Endophytes as sources of antibiotics

Biochemical Pharmacology 134 (2017) 1–17

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Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Review

Endophytes as sources of antibiotics Elena Martinez-Klimova, Karol Rodríguez-Peña, Sergio Sánchez ⇑ Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico

a r t i c l e

i n f o

Article history: Received 25 August 2016 Accepted 25 October 2016 Available online 27 October 2016 Keywords: Natural products Antibacterial Antifungal Antimalarial Endophytes

a b s t r a c t Until a viable alternative can be accessible, the emergence of resistance to antimicrobials requires the constant development of new antibiotics. Recent scientific efforts have been aimed at the bioprospecting of microorganisms’ secondary metabolites, with special emphasis on the search for antimicrobial natural products derived from endophytes. Endophytes are microorganisms that inhabit the internal tissues of plants without causing apparent harm to the plant. The present review article compiles recent (2006– 2016) literature to provide an update on endophyte research aimed at finding metabolites with antibiotic activities. We have included exclusively information on endophytes that produce metabolites capable of inhibiting the growth of bacterial, fungal and protozoan pathogens of humans, animals and plants. Where available, the identified metabolites have been listed. In this review, we have also compiled a list of the bacterial and fungal phyla that have been isolated as endophytes as well as the plant families from which the endophytes were isolated. The majority of endophytes that produce antibiotic metabolites belong to either phylum Ascomycota (kingdom Fungi) or to phylum Actinobacteria (superkingdom Bacteria). Endophytes that produce antibiotic metabolites were predominant, but certainly not exclusively, from the plant families Fabaceae, Lamiaceae, Asteraceae and Araceae, suggesting that endophytes that produce antimicrobial metabolites are not restricted to a reduced number of plant families. The locations where plants (and inhabiting endophytes) were collected from, according to the literature, have been mapped, showing that endophytes that produce bioactive compounds have been collected globally. Ó 2016 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 What are endophytes and why research them?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 How to isolate endophytes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Extracts from endophytic isolates with antibiotic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Identified compounds produced by endophytic isolates and their activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5.1. Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5.2. Other organic compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 6. The biosynthetic potential of endophytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 7. Taxonomic classification of endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 8. Taxonomic classification of plants containing endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 9. Global localization of plant collection sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 10. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

⇑ Corresponding author. E-mail addresses: [email protected] (E. Martinez-Klimova), [email protected] (K. Rodríguez-Peña), sersan@biomedicas. unam.mx (S. Sánchez). http://dx.doi.org/10.1016/j.bcp.2016.10.010 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.

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1. Introduction New antibiotics are in need of development to treat infections of pathogens that have evolved resistance to currently available antibiotics [1,2]. Bioprospecting for natural products is a route for the discovery of sources of new drugs via the isolation of bioactive metabolites from living organisms [2,3]. Actinomycetes are well-known for their capacity to produce secondary metabolites applied in medicine and agriculture [4]. Soil is the most common source to isolate actinomycetes, but they have also been isolated from leaf litter and plants [4]. Apart from actinomycetes, other bacteria and fungi with the potential to produce biologically active secondary metabolites have been isolated from plants [5]. The microorganisms residing within plants are increasingly becoming the object of research efforts, especially when the source plant is traditionally used for healing. More and more information is becoming available in the literature on the biodiversity and biosynthetic potential of endophytes.

2. What are endophytes and why research them? In order to understand why endophytes have been attracting much attention in the field of antimicrobial research, it is important to revise the role they are believed to perform in nature. Microbial groups are known to colonize plant tissues as symbionts or pathogens [6]. According to Wilson’s 1995 definition [7], an ‘‘endophyte” is an organism that lives inside a plant (Gr. Endon, within; phyton, plant). The organisms commonly found living inside plants are fungi and bacteria. The plants that host endophytes do not show symptoms of disease, at least during the endophytic phase of their life cycle [7]. The term endophyte has become a synonym of mutualist [8]. However, not all colonizers are harmless; some plant pests often are bacterial or fungal, so phytopathogens might have an endophytic origin [9]. Fungal endophytes have been found in all plant families (including bryophytes and ferns), throughout the world, in all kinds of climates [6,9–11]. Microbes enter plant tissues through wounds or the roots, or alternatively by creating wounds through the secretion of enzymes like cellulases [9]. Endophytes may be facultative or obligate [12]. It is not clear why plants and endophytes coexist or why plants do not defend themselves against the internal colonizers. So far, it seems that the symbiotic relationship between plant and endophyte is of benefit for both parties: it is beneficial for the endophyte due to availability of the plant’s nutrients [7] and it is beneficial for the plant due to protection from pathogens, enhancement of nutrient uptake, promotion of plant growth [2,11,13] and stress tolerance [6]. Bacteria can facilitate the acquisition of essential nutrients from the environment like nitrogen, phosphorous and iron [9,13]. Endophytes can also produce vitamins [9] or modulate phytohormone levels within the plant, since the bacteria are capable of synthesizing auxin, cytokinin and gibberellin, and may cleave the direct precursor of ethylene [11]. Endophytes can increase the amount of roots a plant produces [9] and plant growth enhancement may outcompete cell apoptosis caused by pathogen infection [2]. However, according to Hyde and Soytong [14] much more work needs to be done to confirm the hypotheses about the beneficial traits that endophytes provide to plants. It is believed that plants that contain endophytes are healthier than endophyte-free plants [9]; perhaps because the endophytes produce metabolites that promote plant growth and help defend the plant against insects, pests and pathogens [10,15]. The secondary metabolites produced by endophytes may be implemented in the pharmaceutical and agricultural industries [10] since the natural products from endophytes have activity as antimicrobials,

antifungals, anticarcinogens, immunosuppressants or antioxidants [9]. It is thought that endophytes reduce the severity of plant disease [6], therefore their implementation as biocontrol agents has been suggested to minimize the use of chemicals [6]. Endophytes interact with other microbial plant colonizers [6,15]. The survival of endophytes within plant tissues is a continuous struggle between the established endophytes and new invading pathogens and pests [16]. Therefore, it is believed that colonization of the plant tissue by endophytes prevents pathogenic organisms from colonizing plant tissues [15]. Endophytes have adapted themselves to overcome the host immune system [17]. It is known that bacterial endophytes are able to interact with plant cells by chemotaxis [11]. Quorum sensing is a cell-to-cell communication system mediated by autoinducers. According to Kusari et al. ‘‘Quorum sensing is responsible for the regulation of virulence factors, infections, invasion, colonization, biofilm formation and antibiotic resistance within bacterial populations” [16]. Endophytes can also impede communication between invading pathogens to stop them from colonizing plants [16], so quorum sensing is a phenomenon that is relevant for bacterial colonization, pathogenesis and development of resistance to antibiotics. Quorum sensing has also been observed in fungi and in plants, which seems to indicate that it is involved in communication between bacteria, fungi and plants [16]. Endophytes are known to be able to biosynthesize some of the same chemical compounds as their host plant, possibly as an adaptation to the host’s microenvironment [9]. The most famous example is that of taxol, but there are several anticancer compounds also reported, such as camptothecin and podophyllotoxin [2]. Endophytes secrete antibiotics or hydrolytic enzymes to prevent colonization of microbial plant pathogens or to prevent insects or nematodes from infecting the plants they inhabit. It is thought that endophytes release metabolites that have the capacity of activating the plant’s defense mechanism or promoting plant growth in an attempt to outcompete cell apoptosis of infected tissues [2]. Endophytes have been attracting attention as sources of potentially valuable compounds. Several reviews have been published recently on the matter. For instance, a 2015 review by Golinska et al. [10] is focused on the diversity of endophytic actinobacteria from medicinal plants, the metabolites they produce, as well as the reported bioactivities of the metabolites: antimicrobial, antiviral, larvicidal, antimalarial, citotoxic, antidiabetic and as plant growth promoters. Likewise, in 2014, Abdalla and Matasyoh [15] published a review on peptide compounds isolated from endophytes. For a review on regulation of secondary metabolic pathways please consult the 2016 work of Deepika et al. [17]. The 2014 work by Alvin et al. [2] is a comprehensive review on antimycobacterial metabolites from endophytes. In the present review article, research papers from the last ten years have been compiled about the antimicrobial properties of metabolites synthesized by endophytes isolated from plants. In some cases, the metabolites have been identified and in other cases, the extracts have been tested for antibiotic activity. We present an overview of the variety of plants that endophytes have been isolated from, as well as the different kinds of endophytic organisms already identified.

3. How to isolate endophytes? The most common technique to isolate endophytes involves surface sterilization, followed by fractionation of the plant material into small pieces with a sterile blade for plating onto agar [10]. The technique can be applied to any plant section [15]. Other techniques involve the use of vacuum [18] but are not recommendable for soft non-woody plant tissues [15]. Surface sterilization is

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necessary to ensure that the isolated strains are endophytes and not epiphytes [19]. The endophytes isolated depend not only on the plant, but also on the tissue of the plant [10]. The isolation procedures must be carried out while the tissues are still fresh. If it is not possible to carry out the isolation procedures within 24 h of harvesting the plant material, it is recommendable to refrigerate it [10] or store it on ice to prevent death of the microorganisms. Surface sterilization must be done carefully, if the solutions are too concentrated, the exposure time too long or the tissue too permeable, then internal microorganisms may die as well [10]. Plant selection and the thoroughness of the sterilization techniques used are of the utmost importance. The selection of plant that appears healthy suggests that the isolate is an endophyte and not a pathogen. Likewise, complete sterilization of the surface of sampled tissues is necessary to reassure that the isolate comes from the inner structures of the plant and is not a contaminant from soil, air or water. Washing the plant tissues under running tap water to remove soil particles is a first-instance requirement [20–23] taking into consideration that it is most common to find actinomycetes in root tissues compared to other plant organs [4,24]. Gohain et al. [24] rinsed the samples with Tween20 (0.1%). Coombs et al. [20] isolated Actinobacteria from roots by surface-sterilizing the root tissues by washing with 99% ethanol, followed by a wash with sodium hypochlorite (3.125% NaOCl), followed by another wash with 99% ethanol and a final rinse with sterile Reverse Osmosis (RO)-treated water. Cao et al. [21,22] immersed plant tissues in 70% ethanol and sodium hypochlorite (0.9% w/v chlorine). Samples were washed thoroughly in sterile water. Qin et al. [25] carried out a ‘‘five-step surface sterilization procedure” consisting of a wash in sodium chlorate (2.5% NaClO3), followed by a wash in sodium thiosulfate (2.5% Na2S2O3), followed by a wash in 75% ethanol, followed by a wash in sodium bicarbonate (10% NaHCO3) and a final wash in sterile water. The fragments can be any size, but smaller is more desirable since it increases surface exposure to the agar [10]. Surface sterilized tissue was aseptically cut into 1 cm fragments and each fragment was placed on media for the isolation of endophytes [20]. Qin et al. [25] crumbled the surface-sterilized roots aseptically into small fragments. Gohain et al. [24] disrupted the sterilized tissued in a blender and further ground the samples with mortar and pestle. Coombs et al. [20] used several isolation media for the isolation of endophytic Actinobacteria, which included (1) yeast extract, K2HPO4 and agar, (2) humic acid, vitamin B and agar, (3) flour, yeast extract, sucrose, casein hydrolysate and agar, (4) flour, calcium carbonate and agar or (5) yeast extract, casein hydrolysate and agar. Cao et al. [22] used a medium containing dextrose, casein hydrolysate, K2HPO4, MgSO4, CaCl2, ferric citrate, CoSO4, CuSO4, H3BO3, MnSO4, (NH4)6Mo7O24 and ZnSO4 for the isolation of endophytic Streptomyces. Incubation to isolate endophytic actinobacteria was carried out at 27 °C for up to four weeks [20]. Incubation to isolate endobacteria was carried out at 25 °C for 5 days [23]. To prevent the growth of fungi [20], supplemented with Benomyl (DuPont). Alternatively, Cao et al. [21,22] immersed sterilized plant tissued in sodium bicarbonate (10% NaHCO3) to prevent the growth of fungi and nalidixic acid to prevent the growth of nonstreptomycete bacteria. Izumi et al. [23] added cycloheximide. To favor the selection of actinomycetes, Gohain et al. [24] added nalidixic acid as well as nystatin to the isolation media. In contrast, to select for endophytic fungi and prevent the growth of endophytic bacteria, Melo et al. [26] supplemented the isolation media with chloramphenicol. Streptomycin has been added for the isolation of slow-growing fungi [27,28].

3

To characterize the isolates [25], for example, observed characteristics of the cultures such as colony color, morphological characteristics of spores and mycelia, tolerance to NaCl concentrations, utilization of sole carbon sources for growth, acid production from carbohydrates and decomposition of test substances, identification of cell-wall amino acids and sugars in whole cell hydrolysates, analysis of menaquinones, polar lipids and fatty acids. Amplification and sequencing of the 16S rRNA gene were used for phylogenetic analysis. In work [24], BOX fingerprinting, a technique used to study bacterial diversity, was compared to 16S rDNA sequences. In order to corroborate if the growth obtained on the plates is from endophytes or contaminants growing in the surface of the tissues, it is recommendable to do a control plate inoculating a sample of the water after the final wash [10] or to do a leaf imprint on the agar surface [14]. It is recommended to consult the 2006 review by Zhang, Song and Tan [9] for methods to isolate and identify endophytes.

4. Extracts from endophytic isolates with antibiotic activity The antibiotic properties of organic extracts obtained from different endophytes with the prospect of finding microorganisms that are producers of novel bioactive compounds have been evaluated during the last few decades, finding a variety of extracts with MIC values suitable to justify further studies. In 2008, Guimarães et al. [29] reported on an endophytic isolate from the medicinal plant Viguiera arenaria collected in Brazil. The endophytic isolate was identified as Diaporthe phaseolorum. The ethyl acetate extract of the endophytic isolate’s fermentation broth showed strong (95%) inhibition of the gGAPDH enzyme of Trypanosoma cruzi. The same extract also showed 60.7% inhibition of the Leishmania tarentolae adenine phosphoribosyltransferase (APRT) enzyme. The gGADPH and APRT assays were used to test for antiparasitic activity. The extract of another endophyte that had most similarity to Phomopsis sp. also showed similar inhibition (61.4%) of the Leishmania tarentolae APRT enzyme as the extract from the endophytic Diaporthe phaseolorum. In 2009, Verma et al. [19] reported on endophytic isolates from the ‘‘neem tree” Azadirachta indica, collected in India. The endophytic isolates were identified as Streptomyces sp., Streptosporangium sp. and Nocardia sp. The assays were done by a modified Bauer–Kirby method using paper discs impregnated in the methanol extract of the isolates’ fermentation broth. The extracts showed activity against Pseudomonas fluorescens, B. subtilis, S. aureus, E. coli, C. albicans, Trichophyton sp., Microsporum sp., Aspergillus sp., Pythium sp. and Phytophthora sp. In 2011, Arivudainambi et al. [30] reported on an endophytic isolate from the medicinal plant Vitex negundo collected in India. The isolate was identified as Colletotrichum gloeosporioides. The methanol extract of the isolate’s fermentation broth had activity against S. aureus MTCC 3160, B. subtilis MTCC 619, E. coli MTCC 4296, P. aeruginosa MTCC 2488 and C. albicans MTCC 3018. Activity was also observed against clinical multidrug-resistant strains of S. aureus. Arivudainambi et al. reported that the methanol extract of C. gloeosporioides in combination with antibiotics had a stronger activity against multidrug resistant bacteria than the extract alone. Buatong et al. [3] in the same year, reported on endophytic isolates from twelve mangrove plant species: Aegiceras corniculatum, Avicennia alba, Avicennia officinalis, Bruguiera gymnorrhiza, Bruguiera parviflora, Lumnitzera littorea, Rhizophora apiculate, Rhizophora mucronata, Sonneratia caseolaris, Scyphiphora hydrophyllacea, Xylocarpus granatum, and Xylocarpus moluccensis; collected in Thailand. The endophytic isolates were identified as Acremonium sp., Diaporthe sp., Hypoxylon sp., Pestalotiopsis sp., Phomopsis sp., and Xylaria sp. The assays were done by testing crude, hexane

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and ethyl acetate extracts in a colorimetric microdilution method. The authors reported that the hexane extracts from fungal mycelia had the highest inhibitory activity, followed by ethyl acetate extracts from the mycelia and ethyl acetate extracts from the fermentation broths. The isolates showed activity against the bacteria S. aureus ATCC25923 and MRSA-SK1, Microsporum gypseum, C. neoformans, C. albicans and P. aeruginosa. Also in 2011, Cui et al. [31] reported on endophytic isolates from the plant Aquilaria sinensis, collected in China. The endophytic isolates were identified as Fusarium equiseti, Phaeoacremonium rubrigenum, Fusarium avenaceum, Phaeoacremonium rubrigenum and Chaetomium globosum. The assays were done by the agar well diffusion method. The isolates showed activity against S. aureus, E. coli, B. subtilis and A. fumigatus. In 2012, Miller et al. [27] reported on endophytic isolates from medicinal plants used traditionally for the treatment of cancer, such as Pinellia ternata, Lycium chinense, Digitalis purpurea, Leonurus heterophyllus, Bletilla striata, Belamcanda chinensis, Pinellia pedatisecta and Taxus yunnanensis; collected in China. The endophytic isolates were identified as Cochliobolus sp., Cladosporium sp., Fusarium sp., Mycosphaerella sp., Phoma sp., Pestalotiopsis sp., Paenibacillus sp., Pseudomonas sp., Pseudomonas sp. B42 and Bacillus sp. The assays were done by incubating cells extracts together with a culture of the test bacteria or fungi in a 96-well microtitre plate. The isolates showed growth inhibitory activity against S. aureus NCTC 6571, E. coli NCTC 10418 and Cryptococcus albidus ATCC 10666. The extract of Pseudomonas sp. B42 inhibited the growth of S. aureus NCTC 6571 by 72.5%. In 2013, Ding et al. [32] reported on endophytic isolates from the medicinal plant Camptotheca acuminata, collected in China. The endophytic isolates were identified as Rhizopus sp. X65, Botryosphaeria dothidea X4, Fusarium proliferatum X25 and Aschersonia sp. M63. The isolates’ fermentation broth supernatants showed activity against B. subtilis, E. coli, Fusarium solani and Verticillium dahliae. In 2014, Machavariani et al. [33] reported on endophytic isolates from the medicinal plants Aloe arborescens, Mentha arvensis, Lysimachia nummularia, Fragaria vesca and Arctium lappa; collected in Russia. The endophytic isolates were identified as Nocardiopsis, Streptomyces and Micromonospora. The assays were done by the well-diffusion method. The isolates showed activity against the bacteria S. aureus FDA 209P, S. aureus 209P/UF-2, S. aureus MRSA, Micrococcus luteus ATCC 9341, B. subtilis ATCC 6633, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and the fungus S. cerevisiae Y1334. In the same year, Melo et al. [26] reported on an endophytic isolate from the plant Schistidium antarctici, collected in Antarctica. The endophytic isolate was identified as Mortierella alpina. The isolate’s extract showed activity against E. coli CCMA 104 (MIC of 26.9 lg/mL), P. aeruginosa LMA 01 (MIC of 107.6 lg/mL), E. faecalis CCMA 292 (MIC of 107.6 lg/mL), Salmonella typhi CCMA 74 (MIC of 215.3 lg/mL), Klebsiella pneumoniae CCMA 291 (MIC of 215.3 lg/mL) and S. aureus CCMA 190 (MIC of 215.3 lg/mL). The success obtained in terms of the bioactivity of organic extracts from endophytes has caused an increase in the number of studies in recent years. In 2015 several studies were published, as performed by Akinsanya et al. [34] reported on endophytic isolates from the medicinal plant Aloe vera, collected in Malaysia. The isolates were identified as Bacillus tequilensis ALR-2, Chryseobacterium indologenes ALR-13, Pseudomonas entomophila ALR-12 and Bacillus aerophilus ALS-8. The crude and ethyl acetate extracts of the isolates’ fermentation broths showed activity against P. aeruginosa, S. aureus, B. cereus, Proteus vulgaris, Klebsiella pneumoniae, E. coli, Streptococcus pyogenes and C. albicans. The inhibition zones ranged from 6.0 ± 0.57 to 16.6 ± 0.57 mm. Bezerra et al. [35] reported on endophytic isolates from the plant Bauhinia forficata, collected in Brazil. The isolates were

identified as Penicillium commune, Gibberella baccata, Penicillium glabrum, Aspergillus ochraceus and Khuskia oryzae. The assays were done by the disc diffusion method. The isolates showed activity against S. aureus UFPEDA02, Streptococcus pyogenes UFPEDA07, Mycobacterium smegmatis UFPEDA71, B. subtilis UFPEDA86, E. faecalis UFPEDA138, Salmonella typhi UFPEDA478, P. aeruginosa UFPEDA735, Proteus vulgaris UFPEDA740, and E. coli UFPEDA224. Mani et al. [8] reported on endophytic isolates from the medicinal tree Aegle marmelos, collected in India. The endophytic isolates were identified as Curvularia australiensis, Alternaria citrimacularis, Alternaria alternata, Cladosporium cladosporioides and Aspergillus niger. The isolates showed activity against S. aureus, K. pneumoniae, S. epidermis, P. aeruginosa, E. faecalis, B. subtilis, E. coli, Pseudomonas mirabilis, Shigella sp., S. typhi, C. albicans and A. niger. Chowdhary and Kaushik [36] reported on an endophytic isolate from the medicinal plant Ocimum sanctum, collected in India. The endophytic isolate was identified as Macrophomina phaseolina. The isolate’s extract showed activity against Sclerotinia sclerotiorum (IC50 of 0.38 mg/mL). The need for a better knowledge on the compounds present in the organic extracts likely responsible for the biological activity promoted studies to elucidate their chemical structures. Some of these studies resulted in novel compounds or, with biological activities not described previously. Such is the case of the study developed by Mousa et al. [37] who reported on an endophytic isolate from the plant Eleusine coracana. The endophytic isolate was identified as Phoma sp. The isolate’s extract showed activity against Fusarium graminearum (6.5 mm), Fusarium lateritium (6 mm), Fusarium sporotrichioides (4 mm), Fusarium avenaceum (4.5 mm), Trichoderma longibrachiatum (8.5 mm), Aspergillus flavus (4 mm) and Alternaria alternata (3 mm). The compounds identified from the extract of Phoma sp. WF4 isolate were: viridicatol, tenuazonic acid, alternariol and alternariol monomethyl ether. These four compounds had not been previously reported to have activity against Fusarium spp. The most recent studies published in 2016 continue to demonstrate the great ability of endophytes to produce antibiotic compounds. For instance, Alvin et al. [38] reported on endophytic isolates from the medicinal plant Tradescantia spathacea (synonym: Rhoeo spathacea) traditionally used against respiratory disease; collected in Indonesia. The endophytic isolates were identified as Colletotrichum sp., Fusarium sp., Guignardia sp., Phomopsis sp., Phoma sp. and Microdochium sp. Ethyl acetate extracts of the endophytic isolates showed activity against P. aeruginosa NCTC 10490, S. aureus NCTC 6571 and E. coli NCTC 10418. The crude extract of Fusarium sp. showed the best antibacterial activity and was further investigated, leading to the discovery of ‘‘javanicin” (7). Tonial et al. [39] reported on endophytic isolates from the medicinal plant Schinus terebinthifolius, collected in an unspecified location in South America. The endophytic isolates were identified as Alternaria sp. Sect. alternata, Bjerkandera sp., Xylaria sp., Diaporthe sp. and Penicillium sp. The isolates’ fermentation broth extracts showed activity against S. aureus ATCC 6538, P. aeruginosa ATCC 27853 and C. albicans ATCC 10231. The extract of Alternaria sp. Sect. alternata showed the highest activity against methicillinresistant S. aureus (MIC of 18.52 lg/mL). The compounds E-2-hexyl-cinnamaldehyde and two pyrrolopyrazine alkaloids were identified in the extract. Tanvir et al. [40] reported on an endophytic isolate from the plant Sonchus oleraceus, collected in Pakistan. The isolate was identified as Nocardia caishijiensis SORS 64b. The isolate’s extract showed activity against methicillin-resistant S. aureus MRSA (14 mm), E. coli ATCC 25922 (14 mm), K. pneumoniae ATCC 706003 (13 mm), S. aureus ATCC 25923 (11 mm) and C. tropicalis (20 mm). In addition, the N. caishijiensis extract gave an LC50 value of 570 lg/mL in the brine shrimp cytotoxicity assay, a value which

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Table 1 Summary of bioactive extracts from endophytes, showing type of extract, endophyte it was obtained from and source plant, as well as activity observed against selected test strains. Name of source plants

Name of endophytes

Type of extract

Activity observed against test strains

Reference

Aloe vera

Bacillus tequilensis, Chryseobacterium indologenes, Pseudomonas entomophila and Bacillus aerophilus. Colletotrichum gloeosporioides

Crude and ethyl acetate

[34]

Aegiceras corniculatum, Avicennia alba, A. officinalis, Bruguiera gymnorrhiza, B. parviflora, Lumnitzera littorea, Rhizophora apiculata, R. mucronata, Sonneratia caseolaris, Scyphiphora hydrophyllacea, Xylocarpus granatum and X. moluccensis Azadirachta indica

Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis and Xylaria

Crude, hexane and ethyl acetate

Antibacterial and antifungal: Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Streptococcus pyogenes and Candida albicans Antibacterial and antifungal: S. aureus, Bacillus subtilis, E. coli, P. aeruginosa and C. albicans Antibacterial and antifungal: S. aureus, Microsporum gypseum, Cryptococcus neoformans, C. albicans and P. aeruginosa

Streptomyces, Streptosporangium and Nocardia

Methanol

[19]

Pinellia ternata, Lycium chinense, Digitalis purpurea, Leonurus heterophyllus, Bletilla striata, Belamcanda chinensis, Pinellia pedatisecta and Taxus yunnanensis Tradescantia spathacea

Cochliobolus, Cladosporium, Fusarium, Mycosphaerella, Phoma, Pestalotiopsis, Paenibacillus, Pseudomonas and Bacillus. Colletotrichum, Fusarium, Guignardia, Phomopsis, Phoma and Microdochium Diaporthe phaseolorum

Crude

Antibacterial and antifungal: Pseudomonas fluorescens, S. aureus, E. coli, B. subtilis, C. albicans, Trichophyton, Microsporum and Aspergillus, Pythium and Phytophthora Antibacterial and antifungal: S. aureus, E. coli and Cryptococcus albidus

Vitex negundo

Viguiera arenaria Ocimum sanctum Camptotheca acuminata

Schinus terebinthifolius

Macrophomina phaseolina Rhizopus, Botryosphaeria dothidea, Fusarium proliferatum and Aschersonia Alternaria alternata, Bjerkandera, Xylaria, Diaporthe and Penicillium

Methanol

[3]

[28]

Ethyl acetate

Antibacterial: P. aeruginosa, S. aureus and E. coli

[38]

Ethyl acetate

Antiprotozoan: Trypanosoma cruzi, Leishmania tarentolae Antifungal: Sclerotinia sclerotiorum Antibacterial and antifungal: B. subtilis, E. coli, Fusarium solani and Verticillium dahliae.

[29]

Antibacterial and antifungal: S. aureus, P. aeruginosa and C. albicans.

[39]

Antibacterial and antifungal: S. aureus, E. coli, K. pneumoniae, S. aureus and Candida tropicalis Antibacterial and antifungal: B. subtilis, C. tropicalis, S. aureus and E. coli Antifungal: Fusarium graminearum, Fusarium lateritium, Fusarium sporotrichioides, Fusarium avenaceum, Trichoderma longibrachiatum, Aspergillus flavus and Alternaria alternata Antibacterial and antifungal: E. coli, Salmonella, B. subtilis, Enterococcus faecium, S. aureus and C. albicans

[40]

Hexane Supernatants

Sonchus oleraceus

Nocardia caishijiensis

Ethyl acetate and methanol Crude

Ageratum conyzoides

Pseudonocardia carboxydivorans

Crude

Eleusine coracana

Phoma sp.

Methanol

Polygonum cuspidatum

Streptomyces sp.

Ethyl acetate

falls within the ‘‘toxic” range. The extract of N. caishijiensis was believed to contain stenothricin and bagremycin A, according to analytical results. In the same study, the authors reported an endophytic isolate from the plant Ageratum conyzoides, collected in Pakistan. The isolate was identified as Pseudonocardia carboxydivorans AGLS 2. The isolate’s extract showed activity against B. subtilis DSM 10 ATCC 6051 (21 mm), C. tropicalis (20 mm), S. aureus ATCC 25923 (17 mm), S. aureus MRSA (17 mm), E. coli K12 W1130 (16 mm) and Chlorella vulgaris (10 mm). In addition, the P. carboxydivorans extract gave an LC50 value of 1100 lg/mL in the brine shrimp cytotoxicity assay, which is considered non-toxic. The extract of P. carboxydivorans was determined to contain borrelidin and a-pyrone, as well as long-chained amide derivatives such as 7octadecenamide and 9, 12 octadecandienamide. In 2016, Wang et al. [41] reported on an endophytic isolate from the plant Polygonum cuspidatum, collected in China. The endophytic isolate was identified as Streptomyces sp. A0916. The isolate’s extract showed activity against E. coli S11A1006 (MIC of 4 lg/mL), Salmonella sp. S11A235 (MIC of 125 lg/mL), B. subtilis S11A1007 (MIC of 4 lg/mL), E. faecium S11A843 (MIC of 16 lg/mL), S. aureus S11A1005 (MIC of 8 lg/mL) and C. albicans S11A2331 (MIC of 16 lg/mL). The compounds identified from the extract of Streptomyces sp. A0916 were: 3-methyl-1-butanol,

[30]

[36] [32]

[40] [37]

[41]

4-methyl-1-pentanol, 1-nonanal, 6-methyl-2-oxiranyl-hept-5-en2-ol, 2, 6, 11, 15-tetramethylhexadecane, among several others. Table 1, included below, shows a summary of the bioactivities seen after testing extracts of endophytic culture broths on selected microorganisms. 5. Identified compounds produced by endophytic isolates and their activities Diverse types of compounds showing different levels of antibiotic activity have been isolated and identified from endophytes. Although some of them were previously identified in other microorganisms, others were reported as novel compounds. 5.1. Peptides Bioactive peptides have been found in some endophytes of Kennedia nigricans, that is a native Australian plant traditionally used to disinfect skin wounds. In a 2002 study, Castillo et al. [42] reported on the finding of two peptides named munumbicins A and B, produced by the endophyte Streptomyces NRRL 30562. It was later reported that munumbicins A and B were misidentified

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and actually correspond to actinomycin X2 and actinomycin D, respectively [43]. Castillo et al. reported that actinomycin X2 showed activity against Cryptococcus neoformans (MIC of 10 lg/mL), Aspergillus fumigatus (MIC of 20 lg/mL) and Enterococcus faecalis ATCC 51299 (MIC of 8 lg/mL). Ten micrograms (10 lg) of actinomycin X2 inhibited the growth of Neisseria gonorrhoeae 49226 (inhibition zone of 9 mm) and Bacillus anthracis K8902 (inhibition zone of 9.5 mm). Actinomycin D showed activity against methicillin-resistant Staphylococcus aureus ATCC 33591 (MIC of 2.5 lg/mL), multidrug-resistant Mycobacterium tuberculosis MDRP (IC50 of 10 lg/mL) and multidrug-sensitive M. tuberculosis H37Rv ATCC 25618 (IC50 of 46 lg/mL), as well as against C. neoformans (MIC of 10 lg/mL) and Aspergillus fumigatus (MIC of 20 lg/mL). Ten micrograms (10 lg) of actinomycin D also inhibited the growth of Vibrio fischeri PIC 345 (inhibition zone of 16 mm), E. faecalis 29212 (inhibition zone of 18 mm), S. aureus 29213 (inhibition zone of 15 mm), N. gonorrhoeae 49226 (inhibition zone of 14 mm), S. pneumoniae 49619 (inhibition zone of 17 mm) and B. anthracis K8902 (inhibition zone of 18 mm. In the same study [42], two munumbicins named munumbicins C and D, novel peptides never described before were produced also by Streptomyces NRRL 30562. Munumbicin C showed activity against drug-resistant Mycobacterium tuberculosis MDR-P (IC50 > 125 lg/mL) and drug-sensitive M. tuberculosis H37Rv ATCC 25618 (IC50 > 150 lg/mL), Cryptococcus neoformans (MIC of 10 lg/mL), Candida albicans (MIC > 10 lg/mL), Aspergillus fumigatus (MIC of 20 lg/mL), vancomycin-sensitive Staphylococcus aureus MH II Eli Lilly Co. (MIC of 0.4 lg/mL) and vancomycin-resistant ciprofloxacin-sensitive Enterococcus faecalis ATCC 51299 (MIC of 16 lg/mL). Ten micrograms (10 lg) of munumbicin C inhibited the growth of Vibrio fischeri PIC 345 (inhibition zone of 9 mm), Neisseria gonorrhoeae 49226 (inhibition zone of 8 mm) and Streptococcus pneumoniae 49619 (inhibition zone of 7 mm). Munumbicin D showed activity against C. neoformans (MIC of 10 lg/mL), C. albicans (MIC > 10 lg/mL), A. fumigatus (MIC of 20 lg/mL), S. aureus MH II Eli Lilly Co. (MIC of 0.4 lg/mL) and E. faecalis ATCC 51299 (MIC of 16 lg/mL). Ten micrograms (10 lg) of munumbicin D inhibited the growth of V. fischeri PIC 345 (inhibition zone of 12 mm), E. faecalis 29212 (inhibition zone of 16 mm), S. aureus 29213 (inhibition zone of 13 mm), N. gonorrhoeae 49226 (inhibition zone of 9 mm) and S. pneumoniae 49619 (inhibition zone of 16 mm). In addition, both munumbicins C and D had particularly good activity against Plasmodium falciparum (IC50 6.5 ng/mL for C and 4.5 ng/mL for D). Munumbicin D was reported to be more effective than chloroquine, the gold-standard antimalarial drug because the IC50 of munumbicin D was about 50% below that of chloroquine. It is important to mention that munumbicins C and D did not cause lysis of human red blood cells at concentrations of up to 80 ng/mL; as reported in [42]. To the best of our knowledge, the structures of munumbicins C and D have not been published. Munumbicins are believed to contain aspartic acid or asparagine, glutamic acid or glutamine, proline, threonine, valine, leucine [42]. In 2006, Castillo et al. [43] reported on the discovery of yet another two new munumbicins, named munumbicins E-4 and E-5. The two peptides were produced by Streptomyces NRRL 30562, an endophyte of the plant Kennedia nigricans. Munumbicin E-4 showed activity against Burkholderia thailandensis (MIC of 192 lg/mL), Escherichia coli (MIC of >16 lg/mL), Staphylococcus aureus ATCC 29213 (MIC of 8 lg/mL), S. aureus MRSA 43000 (MIC of 8 lg/mL), S. aureus clinical isolate #1 (MIC of 32 lg/mL), Bacillus subtilis (MIC of 5 lg/mL), Pythium ultimum (MIC of 5 lg/mL), Rhizoctonia solani (MIC of >80 lg/mL) and Plasmodium falciparum (LD50 of 2.94 ± 0.32 lg/mL); as reported in [43]. Munumbicin E-5 showed activity against B. thailandensis (MIC of 256 lg/mL), E. coli (MIC of 16 lg/mL), S. aureus ATCC 29213 (MIC of 4 lg/mL), S. aureus MRSA 43000 (MIC of 16 lg/mL), S. aureus clinical isolate

(MIC of 32 lg/mL), B. subtilis (MIC of 5 lg/mL), P. ultimum (MIC of 5 lg/mL), R. solani (MIC of >80 lg/mL) and P. falciparum (LD50 of 0.50 ± 0.08 lg/mL); as reported in [43]. As a reference, vancomycin showed activity against Burkholderia thailandensis (MIC of >128 lg/mL), Escherichia coli (MIC of 128 lg/mL), Staphylococcus aureus ATCC 29213 (MIC of 2 lg/mL), S. aureus MRSA 43000 (MIC of 2 lg/mL) and S. aureus clinical isolate (MIC of 2 lg/mL). No activity of vancomycin was detected against B. subtilis, P. ultimum, R. solani or P. falciparum; as reported in [43]. To the best of our knowledge, the structures of munumbicins E-4 and E-5 are still unknown [15]. In 2003, Castillo et al. [44] reported on the discovery of a peptide named kakadumycin A produced by Streptomyces NRRL 30566, an endophyte of the plant Grevillea pteridifolia, collected in Australia. Kakadumycin A had activity against the malarial parasite Plasmodium falciparum (IC50 of 7.04 ± 0.12 ng/mL) and against the bacteria Bacillus anthracis 40/BA 100 (MIC of 0.3 ng/mL), B. anthracis 14578 (MIC of 0.55 lg/mL), B. anthracis 28 (MIC of 0.43 lg/mL), B. anthracis 62-8 (MIC of 0.41 lg/mL), Enterococcus faecalis ATCC 29212 (MIC of 0.062 lg/mL), E. faecalis VRE ATCC 51299 (MIC of 0.062 lg/mL), Enterococcus faecium ATCC 49624 (MIC of 0.062 lg/mL), Staphylococcus aureus ATCC 29213 (MIC of 0.25 lg/mL), S. aureus MRSA ATCC 33591 (MIC of 0.5 lg/mL), S. aureus GISA ATCC 700787 (MIC of 0.5 lg/mL), S. aureus ATCC 27734 (MIC of 0.125 lg/mL), Staphylococcus simulans ATCC 11631 (MIC of 0.25 lg/mL), Staphylococcus epidermis ATCC 12228 (MIC of 0.125 lg/mL), Streptococcus pneumoniae ATCC 70674 (MIC of < 0.0325 lg/mL), S. pneumoniae ATCC 70676 (MIC of < 0.0325 lg/mL), Listeria monocytogenes ATCC 19114 (MIC of 0.25 lg/mL), L. monocytogenes ATCC 19115 (MIC of 0.25 lg/mL) and Shigella dysenteriae ATCC 11835 (MIC of 4.0 lg/mL). In general, for most bacteria tested, the MICs for kakadumycin A were lower than MICs required with echinomycin. Kakadumycin A is believed to be structurally similar to echinomycin, a quinoxaline antibiotic. If so, kakadumycin A and echinomycin probably also share their mode of action by inhibiting RNA synthesis after binding to DNA [44]. We have not been able to find any follow up works about kakadumycin A [44] to provide more information about the structure elucidation or further studies to evaluate its potential to become an antimalarial medicine. A complex of peptides named coronamycin were discovered by Ezra et al. [45], produced by the verticillate Streptomyces sp. MSU2110, an endophyte of the plant Monstera sp. collected in Peru. The best bioactivity of coronamycin was observed against Plasmodium falciparum, indicating the potential of coronamycin as an antimalarial drug, especially considering that the IC50 values of coronamycin are comparable to the IC50 values of the gold standard antimalarial drug chloroquine (IC50 of 9 ± 7.3 ng/mL vs. 7 ng/mL) [45]. Coronamycin also showed activity against Pythium ultimum (MIC of 2 lg/mL), Phytophthora cinnamomi (MIC of 16 lg/mL), Aphanomyces cochlioides (MIC of 4 lg/mL), Geotrichum candidum (MIC > 500 lg/mL), Aspergillus fumigatus (MIC > 500 lg/mL), Aspergillus ochraceus (MIC > 500 lg/mL), Fusarium solani (MIC > 500 lg/mL), Rhizoctonia solani (MIC > 500 lg/mL), Cryptococcus neoformans ATCC 32045 (MIC of 4 lg/mL), Candida parapsilopsis ATCC 90018 (MIC > 32 lg/mL), C. parapsilopsis ATCC 22019 (MIC > 32 lg/mL), Candida albicans ATCC 90018 (MIC of 16–32 lg/mL), C. albicans ATCC 24433 (MIC > 32 lg/mL), Saccharomyces cerevisiae ATCC 9763 (MIC > 32 lg/mL), Candida krusei ATCC 6258 (MIC > 32 lg/mL), Candida tropicalis ATCC 750 (MIC > 32 lg/mL); as reported in [45]. As reference, flucytosine showed activity against C. neoformans ATCC 32045 (MIC of 8 lg/mL), C. parapsilopsis ATCC 90018 (MIC of 0.12 lg/mL), C. parapsilopsis ATCC 22019 (MIC of 0.5 lg/mL), C. albicans ATCC 90018 (MIC of 0.5 lg/mL), C. albicans ATCC 24433 (MIC of 1 lg/mL), S. cerevisiae ATCC 9763 (MIC 6 0.06 lg/mL), C. krusei ATCC 6258 (MIC of 16 lg/mL),

E. Martinez-Klimova et al. / Biochemical Pharmacology 134 (2017) 1–17

7

Fig. 1. Chemical structures of epichlicin (1), curvularide B (2), 3-nitropropionic acid (3), phomodione (4), xiamycin A (5), indosespene (6), javanicin (7) and citromycetin (8).

C. tropicalis ATCC 750 (MIC 6 0.06 lg/mL); as reported in [45]. In summary, coronamycin showed lower MIC values than flucytosine against Cryptococcus neoformans ATCC 32045 (MIC of 4 vs. 8 lg/mL). The coronamycin complex of peptides were characterized in work [45]. Coronamycin was named as such because the spanish term ‘‘corona” means crown and the sporophore of the verticillate Streptomyces sp. MSU-2110 resembles a crown. Another interesting property of coronamycin is its reported ability to adhere to glass [45]. In 2007, Seto et al. [46] reported on the discovery of a cyclic peptide named epichlicin (1), produced by Epichloe typhina, an endophyte of the plant Phleum pratense. The structure of epichlicin (1) is shown in Fig. 1. Epichlicin (1) inhibited the spore germination

of Cladosporium phlei (IC50 of 22 nM). Under normal growth conditions, the endophytic isolate E. typhina produced 2 lM of epichlicin (1). Collutellin A, a peptide produced by Colletotrichum dematium, an endophyte of the plant Pteromischum sp. (a genus name not present in NCBI Taxonomy [47]), collected in Costa Rica [40], showed activity against Pythium ultimum (MIC > 100 lg/mL), Trichoderma viride (MIC > 100 lg/mL), Sclerotinia sclerotiorum (MIC of 3.6 lg/mL), Botrytis cinerea (MIC of 3.6 lg/mL), Fusarium solani (MIC of 7.2 lg/mL), Rhizoctonia solani (MIC > 100 lg/mL), Aspergillus fumigatus (MIC of 2.4 lg/mL) and Geotrichum candidum (MIC of 3.6 lg/mL). Collutellin A was found to have chemical and biological similarities to cyclosporin A, a clinically important

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immunosuppressant. As reference, cyclosporin A showed activity against P. ultimum (MIC > 100 lg/mL), T. viride (MIC > 100 lg/mL), S. sclerotiorum (MIC of 0.07 lg/mL), B. cinerea (MIC of 0.07 lg/mL), F. solani (MIC > 100 lg/mL), R. solani (MIC 1.2 lg/mL), A. fumigatus (MIC of 1.2 lg/mL) and G. candidum (MIC > 100 lg/mL); as reported in [48]. In summary, collutellin A showed lower MIC values than the reference cyclosporin A against F. solani (MIC of 7.2 lg/mL vs.>100 lg/mL) and G. candidum (MIC of 3.6 lg/mL vs. >100 lg/mL). In comparison to cyclosporin A, collutellin A showed little or no toxicity towards human blood cells [40]. The discovery of a new hybrid peptide-polyketide named curvularide B (2), was described by Chomcheon et al. [49]. The structure of curvularide B (2) is shown in Fig. 1. Curvularide B (2) was produced by Curvularia geniculata, an endophyte of the plant Catunaregam tomentosa, collected in Thailand, curvularide B (2) showed activity against the fungus Candida albicans ATCC 90028 (inhibition zone of 12.1 mm). The synergic action of curvularide B (2) and fluconazole inhibited the growth of C. albicans almost 2-fold than curvularide B (2) alone. The MIC values that resulted in no visible growth were 1 lg/mL of fluconazole in combination with 16 lg/mL of curvularide B (2). It is important to mention that curvularide B (2) exhibited no cytotoxicity, which highlights its potential to become a fungicide [49]. In 2013, Tejesvi et al. [50] reported on the discovery of a new peptide named trtesin produced by Fusarium tricinctum, an endophyte of the plant Rhododendron tomentosum, collected in Finland. Purified trtesin showed activity against Staphylococcus carnosus, Candida albicans and Candida utilis (MIC of 64 lg/mL) as well as against Fusarium oxysporum (MIC of 100 lg/mL). Trtesin is a peptide of 52 amino acids. The sequence of the trtesin gene was deposited in GenBank under accession number KC466596. It contains six conserved cysteine amino acid residues. The concentrated supernatant of the Fusarium tricinctum culture showed antimicrobial activity when grown in PDB medium but no activity when grown in FG4 medium. The new peptide trtesin was discovered after an analysis of which transcripts were expressed by Fusarium tricinctum when grown in PDB and not expressed when grown in FG4. The result was that one of the transcripts, that was later identified to encode the novel peptide trtesin, was expressed > 100-fold by the strain when grown in PDB, the condition that showed antimicrobial activity [50]. Interestingly, patented transgenic plants have been developed that express a peptide similar to trtesin due to the antifungal properties of this close homolog [50]. 5.2. Other organic compounds The variety of chemical compounds produced by endophytes is widely spread. Therefore, in addition to the peptides described above, hybrid peptides and organic acids can be mentioned among many others. Taechowisan et al. [51] described two compounds: 5,7dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy4-phenylcoumarin, produced by Streptomyces aureofaciens CMUAc130, an endophyte of the plant Zingiber officinale. 5,7-Dime thoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4phenylcoumarin had activity against the fungi Colletotrichum musae and Fusarium oxysporum at MICs of 120 and 150 lg/mL, respectively. In 2005, Chomcheon et al. [52] reported on the compound 3nitropropionic acid (3). The structure of 3-nitropropionic acid (3) is shown in Fig. 1. 3-Nitropropionic acid (3) was produced by Phomopsis sp. an endophyte of medicinal plants like Urobotrya siamensis, Grewia sp., Mesua ferrea, Rhododendron ciliicalyx subsp. lyi (synonym Rhododendron lyi), Tadehagi sp. and Gmelina ‘‘elliptica” (a name not present in the nomenclatural database NCBI Taxonomy [47]); collected in Thailand. 3-Nitropropionic acid (3) showed

activity against Mycobacterium tuberculosis H37Ra (MIC of 3.3 lM) [52]. Ganihigama et al. in 2015 [53] showed that 3-nitropropionic acid (3) had activity against M. tuberculosis H37Rv (MIC 12.5 lg/ mL) and H37Ra (MIC 50 lg/mL) strains [53]. 3-Nitropropionic acid (3) is commercially available but is a potent neurotoxic agent; this compound inhibits the enzyme succinate dehydrogenase, located in the mitochondria [52]. Martinez-Luis et al. [54] reported the discovery of five compounds named preussomerin EG1, palmarumycin CP2, palmarumycin CP17, palmarumycin CP18 and CJ-12,371, produced by Edenia sp., an endophyte of the plant Petrea volubilis. The five compounds inhibited the growth of the parasite Leishmania donovani, with IC50 values of 0.12, 3.93, 1.34, 0.62, and 8.40 lM, respectively. Also, the five compounds showed weak cytotoxicity towards Vero cells, indicating that the compounds have stronger antileishmanial than citotoxic activity. These compounds were inactive when tested against Plasmodium falciparum or Trypanosoma cruzi indicating that they have selective activity against Leishmania parasites. The therapeutic window of these compounds is significant since their antileishmanial activity was 130 or 245 times stronger than their cytotoxic properties [5,54]. A metabolite named phomoenamide reported by Rukachaisirikul et al. [55] was produced by Phomopsis sp., an endophyte of the plant Garcinia dulcis. Phomoenamide showed activity against Mycobacterium tuberculosis H37Ra (MIC of 6.25 lg/mL). In 2008, Aly et al. [5] reported on the finding of two compounds: 3-O-methylalaternin and the tetrahydroanthraquinone altersolanol A, produced by Ampelomyces sp., an endophyte of the medicinal plant Urospermum picroides. U. picroides is eaten throughout the Mediterranean and was collected in Egypt. 3-Omethylalaternin showed activity against the bacteria S. aureus, S. epidermis and E. faecalis at a MIC of 12.5 lg/mL for all three. Altersolanol A showed activity against S. epidermis and E. faecalis at a MIC of 12.5 lg/mL and also against S. aureus at a MIC of 25 lg/mL. Altersolanol A inhibits bacterial growth because it acts as an electron acceptor in the bacterial membrane [56]. Li et al. [57] reported on the discovery of a new compound named pestalotheol C produced by Pestalotiopsis theae, an endophyte of an unknown plant collected in China. Pestalotheol C showed inhibitory activity against the replication of HIV-LAI virus in C8166 cells (IC50 of 16.1 lM). The discovery of a furandione named phomodione (4), produced by Phoma sp., was reported by Hoffman et al. [58]. The structure of phomodione (4) is shown in Fig. 1. Phoma sp. was an endophyte of the plant Saurauia scaberrinae, collected in Papua New Guinea. Phomodione (4) showed activity against S. aureus (MIC of 1.6 lg/mL), P. ultimum (4–5 lg/mL), S. sclerotiorum (3–5 lg/mL) and R. solani (5–8 lg/mL). As reference, the activity of phomodione (4) was compared to the activity of cercosporamide due to structure similarity. Cercosporamide is another natural product biosynthesized by the Phoma sp. endophytic isolate. Cercosporamide showed activity against S. aureus (MIC of 2 lg/mL), P. ultimum (3–4 lg/mL), S. sclerotiorum (5–8 lg/mL) and R. solani (8–10 lg/mL). In 2009, Gu et al. [59] reported on the compounds altechromone A and herbarin A produced by Alternaria brassicicola ML-P08, an endophyte of the plant Malus halliana, collected in China. Altechromone A showed activity against B. subtilis (MIC of 3.9 lg/mL), E. coli (MIC of 3.9 lg/mL), Pseudomonas fluorescens (MIC of 1.8 lg/mL) and C. albicans (MIC of 3.9 lg/mL). Herbarin A showed activity against Trichophyton rubrum (MIC of 15.6 lg/mL) and C. albicans (MIC of 15.6 lg/mL). In 2010, Lim et al. [60] reported on the compounds dicerandrol A, dicerandrol B, dicerandrol C, deacetylphomoxanthone B and fusaristatin A. The five compounds were produced by Phomopsis longicolla S1B4, an endophyte of an unspecified plant, collected in

E. Martinez-Klimova et al. / Biochemical Pharmacology 134 (2017) 1–17

South Korea. Dicerandrol A, dicerandrol B, dicerandrol C, deacetylphomoxanthone B and fusaristatin A showed activity against Xanthomonas oryzae KACC 10331 (MICs of 8, 16,>16, 4 and 128 lg/mL, respectively). Dicerandrol A also showed activity against S. aureus KCTC 1916 (MIC of 0.25 lg/mL), B. subtilis KCTC 1021 (MIC of 0.125 lg/mL), Clavibacter michiganesis KACC 20122 (MIC of 1 lg/mL), Xanthomonas oryzae KACC 10859, 10874, 10875, 10876, 10882, 10884 and 10885 (MICs 2–64 lg/mL), E. coli KCTC 1924 (MIC > 128 lg/mL), Pseudomonas syringae KACC 10134 (MIC > 128 lg/mL), Salmonella enterica KACC 10763 (MIC > 128 lg/mL), Erwinia amylovora KACC 10060 (MIC of 32 lg/mL), Ralstonia solanacearum KACC 10149 (MIC > 128 lg/mL), Candida albicans KCTC 7965 (MIC of 2 lg/mL) and Aspergillus oryzae KCTC 6377 (MIC > 128 lg/mL). For comparison purposes, the MIC values obtained against the previously listed test strains with neomycin, kanamycin, ampicillin, DAPG and amphotericin B ranged between 0.25–128 lg/mL; as reported in [60]. Two alkaloids classified as multicyclic indolosesquiterpenes: a known one named xiamycin A (5) and a new one named indosespene (6) were reported by Ding et al. [61]. The structures of xiamycin A (5) and indosespene (6) are shown in Fig. 1. The two compounds were produced by Streptomyces sp. HKI0595, an endophyte of the mangrove tree Kandelia candel, collected in China. Xiamycin A (5) showed selective anti-HIV activity as well as activity against P. aeruginosa, B. subtilis, Mycobacterium vaccae, S. aureus, methicillin-resistant S. aureus and vancomycin-resistant E. faecalis; whereas indosespene (6) showed activity against S. aureus, B. subtilis, M. vaccae, methicillin-resistant S. aureus and vancomycin-resistant E. faecalis. Indolosesquiterpenes are a group of alkaloids also found in plants [61]. In 2011, Sun et al. [62] reported on three new compounds named massarigenin D, spiromassaritone and paecilospirone. The compounds contain a spiro-5,6-lactone ring skeleton. The compounds were produced by Massrison sp. (a genus name not present in NCBI Taxonomy [47]), an endophyte of the medicinal plant Rehmannia glutinosa, collected in China. Massarigenin D showed activity against Cryptococcus neoformans (MIC of 16 lg/mL) and Trichophyton rubrum (MIC of 2 lg/mL). Spiromassaritone showed activity against Candida albicans (MIC of 2 lg/mL), C. neoformans (MIC of 4 lg/mL), Trichophyton rubrum (MIC of 0.25 lg/mL) and Aspergillus fumigatus (MIC of 1 lg/mL). Paecilospirone showed activity against C. albicans (MIC of 8 lg/mL), C. neoformans (MIC of 16 lg/mL), T. rubrum (MIC of 2 lg/mL) and A. fumigatus (MIC of 4 lg/mL). In 2011, Sonaimuthu et al. [63] reported on the compound tenuazonic acid produced by Alternaria alternata, an endophyte of the medicinal plant Indigofera ‘‘enneaphylla” (a name not present in NCBI Taxonomy [47]), collected in India. Tenuazonic acid showed activity against Mycobacterium tuberculosis H37Rv (MIC of 250 lg/mL). In the same year, Inahashi et al. [64] reported on the discovery of two new alkaloids of the pyochelin family, named spoxazomicins A and B. The two compounds were produced by Streptosporangium oxazolinicum sp. nov. strain K07-0460 [65], an endophyte of an orchid which scientific or common name was not specified, collected in Japan. Spoxazomicins A showed strong antitrypanosomal activity against Trypanosoma brucei brucei strain GUTat 3.1, 14–21 times more potent than the clinically used antitrypanosomal drugs suramin and eflornithine (IC50 of 0.11 lg/mL). Spoxazomicins B also showed strong antitrypanosomal activity against Trypanosoma brucei brucei strain GUTat 3.1 (IC50 of 0.55 lg/mL). In 2012, Johann et al. [66] reported on the biphenyl derivative compound altenusin, produced by the endophyte Alternaria sp. Altenusin showed activity against Paracoccidioides brasiliensis strains (MIC of 1.9–31.2 lg/mL) and Schizosaccharomyces pombe

9

(MIC of 62.5 lg/mL). Altenusin was 50-fold more active against P. brasiliensis than the reference trimethroprim/sulfamethoxazole compounds. It was suggested that altenusin possibly affects cell wall synthesis or assembly [66]. In 2013, Wijeratne et al. [67] reported on the discovery of three new alkaloid compounds named phomapyrrolidones A, B and C. Phomapyrrolidones A, B and C were produced by Phoma sp. NRRL 46751, an endophyte of the plant Saurauia scaberrinae, collected in Papua New Guinea. We searched the species name ‘‘Saurauia scaberrinae” in the ‘‘NCBI Taxonomy” nomenclature database [47], but it does not seem to be an accepted species name. Phomapyrrolidones A, B and C showed activity against Mycobacterium tuberculosis H37Pv (MICs of 5.2–41.1 lg/mL). In 2015, Forcina et al. 2015 [68] reported on the discovery of two new compounds classified as sesquiterpene-polyol conjugates named stelliosphaerols A and B, produced by Stelliosphaera formicum n. gen., n. sp., an endophyte of the plant Duroia hirsuta, collected in Ecuador. D. hirsuta is a tree species that also has a symbiosis with ants that kill other plants using formic acid as an herbicide [69]. Stelliosphaerols A and B showed activity against S. aureus (approximate MIC values of 250 lg/mL). Stelliophaerols A and B are structurally related to chalmicrin, a compound from Chalara microspora (Ascomycota, Pleosporales) [68]. The closest relative of Stelliosphaera formicum n. gen. n. sp. is Splanchnonema platani, a plant-pathogenic fungus. In 2015, Zhang et al. [70] reported on three compounds: a new compound classified as a sesquiterpenoid ether named fusartricin and the two known compounds fusarielin B and enniatin B. The three compounds were produced by Fusarium tricinctum Salicorn 19, an endophyte of the plant Salicornia bigelovii, collected in China. Fusartricin showed activity against Enterobacter aerogenes, Micrococcus tetragenus ATCC 35098 and C. albicans CMCC 98001 (MICs of 19, 19 and 19 lM, respectively). Fusarielin B showed activity against Mycobacterium smegmatis CMCC 93321, B. subtilis ATCC 6633, Mycobacterium phlei AS 4.1180 and E. coli ATCC 25922 (MICs of 19, 19, 10 and 10 lM, respectively). Enniatin B showed activity against B. subtilis ATCC 6633, E. aerogenes and Micrococcus tetragenus ATCC 35098 with MIC values 13, 13 and 6 lM, respectively. The structure of fusartricin was highlighted to be extremely rare by the authors. In the same year, Shiono et al. [71] reported on the discovery of four new sesquiterpene compounds named phomadecalin F, 8a-monoacetoxyphomadecalin D, 3-epi-phomadecalin D, and 13-hydroxylmacrophorin A. The four new compounds were produced by Microdiplodia sp., an endophyte of an oak tree [Quercus sp.], collected in Japan. The four compounds showed activity against Pseudomonas aeruginosa ATCC 15442 and S. aureus NBRC 13276, yielding inhibition zones of 8–15 mm using 50 lg of the compounds [71]. Eight new polyketides named koningiopisins A-H were reported by Liu et al. [72]. The compounds were produced by Trichoderma koningiopsis YIM PH 30 002, an endophyte of the plant Panax notoginseng, collected in China. A mixture of koningiopisins D-H, in combination with the known polyketide trichodermaketone C (also produced by the endophytic isolate), showed activity against Plectosphaerella cucumerina (MIC of 16 lg/mL). Koningiopisin C on its own showed strong activity against P. cucumerina (MIC of 16 lg/mL), Fusarium solani (MIC of 32 lg/mL) and Fusarium oxysporum (MIC of 32 lg/mL). Koningiopisin B showed activity against Alternaria panax (MIC 64 lg/mL). As a reference, nystatin showed activity against P. cucumerina (MIC of 4 lg/mL), F. solani (MIC of 16 lg/mL), A. panax (MIC of 8 lg/mL) and F. oxysporum (MIC of 8 lg/mL). Kanamycin showed activity against S. aureus (MIC of 8 lg/mL) and [Acinetobacter] baumannii (MIC of 16 lg/mL). Ibrahim et al. [73] reported on the discovery of a new butyrolactone named aspernolide F produced by Aspergillus terreus, an

Name of source plant

Name of endophyte

Compound

Activity observed against test strains

Reference

Carthamus lanatus

Aspergillus terreus

(22E,24R)-stigmasta-5,7,22trien-3-b-ol

[73]

Urobotrya siamensis, Grewia sp., Mesua ferrea, Rhododendron ciliicalyx subsp. lyi, Tadehagi sp. and Gmelina ‘‘elliptica” Urospermum picroides Zingiber officinale

Phomopsis sp.

3-nitropropionic acid (3)

Antifungal: C. neoformans (IC50 of 4.38 lg/mL) Antibacterial: S. aureus (IC50 of 0. 94–11.7 lg/mL) Antiprotozoal: P. falciparum (IC50 of 751.8–997.7 lg/mL) and L. donovani (IC50 of 4.61 lg/mL) Antibacterial: M. tuberculosis (MIC of 3.3 lM) and M. tuberculosis (MIC 12.5–50 lg/mL)

Ampelomyces sp. Streptomyces aureofaciens

Antibacterial: S. aureus, S. epidermis and E. faecalis (MICs of 12.5 lg/mL) Antifungal: Colletotrichum musae and Fusarium oxysporum (MICs of 120 and 150 lg/mL, respectively)

[56] [51]

Urospermum picroides Carthamus lanatus

Ampelomyces sp. Aspergillus terreus

Altersolanol A Aspernolide F

Pteromischum sp.

Colletotrichum dematium

Collutellin A

Monstera sp.

Streptomyces sp.

Coronamycin

Catunaregam tomentosa

Curvularia geniculata Phomopsis longicolla

Curvularide B (2)

Antibacterial: B. subtilis (MIC of 3.9 lg/mL), E. coli (MIC of 3.9 lg/mL), Pseudomonas fluorescens (MIC of 1.8 lg/mL) Antifungal: C. albicans (MIC of 3.9–15.6 lg/mL), Trichophyton rubrum (MIC of 15.6 lg/mL) Antifungal: Paracoccidioides brasiliensis strains (MIC of 1.9–31.2 lg/mL) and Schizosaccharomyces pombe (MIC of 62.5 lg/mL) Antibacterial: S. epidermis, E. faecalis (MICs of 12.5 lg/mL) and S. aureus (MIC of 25 lg/mL) Antifungal: C. neoformans (IC50 of 5.19 lg/mL) Antibacterial: S. aureus (IC50 of 6.39–7.49 lg/mL) (IC50 of lg/mL) Antiprotozoal: P. falciparum (IC50 of 1938.3–4109 lg/mL) and Leishmania donovani (IC50 of 36.8 lg/mL) Antifungal: Pythium ultimum (MIC > 100 lg/mL), Trichoderma viride (MIC > 100 lg/mL), Sclerotinia sclerotiorum (MIC of 3.6 lg/mL), Botrytis cinerea (MIC of 3.6 lg/mL), Fusarium solani (MIC of 7.2 lg/mL), Rhizoctonia solani (MIC > 100 lg/mL), Aspergillus fumigatus (MIC of 2.4 lg/mL) and Geotrichum candidum (MIC of 3.6 lg/mL). Antiprotozoal: Plasmodium falciparum (IC50 of 9 ± 7.3 ng/mL) Antifungal: Pythium ultimum (MIC of 2 lg/mL), Phytophthora cinnamomi (MIC of 16 lg/mL), Aphanomyces cochlioides (MIC of 4 lg/mL), Geotrichum candidum (MIC > 500 lg/mL), Aspergillus fumigatus (MIC > 500 lg/mL), Aspergillus ochraceus (MIC > 500 lg/mL), Fusarium solani (MIC > 500 lg/mL), Rhizoctonia solani (MIC > 500 lg/mL), Cryptococcus neoformans ATCC 32045 (MIC of 4 lg/mL), Candida parapsilopsis ATCC 90018 (MIC > 32 lg/mL), Candida albicans ATCC 90018 (MIC of 16–32 lg/mL), Saccharomyces cerevisiae ATCC 9763 (MIC > 32 lg/mL), Candida parapsilopsis ATCC 22019 (MIC > 32 lg/mL), Candida albicans ATCC 24433 (MIC > 32 lg/mL), Candida krusei ATCC 6258 (MIC > 32 lg/mL), Candida tropicalis ATCC 750 (MIC > 32 lg/mL) Antifungal: Candida albicans (MIC not specified).

[59]

Trixis vauthieri

Alternaria brassicicola Alternaria sp.

3-O-methylalaternin 5,7-dimethoxy-4-pmethoxylphenylcoumarin and 5,7-dimethoxy-4phenylcoumarin Altechromone A and herbarin A Altenusin

Malus halliana

Oryza sativa

Streptomyces sp.

Salicornia bigelovii

Fusarium tricinctum Epichloe typhina Fusarium tricinctum Fusarium tricinctum Streptomyces sp. Fusarium sp.

Phleum pratense Salicornia bigelovii Salicornia bigelovii Kandelia candel Tradescantia spathacea

[52] [53]

[66] [56] [73]

[48]

[45]

[49]

Antibacterial: Xanthomonas oryzae (MICs of 8, 16, >16, 4 and 128 lg/mL, respectively), S. aureus (MIC of 0.25 lg/mL), B. subtilis (MIC of 0.125 lg/mL), Clavibacter michiganesis (MIC of 1 lg/mL), E. coli (MIC > 128 lg/mL), Pseudomonas syringae (MIC > 128 lg/mL), Salmonella enterica (MIC > 128 lg/mL), Erwinia amylovora (MIC of 32 lg/mL), Ralstonia solanacearum (MIC > 128 lg/mL) Antifungal: C. albicans (MIC of 2 lg/mL) and Aspergillus oryzae (MIC > 128 lg/mL) Antiprotozoal: Plasmodium falciparum (IC50 of 1.40–5.23 lg/mL).

[74]

Antibacterial: B. subtilis, E. aerogenes and Micrococcus tetragenus (MICs of 13, 13 and 6 lM, respectively).

[70] [46] [70]

Fusartricin

Antifungal: Cladosporium phlei (IC50 of 22 nM) Antibacterial: Mycobacterium smegmatis, B. subtilis, Mycobacterium phlei and E. coli (MICs of 19, 19, 10 and 10 lM, respectively). Antibacterial: Enterobacter aerogenes, Micrococcus tetragenus (MICs of 19 lM).Antifungal: C. albicans (MIC of 19 lM)

Indosespene (6) Javanicin (7)

Antibacterial: S. aureus, B. subtilis, M. vaccae and E. faecalis (MICs not specified). Antibacterial: Mycobacterium tuberculosis (MIC 25 lg/mL) and Mycobacterium phlei (50 lg/mL)

[61] [38]

Dicerandrol A, dicerandrol B, dicerandrol C, deacetylphomoxanthone B and fusaristatin A Efomycins M and G, oxohygrolidin, abierixin and 29-O-methylabierixin Enniatin B Epichlicin (1) Fusarielin B

[60]

[70]

E. Martinez-Klimova et al. / Biochemical Pharmacology 134 (2017) 1–17

Unspecified

10

Table 2 Summary of bioactive compounds obtained from endophytes, showing names of compound arranged alphabetically, endophyte of precedence and source plant, as well as activity observed against selected test strains.

Table 2 (continued) Name of source plant

Name of endophyte

Compound

Activity observed against test strains

Reference

Grevillea pteridifolia Panax notoginseng

Streptomyces sp. Trichoderma koningiopsis

Kakadumycin A Koningiopisins A-H

[44] [72]

Rehmannia glutinosa

Massrison sp.

Kennedia nigricans

Streptomyces sp.

Massarigenin D, spiromassaritone and paecilospirone Munumbicin C

Antiprotozoal: Plasmodium falciparum (IC50 of 7.04 ± 0.12 ng/mL) Antibacterial: Bacillus anthracis (MIC of 0.3–0.55 ng/mL), Enterococcus faecalis (MIC of 0.062 lg/mL), Enterococcus faecium (MIC of 0.062 lg/mL), Staphylococcus aureus (MIC of 0.125–0.5 lg/mL), Staphylococcus simulans (MIC of 0.25 lg/mL), Staphylococcus epidermis (MIC of 0.125 lg/mL), Streptococcus pneumoniae (MIC of < 0.0325 lg/mL), Listeria monocytogenes (MIC of 0.25 lg/mL) and Shigella dysenteriae (MIC of 4.0 lg/mL) Antifungal: Plectosphaerella cucumerina (MIC of 16 lg/mL), Fusarium solani (MIC of 32 lg/mL) and Fusarium oxysporum (MIC of 32 lg/mL), Alternaria panax (MIC 64 lg/mL) Antifungal: Cryptococcus neoformans (MIC of 16 lg/mL), Trichophyton rubrum (MIC of 0.25–2 lg/mL), C. albicans (MIC of 2–8 lg/mL), C. neoformans (MIC of 4–16 lg/mL), Aspergillus fumigatus (MIC of 1–4 lg/mL)

[42]

Kennedia nigricans

Streptomyces sp.

Munumbicin D

Kennedia nigricans

Streptomyces sp.

Munumbicin E-4

Kennedia nigricans

Streptomyces sp.

Munumbicin E-5

Garcinia nobilis

Penicillium sp.

Unspecified Quercus sp.

Pestalotiopsis theae Microdiplodia sp.

Antiviral: HIV-LAI virus in C8166 cells (IC of 16.1 lM) Antibacterial: P. aeruginosa and S. aureus (MICs not specified)

[57] [71]

Saurauia scaberrinae

Phoma sp.

Antibacterial: M. tuberculosis (MICs of 5.2–41.1 lg/mL)

[67]

Saurauia scaberrinae

Phoma sp.

Penialidin C and citromycetin (8) Pestalotheol C Phomadecalin F, 8amonoacetoxyphomadecalin D, 3-epi-phomadecalin D, and 13hydroxylmacrophorin A. Phomapyrrolidones A, B and C Phomodione (4)

Antiprotozoal: Plasmodium falciparum (IC50 6.5 ng/mL) Antibacterial: Mycobacterium tuberculosis (IC50 > 125–150 lg/mL) S. aureus (MIC of 0.4 lg/mL) and Enterococcus faecalis (MIC of 16 lg/mL) Antifungal: Candida albicans (MIC > 10 lg/mL), Aspergillus fumigatus (MIC of 20 lg/mL) and Cryptococcus neoformans (MIC of 10 lg/mL) Antiprotozoal: Plasmodium falciparum (IC50 4.5 ng/mL) Antifungal: Cryptococcus neoformans (MIC of 10 lg/mL), Candida albicans (MIC > 10 lg/mL), Aspergillus fumigatus (MIC of 20 lg/mL), S. aureus (MIC of 0.4 lg/mL) and Enterococcus faecalis (MIC of 16 lg/mL) Antiprotozoal: Plasmodium falciparum (LD50 of 2.94 ± 0.32 lg/mL) Antifungal: Burkholderia thailandensis (MIC of 192 lg/mL), Pythium ultimum (MIC of 5 lg/mL), Rhizoctonia solani (MIC of >80 lg/mL) Antibacterial: E. coli (MIC of >16 lg/mL), S. aureus (MIC of 8–32 lg/mL), B. subtilis (MIC of 5 lg/mL) Antiprotozoal: P. falciparum (LD50 of 0.50 ± 0.08 lg/mL) Antifungal: B. thailandensis (MIC of 256 lg/mL), P. ultimum (MIC of 5 lg/mL) and R. solani (MIC of >80 lg/mL) Antibacterial: E. coli (MIC of 16 lg/mL), S. aureus (MIC of 4–32 lg/mL), B. subtilis (MIC of 5 lg/mL) Antibacterial: Mycobacterium smegmatis (MICs of 15.6 lg/mL and 31.2 lg/mL, respectively)

[58]

Garcinia dulcis Petrea volubilis

Phomopsis sp. Edenia sp.

Antibacterial: S. aureus (MIC of 1.6 lg/mL) Antifungal: P. ultimum (4–5 lg/mL), S. sclerotiorum (3–5 lg/mL) and R. solani (5–8 lg/mL) Antibacterial: M. tuberculosis (MIC of 6.25 lg/mL) Antiprotozoal: Leishmania donovani (IC50 values of 0.12, 3.93, 1.34, 0.62, and 8.40 lM, respectively)

[55] [54]

Unspecified orchid

Streptosporangium oxazolinicum Stelliosphaera formicum Alternaria alternata Fusarium tricinctum

Antiprotozoal: Trypanosoma brucei brucei (IC50 of 0.11–0.55 lg/mL)

[64]

Stelliosphaerols A and B

Antibacterial: S. aureus (MICs of 250 lg/mL)

[68]

Tenuazonic acid Trtesin

Antibacterial: Mycobacterium tuberculosis H37Rv (MIC of 250 lg/mL) Antibacterial: Staphylococcus carnosus (MIC of 64 lg/mL) Antifungal: Candida albicans and Candida utilis (MICs of 64 lg/mL) as well as against Fusarium oxysporum (MIC of 100 lg/mL) Antiviral: anti-HIV Antibacterial: P. aeruginosa, B. subtilis, Mycobacterium vaccae, S. aureus and E. faecalis (MICs not specified)

[63] [50]

Indigofera ‘‘enneaphylla” Rhododendron tomentosum

Kandelia candel

Streptomyces sp.

Xiamycin A (5)

[42]

[43]

[43]

[75]

E. Martinez-Klimova et al. / Biochemical Pharmacology 134 (2017) 1–17

Duroia hirsuta

Phomoenamide Preussomerin EG1, palmarumycin CP2, palmarumycin CP17, palmarumycin CP18 and CJ12,371 Spoxazomicins A and B

[62]

[61]

11

12

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endophyte of the plant Carthamus lanatus, collected in Egypt. Aspernolide F showed activity against C. neoformans ATCC 90113 (IC50 of 5.19 lg/mL), S. aureus ATCC 2921 (IC50 of 7.49 lg/mL) and methicillin-resistant S. aureus ATCC 33591 (IC50 of 6.39 lg/ mL), chloroquine-sensitive P. falciparum D6 (Sierraleon) clone (IC50 of 1938.3 lg/mL), chloroquine-resistant P. falciparum (IndoChina) clone (IC50 of 4109 lg/mL) and Leishmania donovani (IC50 of 36.8 lg/mL). The endophyte A. terreus also produced the stigmasterol derivative (22E,24R)-stigmasta-5,7,22-trien-3-b-ol, which showed activity against C. neoformans ATCC 90113 (IC50 of 4.38 lg/mL), S. aureus ATCC 2921 (IC50 of 11.7 lg/mL) and methicillin-resistant S. aureus ATCC 33591 (IC50 of 0. 94 lg/mL), chloroquine-sensitive P. falciparum D6 (Sierraleon) clone (IC50 of 751.8 lg/mL), chloroquine-resistant P. falciparum D2 (Indo-China) clone (IC50 of 997.7 lg/mL) and L. donovani (IC50 of 4.61 lg/mL). In 2016, Alvin et al. [38] reported on the discovery of a polyketide named javanicin (7), produced by a Fusarium sp., an endophyte of the medicinal plant Tradescantia spathacea (synonym: Rhoeo spathacea) traditionally used against respiratory disease; collected in Indonesia. The structure of javanicin (7) is shown in Fig. 1. Javanicin (7) showed strong activity against Mycobacterium tuberculosis H37Ra ATCC 25177 (MIC 25 lg/mL) and Mycobacterium phlei ATCC 11758 (50 lg/mL) [38]. The crude extract of the endophytic isolate showed no activity against Mycobacterium

avium or Mycobacterium smegmatis [38], suggesting the possibility of target specificity. However, our understanding is that the bioactivity test against M. avium or M. smegmatis was carried out exclusively with the crude extract of the fermentation broth of the endophytic isolate Fusarium sp. and not with purified javanicin (7). The biosynthetic route for javanicin (7) has not yet been elucidated, but is thought to be a PKS pathway [38]. Supong et al. [74] reported on five compounds: three macrolides, efomycins M, G and oxohygrolidin, along with two polyethers, abierixin and 29-O-methylabierixin. The compounds were produced by Streptomyces sp. BCC72023, an endophyte of the rice plant Oryza sativa, collected in Thailand. All five compounds showed activity against Plasmodium falciparum, with IC50 values ranging from 1.40 to 5.23 lg/mL. Efomycin G, oxohygrolidin and 29-O-methylabierixin also showed activity against Mycobacterium tuberculosis H37Ra (MICs of 12.0–50.0 lg/mL). Efomycin G showed strong activity against Bacillus cereus (MIC of 3.13 lg/mL). 29-O-methylabierixin showed activity against Colletotrichum capsici (MIC of 25 lg/mL). Finally, Jouda et al. [75] reported on the compounds penialidin C and citromycetin (8) produced by Penicillium sp., an endophyte of the plant Garcinia ‘‘nobilis” (a name not present in NCBI Taxonomy [47]), collected in Cameroon. The structure of citromycetin (8) is shown in Fig. 1. Penialidin C and citromycetin (8) showed

Table 3 Taxonomic classification of endophytes. The endophytic microorganisms that were isolated from plants and identified up to genus and/or species level in the above reports have been listed. The scientific name of the endophyte was searched in the ‘‘NCBI Taxonomy” database [47] to list the hierarchical classification of each genus. Genus

Superkingdom

Kingdom

Phylum

Class

Reference

Micromonospora Nocardia Nocardiopsis Pseudonocardia Streptomyces Streptosporangium Chryseobacterium Bacillus Paenibacillus Pseudomonas Alternaria Ampelomyces Botryosphaeria Cladosporium Cochliobolus Curvularia Edenia Guignardia Macrophomina Microdiplodia Mycosphaerella Phoma Stelliosphaera Aspergillus Penicillium Acremonium Aschersonia Chaetomium Colletotrichum Diaporthe Epichloe Fusarium Gibberella Hypoxylon Khuskia Microdochium Pestalotiopsis Phaeoacremonium Phomopsis Trichoderma Xylaria Bjerkandera Mortierella Rhizopus

Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota Eukaryota

– – – – – – – – – – Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi

Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Bacteroidetes Firmicutes Firmicutes Proteobacteria Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Basidiomycota incertae sedis incertae sedis

Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Flavobacteriia Bacilli Bacilli Gammaproteobacteria Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Eurotiomycetes Eurotiomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Agaricomycetes incertae sedis incertae sedis

[33] [19,40] [33] [40] [42,44,45,51,43,61,74,19,33,41] [64,19] [34] [27,34] [27] [27,34] [59,63,66,8,39] [56] [32] [27,8] [27] [49,8] [54] [38] [36] [71] [27] [58,67,27,37,38] [68] [73,35,8] [75,35,39] [3] [32] [31] [48,30,38] [29,3,39] [46] [50,70,38,31,27,32] [35] [3] [35] [38] [57,3,27] [31] [52,55,60,3,38] [72] [3,39] [39] [26] [32]

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activity against Mycobacterium smegmatis (MICs of 15.6 lg/mL and 31.2 lg/mL, respectively). Table 2, includes a summary of the identified compounds included in this review chapter purified from extracted fermentation broths of cultured endophytes.

6. The biosynthetic potential of endophytes Janso and Carter [4] in 2010 were able to isolate endophytes from plants native to tropical Papua New Guinea and Mborokua Island (Solomon Islands). The number of strains isolated was 105, which were classified into 17 genera based on their 16S rRNA sequences, some of which were proposed as new genera. Almost half of the extracts from the isolated endophytes exhibited bioactivity (measured as growth inhibition) against methicillinsensitive Staphylococcus aureus, C. albicans and/or E. coli. In addition, Janso and Carter [4] observed that, even though some extracts did not display any evident biological activity, the chemical profiles obtained by LC-MS-DAD-ELSD showed signals characteristic of secondary metabolites. The actinomycete families that produced the highest amount of secondary metabolites were Pseudonocardiaceae and Streptomycetaceae. In works [4,24] PCR amplification detection tests revealed putative type I and II polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes within the genomic sequences of the isolates, suggesting that the endophytic isolates screened have significant potential for the biosynthesis of secondary metabolites Janso and Carter [4] suggested that it is possible that horizontal gene transfer occurs between the cells of a plant and the microorganisms that inhabit the plant. It has been under debate by the scientific community whether horizontal gene transfer and/or other processes of exchange of genetic material are responsible for phenotypes where the endophytic microbe produces compounds that are attributed to the plant. However, as Janso and Carter pointed out [4]: processes such as hybridization and shuffling of genes are indeed exciting possibilities for the creation of novel metabolic products.

7. Taxonomic classification of endophytes Table 3 contains a list the taxonomic classification of the microorganisms that were isolated as endophytes from the inner tissues of plants, as reported above. Only the endophytes that produced metabolites that had antibacterial, antifungal and antiparasitic activity were included in this list. Fig. 2 shows the frequency of each phylum within the list presented in Table 3. It is possible to observe that the majority of endophytes collected are eukaryotes, belonging to kingdom Fungi, phylum Ascomycota; followed by Bacteria from phylum Actinobacteria. Streptomyces, Alternaria, Phoma, Fusarium and Phomopsis were the genera most commonly found as endophytes of a wide variety of plants.

8. Taxonomic classification of plants containing endophytes Table 4 contains a list of the scientific names of the plants from which endophytes were isolated, as reported in the sections above. Only the plants containing endophytes that produced metabolites that had antibacterial, antifungal and antiparasitic activity were included in this list. Fig. 3 shows the frequency of each plant family within the list presented in Table 4. In total, the plants correspond to 34 plant families. It is possible to observe that the plant families with most representatives are Araceae, Asteraceae, Fabaceae, Lamiaceae, Meliaceae, Rhizophoraceae and Rubiaceae.

Fig. 2. Frequency of phyla of endophytes. Outer circle represents the frequency of each phylum of the endophytes listed in Table 3. Inner circle indicates whether the phyla belong to superkingdom Bacteria or to kingdom Fungi. Numbers indicate the number of genera listed within each phylum.

9. Global localization of plant collection sites Table 4 contains a list of the countries where plants containing endophytes were collected from. Fig. 4 shows a map of approximate locations estimated using ‘‘Google Maps” [76] Ó 2016 Google, based on the information on the collection sites provided in the literature.

10. Concluding remarks Endophytes are microorganisms that inhabit the internal tissues of plants without causing visible harm to the plant, at least during the endophytic phase of the life cycle of these microorganisms. Both endophytes and plants seem to benefit from the association [15] but, the role of endophytes inside their plant hosts requires further clarification [15]. Endophytes have evolved mechanisms that allow them to compete with other microorganisms for the microhabitat inside plants. Therefore, endophytic microorganisms are a good place to search for antimicrobial and antifungal agents. Endophytes have been studied for their ability to produce antibacterial, antiviral, anticancer, antioxidant, antidiabetic and immunosuppressive compounds. This review demonstrates that metabolites with activity against bacteria, fungi and protozoa have been obtained from endophytic microorganisms. Compounds such as peptides, hybrid peptides, polyketides, alkaloids, are examples of different metabolites that have been found in endophytes, some of them are novel while some are known. A potential advantage of researching endophytes, as suggested by [19] is that new microbial lifestyles might increase the likelihood of finding novel bioactive compounds. Endophytes that produce antimicrobial and antifungal agents may be implemented also in agriculture as an environmentally-friendly, selfpropagating pest control. Scientific discoveries contribute to enhance the value of biodiversity: new bioactive drugs and new microorganisms are waiting to be discovered. Important ethnobotanical medicinal knowledge

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Table 4 Taxonomic classification of endophyte-containing plants. The identified plants from which endophytes were isolated are listed. The scientific name of the plant was searched in the NCBI Taxonomy database to annotate the family the plant is classified as. The country where the plant was collected is listed together with the reference. Plant

Family

Country

Reference

Avicennia alba Avicennia officinalis Saurauia ‘‘scaberrinae” Saurauia ‘‘scaberrinae” Monstera sp. Pinellia pedatisecta Pinellia pedatisecta Pinellia ternata Pinellia ternata Panax notoginseng Ageratum conyzoides Arctium lappa Carthamus lanatus Sonchus oleraceus Urospermum picroides Viguiera arenaria Mesua ferrea Salicornia bigelovii Garcinia dulcis Garcinia nobilis Lumnitzera littorea Tradescantia spathacea Camptotheca acuminata Rhododendron ciliicalyx subsp. lyi (synonym Rhododendron lyi) Rhododendron tomentosum Bauhinia forficata Bauhinia forficata Indigofera ‘‘enneaphylla” Kennedia nigricans Kennedia nigricans Tadehagi sp. Belamcanda chinensis Gmelina elliptica Leonurus heterophyllus Leonurus heterophyllus Mentha arvensis Ocimum sanctum Vitex negundo Sonneratia caseolaris Azadirachta indica Xylocarpus granatum Xylocarpus moluccensis Urobotrya siamensis Bletilla striata Bletilla striata Digitalis purpurea Digitalis purpurea Oryza sativa Polygonum cuspidatum Aegiceras corniculatum Lysimachia nummularia Grevillea pteridifolia Rehmannia glutinosa Bruguiera gymnorrhiza Kandelia candel Rhizophora apiculata Fragaria vesca Malus halliana Catunaregam tomentosa Duroia hirsuta Scyphiphora hydrophyllacea Aegle marmelos Lycium chinense Lycium chinense Taxus yunnanensis Aquilaria sinensis Petrea volubilis Aloe arborescens Aloe vera Unspecified

Acanthaceae Acanthaceae Actinidiaceae Actinidiaceae Araceae Araceae Araceae Araceae Araceae Araliaceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Calophyllaceae Chenopodiaceae Clusiaceae Clusiaceae Combretaceae Commelinaceae Cornaceae Ericaceae Ericaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Fabaceae Iridaceae Lamiaceae Lamiaceae Lamiaceae Lamiaceae Lamiaceae Lamiaceae Lythraceae Meliaceae Meliaceae Meliaceae Opiliaceae Orchidaceae Orchidaceae Plantaginaceae Plantaginaceae Poaceae Polygonaceae Primulaceae Primulaceae Proteaceae Rehmanniaceae Rhizophoraceae Rhizophoraceae Rhizophoraceae Rosaceae Rosaceae Rubiaceae Rubiaceae Rubiaceae Rutaceae Solanaceae Solanaceae Taxaceae Thymelaeaceae Verbenaceae Xanthorrhoeaceae Xanthorrhoeaceae –

Thailand Thailand Papua New Guinea Papua New Guinea Peru China China China China China Pakistan Russia Egypt Pakistan Egypt Brazil Thailand China Thailand Cameroon Thailand Indonesia China Thailand Finland Thailand Brazil India Australia Australia Thailand China Thailand China China Russia India India Thailand India Thailand Thailand Thailand China China China China Thailand China Thailand Russia Australia China Thailand China Thailand Russia China Thailand Ecuador Thailand India China China China China – Russia Malaysia South Korea

[3] [3] [58] [67] [45] [27] [27] [27] [27] [72] [40] [33] [73] [40] [56] [29] [52] [70] [55] [75] [3] [38] [32] [52] [50] [3] [35] [63] [42] [43] [52] [27] [52] [27] [27] [33] [36] [30] [3] [19] [3] [3] [52] [27] [27] [27] [27] [74] [41] [3] [33] [44] [62] [3] [61] [3] [33] [59] [49] [68] [3] [8] [27] [27] [27] [31] [54] [33] [34] [60]

is directly linked to the success of finding bioactive molecules as demonstrated by Castillo et al. in 2002 [42] and Alvin et al. in 2016 [38], when ethnobotanical knowledge is taken seriously at

the time of selecting a medicinal plant to collect, the reward frequently is the isolation of endophytes that produce bioactive compounds.

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Fig. 3. Families of plants that endophytes have been isolated from. Size of the wedges is proportional to the number of genera that correspond to each family, as listed in Table 4.

Endophytes may be difficult to grow under laboratory conditions because many of them seem to have developed strong physiological bonds with their host plant, and many of them are species-specific. For optimal growth, some of these endophytes must be grown together with pieces of sterilized, freshly harvested natural plant-host tissues as done by Castillo et al. in 2002 [42]. A common phrase found in works that report on the isolation of endophytes from plants is the endophyte in question was not found in any other plant surrounding the host plant that were not the host plant, but they were found in other plants that were the host plants. For example, in the work of Castillo et al. [42]

15

the authors mention that the Streptomyces NRRL 30562 isolated from the plant Kennedia nigricans was not found in any of the plants in the vicinity of K. nigricans but was isolated from other K. nigricans plants, which is interesting because endophytes seem to be very specific to their plant hosts and at the same time, the same endophyte is found in a number of plants, which raises questions about the methods endophytes use to colonize plant hosts. As Alvin, Miller and Neilan pointed out in 2014 [2] the molecules that endophytes produce are less likely to be toxic for eukaryotes, as they do not harm the plant host. However, it is also possible that the compounds do not harm the plant host because the plant host produces the same or similar compounds and therefore is tolerant to them. Whether that same principle applies to mammalian eukaryotic cells requires testing. Taxonomic names are under constant scrutiny and amendment. ‘‘NCBI Taxonomy” [47] is a very valuable, free database available at http://www.ncbi.nlm.nih.gov/taxonomy that may be consulted when in doubt of the correct spelling of the scientific name of a species or its currently accepted taxonomic positioning. What’s more, the common names are also displayed to minimize confusion. The NCBI Taxonomy database will inform on the full lineage of the species (e.g. kingdom, phylum, class, order, family) and contains links to PubMed entries, as well as nucleotide and protein sequences of the species deposited in NCBI. With all the gathered information from the last years it is not difficult to realize the importance of endophytes in the search for new molecules of pharmacological interest. Many of the papers mentioned here have represented years of study, as well as a huge amount of resources spent by the research groups involved in the field. The coming years in the search of endophytes as source of new molecules with pharmaceutical interest seem to be bright. With the use of latest technologies, sophisticated equipment, collaborations between different areas of research and the use of dereplication processes as a startup parameter, undoubtedly this will be possible. Finally, sequencing of the endophyte genomes and application of genome mining techniques will be determinant to

Fig. 4. Collection sites of plants harboring endophytes. Site locations of collected plants were estimated with ‘‘Google Maps” [76] Ó2016 Google, based on the information provided in the literature. The detailed list of the plant scientific names, references and countries of origin, are listed in Table 4.

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exponentially raise the possibilities of finding new antibiotics and other secondary metabolites of medical interest. Any efforts dealing with alternatives to look for new anti-infective compounds are and will be always necessary to face the necessity for new antibiotics. In the year 2012, the European Union started the European Innovative Medicines Initiative (IMI) [77]. Called the ‘‘New Drugs 4 Bad Bugs” (ND4BB) Project, the project is being supported by government contributions and by European pharmaceutical companies. The program involves pharmaceutical companies, academia and biotechnology organizations. In this way, >500,000 small molecules are being contributed by seven drug companies, academia and small companies [78]. Conflict of interest The authors confirm that they have no conflict of interest to declare for this publication. Acknowledgments This work was supported by the grant IN202216 from PAPIIT, DGAPA, UNAM, México and from the NUATEI Program, Instituto de Investigaciones Biomédicas, UNAM. EMK was supported by a postdoctoral fellowship from Consejo Nacional de Ciencia y Tecnología CONACYT, Mexico, grant CB-2013/219686. KRP was supported by a doctoral scholarship from CONACYT, Mexico. We thank Betsabe Linares-Ferrer for chemical structure draws. We also thank Beatriz Ruiz-Villafán and Marco A. Ortíz-Jiménez for critical reading and manuscript preparation. References [1] S. Sanchez, A.L. Demain, Valuable products from microbes, in: G. Neelam, A. Abhinav (Eds.), Microbes in Process, Nova Scientific Publishers Inc, USA, 2014, pp. 23–57. ISBN: 978-1-63117-127-7. [2] A. Alvin, K.I. Miller, B.A. Neilan, Exploring the potential of endophytes from medicinal plants as sources of antimycobacterial compounds, Microbiol. Res. 169 (7–8) (2014) 483–495, http://dx.doi.org/10.1016/j.micres.2013.12.009. [3] J. Buatong, S. Phongpaichit, V. Rukachaisirikul, J. Sakayaroj, Antimicrobial activity of crude extracts from mangrove fungal endophytes, World J. Microbiol. Biotechnol. 27 (12) (2011) 3005–3008, http://dx.doi.org/10.1007/ s11274-011-0765-8. [4] J.E. Janso, G.T. Carter, Biosynthetic potential of phylogenetically unique endophytic actinomycetes from tropical plants, Appl. Environ. Microbiol. 76 (13) (2010) 4377–4386, http://dx.doi.org/10.1128/AEM.02959-09. [5] A.H. Aly, A. Debbab, P. Proksch, Fungal endophytes: unique plant inhabitants with great promises, Appl. Microbiol. Biotechnol. 90 (6) (2011) 1829–1845, http://dx.doi.org/10.1007/s00253-011-3270-y. [6] S. Larran, M.R. Simón, M.V. Moreno, M.P.S. Siurana, A. Perelló, Endophytes from wheat as biocontrol agents against tan spot disease, Biol. Control 92 (2016) 17–23, http://dx.doi.org/10.1016/j.biocontrol.2015.09.002. [7] D. Wilson, Endophyte: the evolution of a term, and clarification of its use and definition, Oikos 10 (2307/3545919) (1995) 274–276, http://dx.doi.org/ 10.2307/3545919. [8] V.M. Mani, A.P. Soundari, D. Karthiyaini, K. Preeth, Bioprospecting endophytic fungi and their metabolites from medicinal tree Aegle marmelos in Western Ghats, India, Mycobiology 43 (3) (2015) 303–310, http://dx.doi.org/10.5941/ MYCO.2015.43.3.303. [9] H.W. Zhang, Y.C. Song, R.X. Tan, Biology and chemistry of endophytes, Natural Prod. Rep. 23 (5) (2006) 753–771, http://dx.doi.org/10.1039/B609472B. [10] P. Golinska, M. Wypij, G. Agarkar, D. Rathod, H. Dahm, M. Rai, Endophytic actinobacteria of medicinal plants: diversity and bioactivity, Anton Van Leeuwenhoek 108 (2) (2015) 267–289. [11] G. Santoyo, G. Moreno-Hagelsieb, M. Del Carmen Orozco-Mosqueda, B.R. Glick, Plant growth-promoting bacterial endophytes, Microbiol. Res. 183 (2016) 92– 99, http://dx.doi.org/10.1016/j.micres.2015.11.008. [12] P.R. Hardoim, L.S. van Overbeek, J.D. Elsas, Properties of bacterial endophytes and their proposed role in plant growth, Trends Microbiol. 16 (10) (2008) 463– 471, http://dx.doi.org/10.1016/j.tim.2008.07.008. [13] G. Madhurama, D. Sonam, P.G. Urmil, N.K. Ravindra, Diversity and biopotential of endophytic actinomycetes from three medicinal plants in India, African J. Microbiol. Res. 8 (2) (2014) 184–191, http://dx.doi.org/10.5897/ ajmr2012.2452. [14] K. Hyde, K. Soytong, The fungal endophyte dilemma, Fungal Divers 33 (163173) (2008) 2.

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