using Paullinia cupana (Guarana) to treat fatigue and other symptoms of cancer and cancer treatment. In: Watson RR, Preedy VR, editors. Bioactive Nutraceuticals and Dietary Supplements in Neurological and Brain Disease: Prevention and Therapy. Academic Press; 2014. pp. 57–63.–0
  5. 5. Dalonso N, Petkowicz CLO. Guarana powder polysaccharides: Characterisation and evaluation of the antioxidant activity of a pectic fraction. Food Chemistry. 2012;134: 1804–1812. pmid:23442624
  6. 6. Zeidán-Chuliá F, Gelain DP, Kolling EA, Rybarczyk-Filho JL, Ambrosi P, Resende Terra S, et al. Major components of energy drinks (Caffeine, Taurine, and Guarana) exert cytotoxic effects on human neuronal SH-SY5Y cells by decreasing reactive oxygen species production. Oxidative Medicine and Cellular Longevity. Hindawi; 2013;2013: 1–22. pmid:23766861
  7. 7. Bittencourt LS, Machado DC, Machado MM, Dos Santos GFF, Algarve TD, Marinowic DR, et al. The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside. Food and Chemical Toxicology. 2013;53: 119–125. pmid:23220610
  8. 8. Yamaguti-Sasaki E, Ito LA, Canteli VCD, Ushirobira TMA, Ueda-Nakamura T, Dias Filho BP, et al. Antioxidant capacity and in vitro prevention of dental plaque formation by extracts and condensed tannins of Paullinia cupana. Molecules. Molecular Diversity Preservation International; 2007;12: 1950–1963. pmid:17960098
  9. 9. Basile A, Rigano D, Conte B, Bruno M, Rosselli S, Sorbo S. Antibacterial and antifungal activities of acetonic extract from Paullinia cupana Mart. seeds. Natural Product Research. 2013;27: 2084–2090. pmid:23672664
  10. 10. Martins M, Kluczkovski A, de Souza T, Pacheco C, Savi G, Scussel V. Inhibition of growth and aflatoxin production of Aspergillus parasiticus by guarana (Paullinia cupana Kunth) and juc (Libidibia ferrea Mart) extracts. African Journal of Biotechnology. Academic Journals; 2014;13: 131–137.
  11. 11. Fukumasu H, Latorre AO, Zaidan-Dagli ML. Paullinia cupana Mart. var. sorbilis, guarana, increases survival of Ehrlich ascites carcinoma (EAC) bearing mice by decreasing cyclin-D1 expression and inducing a G0/G1 cell cycle arrest in EAC cells. Phytotherapy Research. 2011;25: 11–16. pmid:20564499
  12. 12. Roncon CM, de Almeida CB, Klein T, de Mello JCP, Audi EA. Anxiolytic effects of a semipurified constituent of guarana seeds on rats in the elevated T-maze test. Planta Medica. 2011;77: 236–241. pmid:20845263
  13. 13. Bydlowski SP, Yunker RL, Subbiah MTR. A novel property of an aqueous guarana extract (Paullinia cupana): inhibition of platelet aggregation in vitro and in vivo. Brazilian Journal of Medical and Biological Research. 1988;21: 535–538. pmid:3228635
  14. 14. Bydlowski SP, D’Amico EA, Chamone DAF. An aqueous extract of guarana (Paullinia cupana) decreases platelet thromboxane synthesis. Brazilian Journal of Medical and Biological Research. 1991;24: 421–424. pmid:1823256
  15. 15. Dean R, Van Kan JAL, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, et al. The Top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology. 2012;13: 414–430. pmid:22471698
  16. 16. Souza A das GC de, Sousa NR, Lopes R, Atroch AL, Barcelos E, Cordeiro E, et al. Contribution of the institutions in the Northern region of Brazil to the development of plant cultivars and their impact on agriculture. Crop Breeding and Applied Biotechnology. 2012;12: 47–56.
  17. 17. Soares MA, Nogueira GB, Bazzolli DMS, de Araújo EF, Langin T, de Queiroz MV. PacCl, a pH-responsive transcriptional regulator, is essential in the pathogenicity of Colletotrichum lindemuthianum, a causal agent of anthracnose in bean plants. European Journal of Plant Pathology. Springer Netherlands; 2014;140: 769–785.
  18. 18. Bogas AC, Ferreira AJ, Araújo WL, Astolfi-Filho S, Kitajima EW, Lacava PT, et al. Endophytic bacterial diversity in the phyllosphere of Amazon Paullinia cupana associated with asymptomatic and symptomatic anthracnose. SpringerPlus. 2015;4: 258. pmid:26090305
  19. 19. da Costa WM. Community arrangements, productive systems, scientific and technological inputs for land use and forest resources in Amazon. Resources and Environment. 2012;2: 253–264.
  20. 20. CONAB. Companhia Nacional de Abastecimento [National Supply Company]. [Internet]. 2015. Available:
  21. 21. Soares MA, Li HY, Bergen M, da Silva JM, Kowalski KP, White JF. Functional role of an endophytic Bacillus amyloliquefaciens in enhancing growth and disease protection of invasive English ivy (Hedera helix L.). Plant and Soil. 2016;405: 107–123.
  22. 22. Ji SH, Gururani MA, Chun S-C. Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars. Microbiological Research. 2014;169: 83–98. pmid:23871145
  23. 23. White JF. Widespread distribution of endophytes in the Poaceae. Plant Disease. 1987. pp. 340–342.
  24. 24. Soares MA, Li H-Y, Kowalski KP, Bergen M, Torres MS, White JF. Functional role of bacteria from invasive Phragmites australis in promotion of host growth. Microbial Ecology. Springer US; 2016;72: 407–417. pmid:27260154
  25. 25. Soares MA, Li H-Y, Kowalski KP, Bergen M, Torres MS, White JF. Evaluation of the functional roles of fungal endophytes of Phragmites australis from high saline and low saline habitats. Biological Invasions. Springer International Publishing; 2016;18: 2689–2702.
  26. 26. Dighton J, White J, Oudemans P. The Fungal Community. 4th ed. Boca Raton: CRC Press; 2017.
  27. 27. Wani ZA, Ashraf N, Mohiuddin T, Riyaz-Ul-Hassan S. Plant-endophyte symbiosis, an ecological perspective. Applied Microbiology and Biotechnology. Springer Berlin Heidelberg; 2015. pp. 2955–2965. pmid:25750045
  28. 28. Arnold A, Lutzoni F. Diversity and host range of foliar fungal endophytes: are tropical leaves biodiversity hotspots? Ecology. 2007;88: 541–549. pmid:17503580
  29. 29. de Freitas Sia E, Marcon J, Luvizotto D, Quecine M, Tsui S, Pereira J, et al. Endophytic fungi from the Amazonian plant Paullinia cupana and from Olea europaea isolated using cassava as an alternative starch media source. SpringerPlus. Springer International Publishing; 2013;2: 579. pmid:25674409
  30. 30. Bonatelli ML, Tsui S, Marcon J, Batista BD, Kitajima EW, Pereira JO, et al. Antagonistic activity of fungi from anthracnose lesions on Paullinia cupana against Colletotrichum sp. Journal of Plant Pathology. 2016;98: 197–205.
  31. 31. Silva MCS, Polonio JC, Quecine MC, Almeida TT de, Bogas AC, Pamphile JA, et al. Endophytic cultivable bacterial community obtained from the Paullinia cupana seed in Amazonas and Bahia regions and its antagonistic effects against Colletotrichum gloeosporioides. Microbial Pathogenesis. 2016;98: 16–22. pmid:27343372
  32. 32. Liotti RG, da Silva Figueiredo MI, da Silva GF, de Mendonça EAF, Soares MA. Diversity of cultivable bacterial endophytes in Paullinia cupana and their potential for plant growth promotion and phytopathogen control. Microbiological Research. Urban & Fischer; 2018;207: 8–18. pmid:29458872
  33. 33. Higgins KL, Arnold AE, Coley PD, Kursar TA. Communities of fungal endophytes in tropical forest grasses: highly diverse host- and habitat generalists characterized by strong spatial structure. Fungal Ecology. 2014;8: 1–11.
  34. 34. Polishook JD, Bills GF, Lodge DJ. Microfungi from decaying leaves of two rain forest trees in Puerto Rico. Journal of Industrial Microbiology & Biotechnology. 1996;17: 284–294.
  35. 35. Arnold A, Maynard Z, Gilbert G, Coley P, Kursar T. Are tropical fungal endophytes hyperdiverse? Ecology Letters. 2000;3: 267–274.
  36. 36. Wearn JA, Sutton BC, Morley NJ, Gange AC. Species and organ specificity of fungal endophytes in herbaceous grassland plants. Journal of Ecology. 2012;100: 1085–1092.
  37. 37. Rinnan R, Keinanen MM, Kasurinen A, Asikainen J, Kekki TK, Holopainen T, et al. Ambient ultraviolet radiation in the Arctic reduces root biomass and alters microbial community composition but has no effects on microbial biomass. Global Change Biology. Blackwell Science Ltd; 2005;11: 564–574.
  38. 38. Saucedo-García A, Anaya AL, Espinosa-García FJ, González MC. Diversity and communities of foliar endophytic fungi from different agroecosystems of Coffea arabica L. in two regions of Veracruz, Mexico. Treseder K, editor. PLoS ONE. 2014;9: e98454. pmid:24887512
  39. 39. Manter DK, Delgado JA, Holm DG, Stong RA. Pyrosequencing reveals a highly diverse and cultivar-specific bacterial endophyte community in potato roots. Microbial Ecology. 2010;60: 157–166. pmid:20414647
  40. 40. Feng H, Li Y, Liu Q. Endophytic bacterial communities in tomato plants with differential resistance to Ralstonia solanacearum. African Journal of Microbiology Research. 2013;7: 1311–1318.
  41. 41. Batista BD, de Almeida JR, Bezerra TE, de Azevedo JL, Quecine MC. Describing the unexplored microorganisms associated with Guarana: A typical tropical plant. In: Azevedo JL de Quecine MC, editors. Diversity and Benefits of Microorganisms from the Tropics. 1st ed. Cham: Springer International Publishing; 2017. pp. 293–312.
  42. 42. Schulz B, Boyle C. The endophytic continuum. Mycological Research. Elsevier; 2005. pp. 661–686.
  43. 43. Martínez-Medina A, Alguacil MDM, Pascual JA, Van Wees SCM. Phytohormone profiles induced by Trichoderma isolates correspond with their biocontrol and plant growth-promoting activity on melon plants. Journal of Chemical Ecology. Springer US; 2014;40: 804–815. pmid:25023078
  44. 44. Punja ZK, Utkhede RS. Using fungi and yeasts to manage vegetable crop diseases. Trends in Biotechnology. Elsevier Current Trends; 2003. pp. 400–407. pmid:12948673
  45. 45. Wellensiek BP, Ramakrishnan R, Bashyal BP, Eason Y, Gunatilaka AAL, Ahmad N. Inhibition of HIV-1 replication by secondary metabolites from endophytic fungi of desert plants. The open virology journal. Bentham Science Publishers; 2013;7: 72–80. pmid:23961302
  46. 46. Chen S, Ding M, Liu W, Huang X, Liu Z, Lu Y, et al. Anti-inflammatory meroterpenoids from the mangrove endophytic fungus Talaromyces amestolkiae YX1. Phytochemistry. Pergamon; 2018;146: 8–15. pmid:29197643
  47. 47. Cui JL, Guo TT, Ren ZX, Zhang NS, Wang ML. Diversity and antioxidant activity of culturable endophytic fungi from alpine plants of Rhodiola crenulata, R. angusta, and R. sachalinensis. Berg G, editor. PLoS ONE. Public Library of Science; 2015;10: e0118204. pmid:25768014
  48. 48. Uzor PF, Osadebe PO, Nwodo NJ. Antidiabetic Activity of extract and compounds from an endophytic fungus Nigrospora oryzae. Drug Research. 2017;67: 308–311. pmid:28561223
  49. 49. Wang LW, Wang JL, Chen J, Chen JJ, Shen JW, Feng XX, et al. A novel derivative of (-)mycousnine produced by the endophytic fungus Mycosphaerella nawae, exhibits high and selective immunosuppressive activity on T cells. Frontiers in Microbiology. Frontiers; 2017;8: 1251. pmid:28725220
  50. 50. Brissow ER, da Silva IP, de Siqueira KA, Senabio JA, Pimenta LP, Januário AH, et al. 18-Des-hydroxy Cytochalasin: an antiparasitic compound of Diaporthe phaseolorum-92C, an endophytic fungus isolated from Combretum lanceolatum Pohl ex Eichler. Parasitology Research. 2017;116: 1823–1830. pmid:28497228
  51. 51. Channabasava R, Govindappa M. First report of anticancer agent, lapachol producing endophyte, Aspergillus niger of Tabebuia argentea and its in vitro cytotoxicity assays. Bangladesh Journal of Pharmacology. 2014;9: 129–139.
  52. 52. Zaiyou J, Li M, Xiqiao H. An endophytic fungus efficiently producing paclitaxel isolated from Taxus wallichiana var. mairei. Medicine. Wolters Kluwer Health; 2017;96: e7406. pmid:28682896
  53. 53. Martinez-Klimova E, Rodríguez-Peña K, Sánchez S. Endophytes as sources of antibiotics. Biochemical Pharmacology. Elsevier; 2017. pp. 1–17. pmid:27984002
  54. 54. Guzmán-Trampe S, Rodríguez-Peña K, Espinosa-Gómez A, Sánchez-Fernández RE, Macías-Rubalcava ML, Flores-Cotera LB, et al. Endophytes as a potential source of new antibiotics. In: Sanchez S, Demain AL, editors. Antibiotics: Current Innovations and Future Trends. Norfolk, UK: Caister Academic Press; 2015. pp. 175–204.
  55. 55. Erickson HT, Correa MPF, Escobar JR. Guaraná (Paullinia cupana) as a commercial crop in Brazilian Amazonia. Economic Botany. Springer-Verlag; 1984;38: 273–286.
  56. 56. INMET. Instituto Nacional de Meteorologia [Brazilian National Institute Of Meteorology] [Internet]. 2009 [cited 25 Sep 2017]. Available:
  57. 57. Ramos AM, Santos LAR dos, Fortes LTG. Normais climatológicas do Brasil, 1961–1990. 1st ed. Brasília: Instituto Nacional de Meteorologia—INMET, Ministério da Agricultura, Pecuária e Abastecimento—MAPA; 2009.
  58. 58. Petrini LE, Muller E. Haupt—und Nebenfruchtformen europäischer Hypoxylon-Arten (Xylanaceae, Sphaeriales) und verwandter Pilze. Mycologia Helvética. 1986;1: 501–627.
  59. 59. Liew ECY, Aptroot A, Hyde KD. An evaluation of the monophyly of Massarina based on ribosomal DNA sequences. Mycologia. 2002;94: 803–13. pmid:21156554
  60. 60. Kern ME, Blevins KS. Micologia Médica. 2° Edição. São Paulo: Premier; 1999.
  61. 61. Barnett HL. Illustrated Genera of Imperfect Fungi. Mycologia. Minneapolis, Burgess Publishing Company.; 1955;47: 616.
  62. 62. Seifert KA. Compendium of Soil Fungi. European Journal of Soil Science. London: Academic Press (London) Ltd.; 2008;59: 1007–1007.
  63. 63. Rosa LH, Vaz ABM, Caligiorne RB, Campolina S, Rosa CA. Endophytic fungi associated with the Antarctic grass Deschampsia antarctica Desv. (Poaceae). Polar Biology. 2009;32: 161–167.
  64. 64. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols: A Guide to Methods and Applications. 1st ed. London: Academic Press; 1990. pp. 315–322.
  65. 65. Petrini O. Fungal Endophytes of Tree Leaves. In: Andrews JH, Hirano SS, editors. Microbial ecology of leaves. 1st ed. New York, NY: Springer-Verlag New York; 1991. pp. 179–197.
  66. 66. Shannon C, Weaver W. A Mathematical Theory of Communication. 1st ed. Wiley-IEEE Press; 2009.
  67. 67. Chao A. Estimating the population size for capture-recapture data with unequal catchability. Biometrics. 1987;43: 783–791. pmid:3427163
  68. 68. Simpson EH. Measurement of Diversity. Nature. 1949;163: 688–688.
  69. 69. Hancock JM. Jaccard Distance (Jaccard Index, Jaccard Similarity Coefficient). In: Hancock JM, Zvelebil MJ, editors. Dictionary of Bioinformatics and Computational Biology. 1st ed. Chichester, UK: John Wiley & Sons, Ltd; 2004. pp. 223–270.
  70. 70. Farr DF, Rossman AY. Fungal Databases, U.S. National Fungus Collections, ARS, USDA. [Internet]. 2017 [cited 2 Aug 2017]. Available:
  71. 71. Dufrene M, Legendre P. Species assemblages and indicator species: the need for a flexible assymetrical approach. Ecological Monographs. Ecological Society of America; 1997;67: 345–366.
  72. 72. Blondel VD, Guillaume J-L, Lambiotte R, Lefebvre E. Fast unfolding of communities in large networks. Journal of Statistical Mechanics: Theory and Experiment. IOP Publishing; 2008;2008: P10008.
  73. 73. Phillips J, Hayman D. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society. 1970;55: 158–IN18.
  74. 74. Varma A. Mycorrhiza Manual. Varma A, editor. Heidelberg: Springer Berlin Heidelberg; 1998. p. 541.
  75. 75. Duarte O, Paull RE. Exotic fruits and nuts of the New World. 1st ed. Wallingford, UK: CABI; 2015.
  76. 76. Piernas V, Guiraud JP. Disinfection of rice seeds prior to sprouting. Journal of Food Science. 1997;62: 611–615.
  77. 77. Davis RH, de Serres FJ. Genetic and microbiological research techniques for Neurospora crassa. In: Tabor H, Tabor CW, editors. Methods in Enzymology. 1970. pp. 79–143.
  78. 78. Koske RE, Tessier B. A convenient, permanent slide mounting medium. Mycological Society of America Newsletter. 1983;34: 59.
  79. 79. Hagerman AE, Blau DM, McClure AL. Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores. Analytical Biochemistry. 1985;151: 334–342. pmid:3913330
  80. 80. Sierra G. A simple method for the detection of lipolytic activity of micro-organisms and some observations on the influence of the contact between cells and fatty substrates. Antonie van Leeuwenhoek. 1957;23: 15–22. pmid:13425509
  81. 81. Hankin L, Anagnostakis SL. The use of solid media for detection of enzyme production by fungi. Mycologia. 1975;67: 597–607.
  82. 82. Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry. 1987;160: 47–56. pmid:2952030
  83. 83. Gordon SA, Weber RP. Colorimetric estimation of indoleacetic acid. Plant Physiol. 1951;26: 192–195. pmid:16654351
  84. 84. Rosa LH. Diversity and biological activities of endophytic fungi associated with micropropagated medicinal plant Echinacea purpurea (L.) Moench. American Journal of Plant Sciences. 2012;3: 1105–1114.
  85. 85. Ichikawa T, Date M, Ishikura T, Ozaki A. Improvement of kasugamycin-producing strain by the agar piece method and the prototroph method. Folia Microbiologica. Springer Netherlands; 1971;16: 218–224. pmid:4935426
  86. 86. Padhi S, Tayung K. Antimicrobial activity and molecular characterization of an endophytic fungus, Quambalaria sp. isolated from Ipomoea carnea. Annals of Microbiology. 2013;63: 793–800.
  87. 87. Elek SD, Hilson GRF. Combined agar diffusion and replica plating techniques in the study of antibacterial substances. Journal of Clinical Pathology. 1954;7: 37–44. pmid:13143101
  88. 88. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods. 1983;65: 55–63. pmid:6606682
  89. 89. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research. 1988;175: 184–191. pmid:3345800
  90. 90. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al. Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Mutagenesis. 2000;35: 206–221. pmid:10737956
  91. 91. Speit G, Schütz P, Bausinger J. Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2016;803–804: 22–26. pmid:27265376
  92. 92. Hammer Ø, Harper DAT, Ryan PD. PAST: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica. 2001;4: 1–9.
  93. 93. Dixon P. VEGAN, a package of R functions for community ecology. Journal of Vegetation Science. 2003;14: 927–930.
  94. 94. Leonard M, Graham S, Bonacum D. The human factor: the critical importance of effective teamwork and communication in providing safe care. Quality & Safety in Health Care. 2004;13 Suppl 1: i85–i90. pmid:15465961
  95. 95. Rossman AY, Farr DF, Castlebury LA. A review of the phylogeny and biology of the Diaporthales. Mycoscience. 2007;48: 135–144.
  96. 96. Wu L, Han T, Li W, Jia M, Xue L, Rahman K, et al. Geographic and tissue influences on endophytic fungal communities of Taxus chinensis var. mairei in China. Current Microbiology. 2013;66: 40–48. pmid:23053484
  97. 97. Kang HJ, Chang WB, Yun SH, Lee YW. Roles of ascospores and arthroconidia of Xylogone ganodermophthora in development of yellow rot in cultivated mushroom, Ganoderma lucidum. Plant Pathology Journal. 2011;27: 138–147.
  98. 98. Kang H, Kim Y, Kim T, Jeong TK, Han CU, Nam SY, et al. Suppression of powdery mildew using the water extract of Xylogone ganodermophthora and aqueous potassium phosphonate solution on watermelon under greenhouse conditions. Research in Plant Disease. 2015;21: 309–314.
  99. 99. Murali TS, Suryanarayanan TS, Geeta R. Endophytic Phomopsis species: host range and implications for diversity estimates. Canadian Journal of Microbiology. 2006;52: 673–680. pmid:16917524
  100. 100. Yin J, Chin C, Ye J, Zhao W, Li G. An effective asparagus stem blight management program. Acta Horticulturae. 2012;950: 293–298.
  101. 101. Udayanga D, Liu X, McKenzie EHC, Chukeatirote E, Bahkali AHA, Hyde KD. The genus Phomopsis: biology, applications, species concepts and names of common phytopathogens. Fungal Diversity. 2011;50: 189–225.
  102. 102. de Siqueira KA, Brissow ER, dos Santos JL, White JF, Santos FR, de Almeida EG, et al. Endophytism and bioactivity of endophytic fungi isolated from Combretum lanceolatum Pohl ex Eichler. Symbiosis. 2017;71: 211–222.
  103. 103. Sebastianes FLS, Cabedo N, Aouad N El, Valente AMMP, Lacava PT, Azevedo JL, et al. 3-Hydroxypropionic acid as an antibacterial agent from endophytic fungi Diaporthe phaseolorum. Current Microbiology. 2012;65: 622–632. pmid:22886401
  104. 104. Casella TM, Eparvier V, Mandavid H, Bendelac A, Odonne G, Dayan L, et al. Antimicrobial and cytotoxic secondary metabolites from tropical leaf endophytes: Isolation of antibacterial agent pyrrocidine C from Lewia infectoria SNB-GTC2402. Phytochemistry. 2013;96: 370–377. pmid:24189345
  105. 105. Photita W, Lumyong S, Lumyong P, Hyde KD. Endophytic fungi of wild banana (Musa acuminata) at Doi Suthep Pui National Park, Thailand. Mycological Research. 2001;105: 1508–1513.
  106. 106. Sieber TN. Endophytic fungi in twigs of healthy and diseased Norway spruce and white fir. Mycological Research. Elsevier; 1989;92: 322–326.
  107. 107. Wearn JA, Sutton BC, Morley NJ, Gange AC. Species and organ specificity of fungal endophytes in herbaceous grassland plants. Journal of Ecology. Blackwell Publishing Ltd; 2012;100: 1085–1092.
  108. 108. Schroth G, Krauss U, Gasparotto L, Duarte JA. Pests and diseases in agroforestry systems of the humid tropics. Agroforestry Systems. 2000;50: 199–241.
  109. 109. Cai L, Jeewon R, Hyde KD. Phylogenetic investigations of Sordariaceae based on multiple gene sequences and morphology. Mycological Research. 2006;110: 137–150. pmid:16378718
  110. 110. Arevalo J, Hidalgo-Diaz L, Martins I, Souza JF, Castro JMC, Carneiro RMDG, et al. Cultural and morphological characterization of Pochonia chlamydosporia and Lecanicillium psalliotae isolated from Meloidogyne mayaguensis eggs in Brazil. Tropical Plant Pathology. 2009;34: 158–163.
  111. 111. Cai L, Kurniawati E, Hyde KD. Morphological and molecular characterization of Mariannaea aquaticola sp. nov. collected from freshwater habitats. Mycological Progress. 2010;9: 337–343.
  112. 112. Yeo T, Naming M, Manurung R. Building a discovery partnership with sarawak biodiversity centre: a gateway to access natural products from the rainforests. Combinatorial Chemistry & High Throughput Screening. 2014;17: 192–200.
  113. 113. Kövics GJ, Sándor E, Rai MK, Irinyi L. Phoma-like fungi on soybeans. Critical Reviews in Microbiology. 2014;40: 49–62. pmid:23363325
  114. 114. Rajeshkumar KC, Crous PW, Groenewald JZ, Seifert KA. Resolving the phylogenetic placement of Porobeltraniella and allied genera in the Beltraniaceae. Mycological Progress. 2016;15: 1119–1136.
  115. 115. Kang H-J, Sigler L, Lee J, Gibas CFC, Yun S-H, Lee Y-W. Xylogone ganodermophthora sp. nov., an ascomycetous pathogen causing yellow rot on cultivated mushroom Ganoderma lucidum in Korea. Mycologia. 2010;102: 1167–84. pmid:20943517
  116. 116. Marinelli E, Orzali L, Lotti E, Riccioni L. Activity of some essential oils against pathogenic seed borne fungi on legumes. Asian Journal of Plant Pathology. 2012;6: 66–74.
  117. 117. Crous PW, Shivas RG, Quaedvlieg W, van der Bank M, Zhang Y, Summerell BA, et al. Fungal Planet description sheets: 214–280. Persoonia. 2014;32: 184–306. pmid:25264390
  118. 118. Redman RS, Dunigan DD, Rodriguez RJ. Fungal symbiosis from mutualism to parasitism: who controls the outcome, host or invader? New Phytologist. Blackwell Science Ltd; 2001;151: 705–716.
  119. 119. Zabalgogeazcoa I. Fungal endophytes and their interaction with plant pathogens: a review. Spanish Journal of Agricultural Research. 2008;6: 138.
  120. 120. Brader G, Compant S, Vescio K, Mitter B, Trognitz F, Ma L-J, et al. Ecology and genomic insights into plant-pathogenic and plant-nonpathogenic endophytes. Annual Review of Phytopathology. Annual Reviews 4139 El Camino Way, PO Box 10139, Palo Alto, California 94303–0139, USA; 2017;55: 61–83. pmid:28489497
  121. 121. Tan RX, Zou WX. Endophytes: a rich source of functional metabolites. Natural product reports. 2001;18: 448–459. pmid:11548053
  122. 122. Schlegel M, Münsterkötter M, Güldener U, Bruggmann R, Duò A, Hainaut M, et al. Globally distributed root endophyte Phialocephala subalpina links pathogenic and saprophytic lifestyles. BMC genomics. 2016;17: 1015. pmid:27938347
  123. 123. Mithöfer A, Maffei ME. General mechanisms of plant defense and plant toxins. In: Carlini CR, Ligabue-Braun R, editors. Plant Toxins. 1st ed. Dordrecht: Springer Netherlands; 2016. pp. 3–24.–1
  124. 124. Ballhorn DJ, Kautz S, Heil M, Hegeman AD. Cyanogenesis of wild lima bean (Phaseolus lunatus L.) is an efficient direct defence in nature. Kroymann J, editor. PLoS ONE. Public Library of Science; 2009;4: e5450. pmid:19424497
  125. 125. Herrera J, Poudel R, Khidir HH. Molecular characterization of coprophilous fungal communities reveals sequences related to root-associated fungal endophytes. Microbial Ecology. Springer-Verlag; 2011;61: 239–244. pmid:20842497
  126. 126. Setälä H, McLean MA. Decomposition rate of organic substrates in relation to the species diversity of soil saprophytic fungi. Oecologia. 2004;139: 98–107. pmid:14740289
  127. 127. Jaber LR, Ownley BH. Can we use entomopathogenic fungi as endophytes for dual biological control of insect pests and plant pathogens? Biological Control. 2017;107: 50–59.
  128. 128. Garrido-Jurado I, Resquín-Romero G, Amarilla SP, Ríos-Moreno A, Carrasco L, Quesada-Moraga E. Transient endophytic colonization of melon plants by entomopathogenic fungi after foliar application for the control of Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). Journal of Pest Science. 2017;90: 319–330.
  129. 129. Vega FE, Posada F, Catherine AM, Pava-Ripoll M, Infante F, Rehner SA. Entomopathogenic fungal endophytes. Biological Control. 2008;46: 72–82.
  130. 130. Lopez DC, Zhu-Salzman K, Ek-Ramos MJ, Sword GA. The entomopathogenic fungal endophytes Purpureocillium lilacinum (formerly Paecilomyces lilacinus) and Beauveria bassiana negatively affect cotton aphid reproduction under both greenhouse and field conditions. PloS one. 2014;9: e103891. pmid:25093505
  131. 131. Vidal S, Jaber LR. Entomopathogenic fungi as endophytes: plant–endophyte–herbivore interactions and prospects for use in biological control. Current Science. 2015;109: 46–54.
  132. 132. Gazis R, Chaverri P. Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis) in Peru. Fungal Ecology. 2010;3: 240–254.
  133. 133. Gonzaga LL, Costa LEO, Santos TT, Araújo EF, Queiroz MV. Endophytic fungi from the genus Colletotrichum are abundant in the Phaseolus vulgaris and have high genetic diversity. Journal of Applied Microbiology. 2015;118: 485–496. pmid:25410007
  134. 134. Evans HC, Holmes KA, Thomas SE. Endophytes and mycoparasites associated with an indigenous forest tree, Theobroma gileri, in Ecuador and a preliminary assessment of their potential as biocontrol agents of cocoa diseases. Mycological Progress. Springer-Verlag; 2003;2: 149–160.
  135. 135. Rojas EI, Rehner SA, Samuels GJ, Van Bael SA, Herre EA, Cannon P, et al. Colletotrichum gloeosporioides s.l. associated with Theobroma cacao and other plants in Panamá: multilocus phylogenies distinguish host-associated pathogens from asymptomatic endophytes. Mycologia. Taylor & Francis; 2010;102: 1318–1338. pmid:20943565
  136. 136. Vajna L. Phytopathogenic Fusarium oxysporum Schlecht, as a necrotrophic mycoparasite. Journal of Phytopathology. 1985;114: 338–347.
  137. 137. Joshi BK, Gloer JB, Wicklow DT. Bioactive natural products from a sclerotium-colonizing isolate of Humicola fuscoatra. Journal of Natural Products. American Chemical Society; 2002;65: 1734–1737. pmid:12444718
  138. 138. De Stefano S, Nicoletti R, Milone A, Zambardino S. 3-O-methylfunicone, a fungitoxic metabolite produced by the fungus Penicillium pinophilum. Phytochemistry. 1999;52: 1399–1401.
  139. 139. Tchameni SN, Ngonkeu MEL, Begoude BAD, Wakam Nana L, Fokom R, Owona AD, et al. Effect of Trichoderma asperellum and arbuscular mycorrhizal fungi on cacao growth and resistance against black pod disease. Crop Protection. 2011;30: 1321–1327.
  140. 140. da Mota PR, Ribeiro MS, de Castro GR, Silva GR, de Paula RG, Silva R do N, et al. Expression analysis of the α-1,2-mannosidase from the mycoparasitic fungus Trichoderma harzianum. Biological Control. 2016;95: 1–4.
  141. 141. Ferreira MC, Vieira M de LA, Zani CL, Alves TM de A, Junior PAS, Murta SMF, et al. 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. 2015;59: 36–44.
  142. 142. Krishnamurthy YL, Naik SB, Jayaram S. Fungal communities in herbaceous medicinal plants from the malnad region, southern India. Microbes and Environments. 2008;23: 24–28. pmid:21558683
  143. 143. Hanada RE, Pomella AW V, Costa HS, Bezerra JL, Loguercio LL, Pereira JO. Endophytic fungal diversity in Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) trees and their potential for growth promotion and biocontrol of black-pod disease. Fungal Biology. 2010;114: 901–910. pmid:21036333
  144. 144. Bailey BA, Strem MD, Wood D. Trichoderma species form endophytic associations within Theobroma cacao trichomes. Mycological Research. 2009;113: 1365–1376. pmid:19765658
  145. 145. Bottomly T, Fortnum B, Kurtz H, Kluepfel D. Diversity of endophytic bacteria in healthy and Ralstonia solanacearum infected tobacco seedlings. Agro Phyto Groups, abstract. Kyoto; 2004.
  146. 146. Loughman R, Deverall BJ. Infection of resistant and susceptible cultivars of wheat by Pyrenophora tritici-repentis. Plant Pathology. 1986;35: 443–450.
  147. 147. Kang Z, Buchenauer H. Ultrastructural and immunocytochemical investigation of pathogen development and host responses in resistant and susceptible wheat spikes infected by Fusarium culmorum. Physiological and Molecular Plant Pathology. 2000;57: 255–268.
  148. 148. Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM. The role of root exudates in rhizosphere interactions with plants and other organisms. Annual Review of Plant Biology. 2006;57: 233–266. pmid:16669762
  149. 149. Rudrappa T, Bais HP. Arabidopsis thaliana root surface chemistry regulates in planta biofilm formation of Bacillus subtilis. Plant signaling & behavior. 2007;2: 349–350.
  150. 150. Broeckling CD, Broz AK, Bergelson J, Manter DK, Vivanco JM. Root exudates regulate soil fungal community composition and diversity. Appl Environ Microbiol. 2008;74: 738–744. pmid:18083870
  151. 151. Zereini F, Wiseman CLS. Urban Airborne Particulate Matter. 1st ed. Berlin: Springer Berlin Heidelberg; 2011.
  152. 152. Zhang R, Wang G, Guo S, Zamora ML, Ying Q, Lin Y, et al. Formation of urban fine particulate matter. Chemical Reviews. American Chemical Society; 2015;115: 3803–3855. pmid:25942499
  153. 153. Fröhlich-Nowoisky J, Pickersgill DA, Després VR, Pöschl U. High diversity of fungi in air particulate matter. Proceedings of the National Academy of Sciences of the United States of America. National Academy of Sciences; 2009;106: 12814–9. pmid:19617562
  154. 154. Franzetti A, Gandolfi I, Gaspari E, Ambrosini R, Bestetti G. Seasonal variability of bacteria in fine and coarse urban air particulate matter. Applied Microbiology and Biotechnology. Springer-Verlag; 2011;90: 745–753. pmid:21184061
  155. 155. Brodie EL, DeSantis TZ, Parker JPM, Zubietta IX, Piceno YM, Andersen GL. Urban aerosols harbor diverse and dynamic bacterial populations. Proceedings of the National Academy of Sciences of the United States of America. National Academy of Sciences; 2007;104: 299–304. pmid:17182744
  156. 156. Pashley CH, Fairs A, Free RC, Wardlaw AJ. DNA analysis of outdoor air reveals a high degree of fungal diversity, temporal variability, and genera not seen by spore morphology. Fungal Biology. Elsevier; 2012;116: 214–224. pmid:22289767
  157. 157. Leveau JHJ. Life of Microbes on Aerial Plant Parts. In: Lugtenberg B, editor. Principles of Plant-Microbe Interactions. 1st ed. Cham: Springer International Publishing; 2015. pp. 17–24.
  158. 158. Vega FE, Pava-Ripoll M, Posada F, Buyer JS. Endophytic bacteria in Coffea arabica L. Journal of Basic Microbiology. Wiley‐VCH Verlag; 2005;45: 371–380. pmid:16187260
  159. 159. West ER, Cother EJ, Steel CC, Ash GJ. The characterization and diversity of bacterial endophytes of grapevine. Canadian Journal of Microbiology. 2010;56: 209–216. pmid:20453907
  160. 160. Helander ML. Responses of pine needle endophytes to air pollution. New Phytologist. Blackwell Publishing Ltd; 1995;131: 223–229.
  161. 161. Jumpponen A, Jones KL. Seasonally dynamic fungal communities in the Quercus macrocarpa phyllosphere differ between urban and nonurban environments. New Phytologist. Blackwell Publishing Ltd; 2010;186: 496–513. pmid:20180911
  162. 162. Matsumura E, Fukuda K. A comparison of fungal endophytic community diversity in tree leaves of rural and urban temperate forests of Kanto district, eastern Japan. Fungal Biology. Elsevier; 2013;117: 191–201. pmid:23537876
  163. 163. Carroll G. Forest endophytes: pattern and process. Canadian Journal of Botany. 1995;73: 1316–1324.
  164. 164. Hoffman MT, Arnold AE. Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees. Mycological Research. 2008;112: 331–344. pmid:18308531
  165. 165. U’Ren JM, Lutzoni F, Miadlikowska J, Laetsch AD, Elizabeth AA. Host and geographic structure of endophytic and endolichenic fungi at a continental scale. American Journal of Botany. 2012;99: 898–914. pmid:22539507
  166. 166. IBGE. Instituto Brasileiro de Geografia e Estatística (IBGE) [Brazilian Institute of Geography and Statistics] [Internet]. 2010. Available:
  167. 167. Alvares CA, Stape JL, Sentelhas PC, de Moraes GJL, Sparovek G. Köppen’s climate classification map for Brazil. Meteorologische Zeitschrift. 2013;22: 711–728.
  168. 168. Vega FE, Simpkins A, Aime MC, Posada F, Peterson SW, Rehner SA, et al. Fungal endophyte diversity in coffee plants from Colombia, Hawaii, Mexico and Puerto Rico. Fungal Ecology. 2010;3: 122–138.
  169. 169. Arnold AE, Herre EA. Canopy cover and leaf age affect colonization by tropical fungal endophytes: Ecological pattern and process in Theobroma cacao (Malvaceae). Mycologia. 2003;95: 388–398. pmid:21156627
  170. 170. Jumpponen A. Dark septate endophytes-are they mycorrhizal? Mycorrhiza. 2001;11: 207–211.
  171. 171. Kytoviita M-M, Ruotsalainen AL. Mycorrhizal benefit in two low arctic herbs increases with increasing temperature. American Journal of Botany. Botanical Society of America; 2007;94: 1309–1315. pmid:21636497
  172. 172. Mohan JE, Cowden CC, Baas P, Dawadi A, Frankson PT, Helmick K, et al. Mycorrhizal fungi mediation of terrestrial ecosystem responses to global change: mini-review. Fungal Ecology. Elsevier; 2014;10: 3–19.
  173. 173. de Oliveira AN, de Oliveira LA. Influence of edapho-climatic factors on the sporulation and colonization of arbuscular mycorrhizal fungi in two Amazonian native fruit species. Brazilian Archives of Biology and Technology. 2010;53: 653–661.
  174. 174. de Oliveira AN, de Oliveira LA. Arbuscular mycorrhizal association and foliar nutrient concentrations of cupuassu (Theobroma grandiflorum) and guarana (Paullinia cupana) plants in an agroforestry system in Manaus, AM, Brazil. Revista Brasileira De Ciencia do Solo. 2004;28: 1063–1068.
  175. 175. Elena K. First report of Phomopsis asparagi causing stem blight of asparagus in Greece. Plant Pathology. Blackwell Publishing Ltd; 2006;55: 300–300.
  176. 176. Uecker FA, Johnson DA. Morphology and taxonomy of species of Phomopsis on asparagus. Mycologia. Taylor & Francis, Ltd.; 1991;83: 192.
  177. 177. Herrera J, Poudel R, Bokati D. Assessment of root-associated fungal communities colonizing two species of tropical grasses reveals incongruence to fungal communities of North American native grasses. Fungal Ecology. 2013;6: 65–69.
  178. 178. Vaz ABM, Sampedro I, Siles JA, Vasquez JA, García-Romera I, Vierheilig H, et al. Arbuscular mycorrhizal colonization of Sorghum vulgare in presence of root endophytic fungi of Myrtus communis. Applied Soil Ecology. 2012;61: 288–294.
  179. 179. Porras-Alfaro A, Herrera J, Natvig DO, Lipinski K, Sinsabaugh RL. Diversity and distribution of soil fungal communities in a semiarid grassland. Mycologia. 2011;103: 10–21. pmid:20943560
  180. 180. Loro M, Valero-Jiménez CA, Nozawa S, Márquez LM. Diversity and composition of fungal endophytes in semiarid northwest Venezuela. Journal of Arid Environments. 2012;85: 46–55.
  181. 181. Jumpponen A, Trappe JM. Dark septate endophytes: A review of facultative biotrophic root-colonizing fungi. New Phytologist. 1998;140: 295–310.
  182. 182. Kytöviita M-M. Asymmetric symbiont adaptation to Arctic conditions could explain why high Arctic plants are non-mycorrhizal. FEMS Microbiology Ecology. Oxford University Press; 2005;53: 27–32. pmid:16329926
  183. 183. Mandyam K, Jumpponen A. Seeking the elusive function of the root-colonising dark septate endophytic fungi. Studies in Mycology. 2005;53: 173–189.
  184. 184. Stevens KJ, Wellner MR, Acevedo MF. Dark septate endophyte and arbuscular mycorrhizal status of vegetation colonizing a bottomland hardwood forest after a 100 year flood. Aquatic Botany. 2010;92: 105–111.
  185. 185. Zhang G, Sun S, Zhu T, Lin Z, Gu J, Li D, et al. Antiviral isoindolone derivatives from an endophytic fungus Emericella sp. associated with Aegiceras corniculatum. Phytochemistry. 2011;72: 1436–1442. pmid:21601895
  186. 186. Posada RH, Madriñan S, Rivera EL. Relationships between the litter colonization by saprotrophic and arbuscular mycorrhizal fungi with depth in a tropical forest. Fungal Biology. 2012;116: 747–755. pmid:22749161
  187. 187. Fernandes Júnior PI, Pereira GMD, Perin L, da Silva LM, Baraúna AC, Alvess FM, et al. Diazotrophic bacteria isolated from wild rice Oryza glumaepatula (Poaceae) in the Brazilian Amazon. Revista de biología tropical. 2013;61: 991–9. pmid:23885604
  188. 188. Arivudainambi USE, Anand TD, Shanmugaiah V, Karunakaran C, Rajendran A. Novel bioactive metabolites producing endophytic fungus Colletotrichum gloeosporioides against multidrug-resistant Staphylococcus aureus. FEMS Immunology & Medical Microbiology. Oxford University Press; 2011;61: 340–345. pmid:21219448
  189. 189. Ola ARB, Debbab A, Kurtán T, Brötz-Oesterhelt H, Aly AH, Proksch P. Dihydroanthracenone metabolites from the endophytic fungus Diaporthe melonis isolated from Annona squamosa. Tetrahedron Letters. Pergamon; 2014;55: 3147–3150.
  190. 190. Katoch M, Singh G, Sharma S, Gupta N, Sangwan PL, Saxena AK. Cytotoxic and antimicrobial activities of endophytic fungi isolated from Bacopa monnieri (L.) Pennell (Scrophulariaceae). BMC Complementary and Alternative Medicine. BioMed Central; 2014;14: 52. pmid:24512530
  191. 191. Syed A, Ahmad A. Extracellular biosynthesis of CdTe quantum dots by the fungus Fusarium oxysporum and their anti-bacterial activity. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. Elsevier; 2013;106: 41–47. pmid:23357677
  192. 192. Mandeel Q, Al-Laith A, Mohsen L. Survey of Fusarium species in an arid environment of Bahrain. V. Antimicrobial activity of some local and international Fusarium species. Pharmaceutical Biology. Taylor & Francis; 1999;37: 181–187.
  193. 193. Tayung K, Barik BP, Jha DK, Deka DC. Identification and characterization of antimicrobial metabolite from an endophytic fungus, Fusarium solani isolated from bark of Himalayan yew. Mycosphere. 2011;2: 203–213.
  194. 194. Vieira MLA, Hughes AFS, Gil VB, Vaz ABM, Alves TMA, Zani CL, et al. Diversity and antimicrobial activities of the fungal endophyte community associated with the traditional Brazilian medicinal plant Solanum cernuum Vell. (Solanaceae). Canadian Journal of Microbiology. 2012;58: 54–66. pmid:22182199
  195. 195. Kim J-E, Han K-H, Jin J, Kim H, Kim J-C, Yun S-H, et al. Putative polyketide synthase and laccase genes for biosynthesis of aurofusarin in Gibberella zeae. Applied and Environmental Microbiology. American Society for Microbiology; 2005;71: 1701–1708. pmid:15811992
  196. 196. Smetanina OF, Kuznetsova TA, Gerasimenko A V., Kalinovsky AI, Pivkin M V., Dmitrenok PC, et al. Metabolites of the marine fungus Humicola fuscoatra KMM 4629. Russian Chemical Bulletin. Kluwer Academic Publishers-Consultants Bureau; 2004;53: 2643–2646.
  197. 197. Fukuda T, Sudoh Y, Tsuchiya Y, Okuda T, Fujimori F, Igarashi Y. Marianins A and B, prenylated phenylpropanoids from Mariannaea camptospora. Journal of Natural Products. 2011;74: 1327–1330. pmid:21488655
  198. 198. do Rosário Marinho AM, Rodrigues-Fo E. Dicitrinol, a citrinin dimer, produced by Penicillium janthinellum. Helvetica Chimica Acta. Wiley‐VCH Verlag; 2011;94: 835–841.
  199. 199. Li X, Guo Z, Deng Z, Yang J, Kun Zou. A new α-pyrone derivative from endophytic fungus Pestalotiopsis microspora. Records of Natural Products. 2015;9: 503–508.
  200. 200. Mishra PD, Verekar SA, Deshmukh SK, Joshi KS, Fiebig HH, Kelter G. Altersolanol A: a selective cytotoxic anthraquinone from a Phomopsis sp. Letters in Applied Microbiology. 2015;60: 387–391. pmid:25534717
  201. 201. Kang HJ, Ahn KS, Han CW, Jeong KH, Park SJ, Song IG, et al. Xylogone ganodermophthora strain with antifungal activity, and composition including same for preventing plant diseases. Korea; US 8591911 B2, 2013. p. 16.
  202. 202. Yang C-A, Cheng C-H, Liu S-Y, Lo C-T, Lee J-W, Peng K-C. Identification of antibacterial mechanism of l-amino acid oxidase derived from Trichoderma harzianum ETS 323. FEBS Journal. Blackwell Publishing Ltd; 2011;278: 3381–3394. pmid:21781279
  203. 203. Marfori EC, Kajiyama S, Fukusaki E, Kobayashi A. Trichosetin, a novel tetramic acid antibiotic produced in dual culture of Trichoderma harzianum and Catharanthus roseus callus. Zeitschrift für Naturforschung C. Verlag der Zeitschrift für Naturforschung; 2002;57: 465–470.
  204. 204. Akonda MMR, Himel RM, Ali M, Islam MS. In vitro evaluation of antibiotic performances on Trichoderma harzianum and some crop infecting fungi. Plant Science Today. 2016;3: 267.
  205. 205. Katoch M, Salgotra A, Singh G. Endophytic fungi found in association with Bacopa monnieri as potential producers of industrial enzymes and antimicrobial bioactive compounds. Brazilian Archives of Biology and Technology. 2014;57: 714–722.
  206. 206. Bhatti HN, Rashid MH, Nawaz R, Khalid AM, Asgher M, Jabbar A. Effect of aniline coupling on kinetic and thermodynamic properties of Fusarium solani glucoamylase. Applied Microbiology and Biotechnology. Springer-Verlag; 2007;73: 1290–1298. pmid:17031637
  207. 207. Adsul MG, Bastawde KB, Varma AJ, Gokhale DV. Strain improvement of Penicillium janthinellum NCIM 1171 for increased cellulase production. Bioresource Technology. Elsevier; 2007;98: 1467–1473. pmid:17097876
  208. 208. Mohamed SA, Khan JA, Al-Bar OAM, El-Shishtawy RM. Immobilization of Trichoderma harzianum alpha-Amylase on treated wool: Optimization and characterization. Molecules. 2014;19: 8027–8038. pmid:24932573
  209. 209. Ogórek R. Enzymatic activity of potential fungal plant pathogens and the effect of their culture filtrates on seed germination and seedling growth of garden cress (Lepidium sativum L.). European Journal of Plant Pathology. Springer Netherlands; 2016;145: 469–481.
  210. 210. Barata RA, Andrade MHG, Rodrigues RD, Castro IM. Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini. Journal of Bioscience and Bioengineering. Elsevier; 2002;94: 304–308. pmid:16233307
  211. 211. Lowe RGT, McCorkelle O, Bleackley M, Collins C, Faou P, Mathivanan S, et al. Extracellular peptidases of the cereal pathogen Fusarium graminearum. Frontiers in Plant Science. 2015;6: 962. pmid:26635820
  212. 212. Jie W, Li-jiao C, Liu-bo L. Diversity of culturable extracellular proteases producing marine fungi isolated from the intertidal zone of Naozhou Island in South China Sea. Microbiology China. 2015;42: 238–253.
  213. 213. Chandrasekaran M, Thangavelu B, Chun SC, Sathiyabama M. Proteases from phytopathogenic fungi and their importance in phytopathogenicity. Journal of General Plant Pathology. Springer Japan; 2016;82: 233–239.
  214. 214. Peterson R, Grinyer J, Nevalainen H. Extracellular hydrolase profiles of fungi isolated from koala faeces invite biotechnological interest. Mycological Progress. 2011;10: 207–218.
  215. 215. Shearer JF. A historical perspective of pathogen biological control of aquatic plants. Weed Technology. 2010;24: 202–207.
  216. 216. Maeda RN, Serpa VI, Rocha VAL, Mesquita RAA, Anna LMMS, de Castro AM, et al. Enzymatic hydrolysis of pretreated sugar cane bagasse using Penicillium funiculosum and Trichoderma harzianum cellulases. Process Biochemistry. 2011;46: 1196–1201.
  217. 217. Wood TM. The cellulase of Fusarium solani. Purification and specificity of the β-(1→4)-glucanase and the β-D-glucosidase components. Biochemical Journal. Portland Press Limited; 1971;121: 353–362. pmid:5119766
  218. 218. Xu J, Wang X, Hu L, Xia J, Wu Z, Xu N, et al. A novel ionic liquid-tolerant Fusarium oxysporum BN secreting ionic liquid-stable cellulase: Consolidated bioprocessing of pretreated lignocellulose containing residual ionic liquid. Bioresource Technology. Elsevier; 2015;181: 18–25. pmid:25625459
  219. 219. Maroldi MMC, Vasconcellos VM, Lacava PT, Farinas CS. Potential of mangrove-associated endophytic fungi for production of carbohydrolases with high saccharification efficiency. Applied Biochemistry and Biotechnology. Springer US; 2017; 1–15. pmid:28866806
  220. 220. Cappellini RA, Peterson JL. Production, In vitro, of certain pectolytic and cellulolytic enzymes by fungi associated with corn stalk rot. Bulletin of the Torrey Botanical Club. Torrey Botanical Society; 1966;93: 52.
  221. 221. Zhao Y, Li W, Zhou Z, Wang L, Pan Y, Zhao L. Dynamics of microbial community structure and cellulolytic activity in agricultural soil amended with two biofertilizers. European Journal of Soil Biology. Elsevier Masson; 2005;41: 21–29.
  222. 222. Moubasher AH, Mazen MB. Assay of cellulolytic activity of cellulose-decomposing fungi isolated from Egyptian soils. Journal of Basic Microbiology. Wiley‐VCH; 1991;31: 59–68.
  223. 223. Mandyam K, Loughin T, Jumpponen A. Isolation and morphological and metabolic characterization of common endophytes in annually burned tallgrass prairie. Mycologia. 2010;102: 813–821. pmid:20648749
  224. 224. Peterson RA, Bradner JR, Roberts TH, Nevalainen KMH. Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates. Letters in Applied Microbiology. 2009;48: 218–225. pmid:19141036
  225. 225. Viterbo A, Harel M, Chet I. Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots. FEMS Microbiology Letters. Oxford University Press; 2004;238: 151–158. pmid:15336416
  226. 226. Bech L, Herbst F-A. On-site enzyme production by Trichoderma asperellum for the degradation of duckweed. Fungal Genomics & Biology. OMICS International; 2015;5.
  227. 227. Raghuwanshi S, Deswal D, Karp M, Kuhad RC. Bioprocessing of enhanced cellulase production from a mutant of Trichoderma asperellum RCK2011 and its application in hydrolysis of cellulose. Fuel. Elsevier; 2014;124: 183–189.
  228. 228. Azevedo JL. Endophytic fungi from Brazilian tropical hosts and their biotechnological applications. Microbial Diversity and Biotechnology in Food Security. 1st ed. New Delhi: Springer India; 2014. pp. 17–22.
  229. 229. Gunasekaran M, Weber DJ. Auxin production of three phytopathogenic fungi. Mycologia. 1972;64: 1180. pmid:4673330
  230. 230. Hoyos-Carvajal L, Orduz S, Bissett J. Growth stimulation in bean (Phaseolus vulgaris L.) by Trichoderma. Biological Control. 2009;51: 409–416.
  231. 231. Tsavkelova E, Oeser B, Oren-Young L, Israeli M, Sasson Y, Tudzynski B, et al. Identification and functional characterization of indole-3-acetamide-mediated IAA biosynthesis in plant-associated Fusarium species. Fungal Genetics and Biology. 2012;49: 48–57. pmid:22079545
  232. 232. Jia M, Chen L, Xin H-L, Zheng C-J, Rahman K, Han T, et al. A friendly relationship between endophytic fungi and medicinal plants: a systematic review. Frontiers in Microbiology. Frontiers Media SA; 2016;7: 906. pmid:27375610
  233. 233. Hirota A, Horikawa T, Fujiwara A. Isolation of phenylacetic acid and indoleacetic acid from a phytopathogenic fungus, Glomerella cingulata. Bioscience, Biotechnology, and Biochemistry. Japan Society for Bioscience, Biotechnology, and Agrochemistry; 1993;57: 492–492.
  234. 234. Hoffman MT, Gunatilaka MK, Wijeratne K, Gunatilaka L, Arnold AE. Endohyphal bacterium enhances production of indole-3-acetic acid by a foliar fungal endophyte. Corradi N, editor. PLoS ONE. Public Library of Science; 2013;8: e73132. pmid:24086270
  235. 235. Nutaratat P, Srisuk N, Arunrattiyakorn P, Limtong S. Plant growth-promoting traits of epiphytic and endophytic yeasts isolated from rice and sugar cane leaves in Thailand. Fungal Biology. 2014;118: 683–694. pmid:25110131
  236. 236. Silva HSA, Tozzi PL, Terrasan RF, Bettiol W. Endophytic microorganisms from coffee tissues as plant growth promoters and biocontrol agents of coffee leaf rust. Biological Control. 2012;63: 62–67.
  237. 237. Yang Z, Zhang J, Wang T, Huang X, Hu GB, Wang HC. Does acid invertase regulate the seed development of Litchi chinensis? Acta Horticulturae. 2014; 301–308.
  238. 238. Schwarz M, Köpcke B, Weber RWS, Sterner O, Anke H. 3-Hydroxypropionic acid as a nematicidal principle in endophytic fungi. Phytochemistry. 2004;65: 2239–2245. pmid:15587708
  239. 239. Amade P, Mallea M, Bouicha N. Isolation, structural identification and biological activity of two metabolites produced by Penicillium olsonii Bainer and sartory. The Journal of Antibiotics. 1994;47: 201–208. pmid:8150716
  240. 240. Ayer WA, Browne LM, Lin G. Metabolites of Leptographium wageneri, the causative agent of black stain root disease of conifers. Journal of Natural Products. 1989;52: 119–129.
  241. 241. Zolfaghari M, Drogui P, Seyhi B, Brar SK, Buelna G, Dubé R. Occurrence, fate and effects of Di (2-ethylhexyl) phthalate in wastewater treatment plants: A review. Environmental Pollution. 2014;194: 281–293. pmid:25091800
  242. 242. da Silva IP, Brissow E, Kellner Filho LC, Senabio J, de Siqueira KA, Vandresen Filho S, et al. Bioactive compounds of Aspergillus terreus—F7, an endophytic fungus from Hyptis suaveolens (L.) Poit. World Journal of Microbiology and Biotechnology. 2017;33: 62. pmid:28243983
  243. 243. Muharni M, Fitrya F, Ruliza MO, Susanti DA, Elfita E. Di-(2-ethylhexyl)phthalate and pyranon derivated from endophytic fungi Penicillium sp the leave of kunyit putih (Curcuma zedoaria). Indonesian Journal of Chemistry. 2014;14: 290.
  244. 244. Erythropel HC, Maric M, Nicell JA, Leask RL, Yargeau V. Leaching of the plasticizer di(2-ethylhexyl)phthalate (DEHP) from plastic containers and the question of human exposure. Applied Microbiology and Biotechnology. Springer Berlin Heidelberg; 2014;98: 9967–9981. pmid:25376446
  245. 245. Rodgers K, Rudel R, Just A. Toxicants in food packaging and household plastics. 1st ed. London: Springer London; 2014.
  246. 246. Nguyen DH, Nguyen DTM, Kim E-K. Effects of di-(2-ethylhexyl) phthalate (DEHP) released from laboratory equipments. Korean Journal of Chemical Engineering. Springer US; 2008;25: 1136–1139.
  247. 247. Reid AM, Brougham CA, Fogarty AM, Roche JJ. An investigation into possible sources of phthalate contamination in the environmental analytical laboratory. International Journal of Environmental Analytical Chemistry. Taylor & Francis Group; 2007;87: 125–133.
  248. 248. Mathur SP. Phthalate esters in the environment: pollutants or natural products? Journal of Environment Quality. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America; 1974;3: 189.
  249. 249. Hayasaka Y. Analysis of phthalates in wine using liquid chromatography tandem mass spectrometry combined with a hold-back column: Chromatographic strategy to avoid the influence of pre-existing phthalate contamination in a liquid chromatography system. Journal of Chromatography A. 2014;1372: 120–127. pmid:25465010
  250. 250. Marega M, Grob K, Moret S, Conte L. Phthalate analysis by gas chromatography–mass spectrometry: Blank problems related to the syringe needle. Journal of Chromatography A. 2013;1273: 105–110. pmid:23265992
  251. 251. Jiang X, Meng X, Xian M. Biosynthetic pathways for 3-hydroxypropionic acid production. Applied Microbiology and Biotechnology. 2009;82: 995–1003. pmid:19221732
  252. 252. Kumar V, Ashok S, Park S. Recent advances in biological production of 3-hydroxypropionic acid. Biotechnology Advances. 2013;31: 945–961. pmid:23473969
  253. 253. Qadri M, Deshidi R, Shah BA, Bindu K, Vishwakarma RA, Riyaz-Ul-Hassan S. An endophyte of Picrorhiza kurroa Royle ex. Benth, producing menthol, phenylethyl alcohol and 3-hydroxypropionic acid, and other volatile organic compounds. World Journal of Microbiology and Biotechnology. Springer Netherlands; 2015;31: 1647–1654. pmid:26220851
  254. 254. Preston PN, Will SG, Winwick T, Morley JO. Preparation of 3,4-dihydroanthracen-1(2H)-ones. A synthetic approach to islandicin and digitopurpone via difluoro[anthracen-1(2H)-onato-O 1,O 9]boron chelates. Journal of the Chemical Society, Perkin Transactions 1. The Royal Society of Chemistry; 1983;0: 1001.
  255. 255. Girija S, Duraipandiyan V, Kuppusamy PS, Gajendran H, Rajagopal R. Chromatographic characterization and GC-MS evaluation of the bioactive constituents with antimicrobial potential from the pigmented ink of Loligo duvauceli. International Scholarly Research Notices. 2014;2014: 1–7. pmid:27437466
  256. 256. Driche EH, Belghit S, Bijani C, Zitouni A, Sabaou N, Mathieu F, et al. A new Streptomyces strain isolated from Saharan soil produces di-(2-ethylhexyl) phthalate, a metabolite active against methicillin-resistant Staphylococcus aureus. Annals of Microbiology. 2015;65: 1341–1350.
  257. 257. Habib MR, Karim MR, Szegletes Z, Imai K, Matsumoto T, Yoshikawa M, et al. Antimicrobial and cytotoxic activity of Di-(2-ethylhexyl) phthalate and anhydrosophoradiol-3-acetate isolated from Calotropis gigantea (Linn.) flower. Mycobiology. Roche Scientific Services; 2009;37: 31. pmid:23983504
  258. 258. El-Sayed MH. Di-(2-ethylhexyl) Phthalate, a major bioactive metabolite with antimicrobial and cytotoxic activity isolated from the culture filtrate of newly isolated soil Streptomyces (Streptomyces mirabilis strain NSQu-25). World Applied Sciences Journal. 2012;20: 1202–1212.
  259. 259. Al-Bari MAA, Bhuiyan MSA, Flores ME, Petrosyan P, García-Varela M, Islam MAU. Streptomyces bangladeshensis sp. nov., isolated from soil, which produces bis-(2-ethylhexyl)phthalate. International journal of systematic and evolutionary microbiology. 2005;55: 1973–1977. pmid:16166697
  260. 260. Lee KH, Kim JH, Lim DS, Kim CH. Anti-leukaemic and anti-mutagenic effects of Di(2-ethylhexyl)phthalate isolated from Aloe vera Linne. Journal of Pharmacy and Pharmacology. Blackwell Publishing Ltd; 2000;52: 593–598. pmid:10864149
  261. 261. Su M, Li Y, Ge X, Tian P. 3-Hydroxypropionaldehyde-specific aldehyde dehydrogenase from Bacillus subtilis catalyzes 3-hydroxypropionic acid production in Klebsiella pneumoniae. Biotechnology Letters. 2015;37: 717–724. pmid:25409630
  262. 262. Tickner JA, Schettler T, Guidotti T, McCally M, Rossi M. Health risks posed by use of Di-2-ethylhexyl phthalate (DEHP) in PVC medical devices: A critical review. American Journal of Industrial Medicine. John Wiley & Sons, Inc.; 2001;39: 100–111. pmid:11148020
  263. 263. Roth B, Herkenrath P, Lehmann H-J, Ohles H-D, Hemig HJ, Benz-Bohm G, et al. Di-(2-ethylhexyl)-phthalate as plasticizer in PVC respiratory tubing systems: indications of hazardous effects on pulmonary function in mechanically ventilated, preterm infants. European Journal of Pediatrics. Springer-Verlag; 1988;147: 41–46. pmid:3422189
  264. 264. Celińska E. Debottlenecking the 1,3-propanediol pathway by metabolic engineering. Biotechnology Advances. 2010;28: 519–530. pmid:20362657
  265. 265. Zheng P, Wereath K, Sun J, van den Heuvel J, Zeng A-P. Overexpression of genes of the dha regulon and its effects on cell growth, glycerol fermentation to 1,3-propanediol and plasmid stability in Klebsiella pneumoniae. Process Biochemistry. 2006;41: 2160–2169.
  266. 266. Erexson GL, Periago MV, Spicer CS. Differential sensitivity of Chinese hamster V79 and Chinese hamster ovary (CHO) cells in the in vitro micronucleus screening assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2001;495: 75–80.