Brazilian Plants: An Unexplored Source of Endophytes as Producers of Active Metabolites

Key words

Brazilian biodiversity - medicinal plants - endophytes - secondary metabolites

Introduction

The discovery of new compounds for pharmaceutical and agronomic purposes is now more necessary than ever [1]. In drug discovery programs, nature remains an unlimited source of complex molecules. Plants have served as a repository of medicinal bioactive compounds against numerous diseases for centuries, however, the isolation and purification of plant compounds in an adequate yield remains a major concern [2]. In addition to the low yield, the exploitation of plants for extraction of metabolites is also associated with environmental impacts, and new strategies, such as the use of endophytic microorganisms instead of the plants themselves, have offered compounds with high therapeutic potential [3].

Microorganisms are well known for their ability to produce secondary metabolites that are applied in medicine and agriculture, and endophytes have gained remarkable attention in view of their diversity and biotechnological potential [4]. The long relationship of endophytes with medicinal plants may influence the natural bioactivity of endophytes by acquiring genetic information from the plant to produce host-like metabolites [4]. The hypothesis of genetic exchange involving the endophyte and its host is supported by the presence of host-like genes in the biosynthetic pathways of endophytes, resulting in a huge repertoire of enzymes and the production of complex molecules. A great example of the plant-endophyte relationship is the production of paclitaxel by the endophytic fungus Taxomyces andreanae, a compound produced primarily by the host, Taxus sp. [5]. Despite the large number of studies reporting synergism in the metabolic pathways, our knowledge of the exact mechanisms involving the host-endophyte relationship remains limited [3].

It is estimated that more than one million endophytic species occur in nature, and less than 10% of these species are cataloged [3]. The challenge in exploring endophytes for drug discovery lies in how to access the potential of chemical diversity, since the population of endophytes is highly variable and depends on several components, such as host species and environmental conditions [6]. In this scenario, tropical forests, such as the Brazilian flora that holds about 55 000 species of terrestrial plants [7], may represent an interesting repository of new endophytic species that can be used as a repertoire of new molecules.

An important step in the bioprospecting of microorganisms involves accurate identification of the species to ensure correct reporting of the biochemical potential of each species. The precise identification of microorganisms involves morphological, biochemical, and genetic analyses. However, due to an inadequate interpretation of DNA sequences (using only blast analysis in public databases), several studies have identified endophytes incorrectly or incompletely (only at the genus or family level).

Based on these data, this study reviewed the biodiversity of endophytes of plants found in different Brazilian biomes from 2012 to 2017, including species diversity, species identification challenges, and biotechnological aspects.

Definition of endophytes and strategies for plant colonization

De Bary [8] suggested that endophytes are microorganisms that dwell within plant tissues. Based on the microscopic analysis, Petrini [9] suggested that endophytes are microorganisms that interact with the host plant and as a result of this interaction, no symptoms of disease are observed. The problem with the definitions of endophytes proposed by De Bary [8] and Petrini [9] is related to some latent pathogens that live part of their life inside plants tissues without causing any negative damage. In this context, Hardoim et al. [10] suggested that the term endophyte refers only to habitat and should not be associated with a function, for example, phytopathogenic or non-phytopathogenic, and all microorganisms that throughout all or part of their life colonize the internal tissues of plants are considered endophytes.

The coexistence of plants and endophytes remains unclear [11], and the main question is: Why do plants not defend themselves against internal colonizers? So far, it seems that the momentary symbiotic relationship between the plant and the endophyte is established [12]. The endophytes provide nutrients for the plant, can also facilitate the acquisition of essential nutrients from the environment, such as nitrogen, phosphorous, and iron [13], and produce secondary metabolites that can inhibit an infection by phytopathogens [14]. The main factors that can regulate endophytic colonization within a plant include plant genotype, growth stage, tissue physiology, colonized plant tissue, and environmental conditions. The genetic factors of the host can critically influence the structure and function of the microbiomes associated with the plants. Thus, endophytes seem to have successfully adapted to overcome a host-specific immune system and, thus, to form populations in the internal tissues [4]. To overcome plant defense mechanisms, endophytes produce secondary metabolites or enzymes, such as cellulases, lactases, and proteases, which can damage the plant cell wall and facilitate penetration into the host [14]. Another system also used by endophytes in plant colonization involves opportunistic penetration through wounds or roots [13].

Once within the plant tissues, the endophyte colonizes the tissues without causing symptoms, but at any time, due to environmental changes such as plant development, availability of nutrients, or other factors, that relationship can be broken and the symptoms of the disease can be observed [15].

Plants explored as a source of endophytes in Brazil

The Brazilian diversity is divided into six biomes: the Amazonian rainforest, the Caatinga, the Cerrado (Savannah), the Atlantic Forest, the Pampa, and the Pantanal (Swampland) [16]. For several years, the International Conservation has placed Brazil at the top of the 17 megadiverse countries of the world, with the largest number of plant species, 55 000, representing 22% of the world total (http://www.unesco.org/new/en/brasilia/natural-sciences/environment/biodiversity/). Considering that some endophytes are host-specific, the diversity of plants found in Brazil comprises an extraordinary diversity of habitats, life forms, and biological associations confined to particular environments at different geographic scales. Although plant diversity is well documented, the number and richness of microorganisms in most countries remain unlisted, and Brazil is no exception. In view of species richness, two biomes are recognized as global biodiversity hot spots, the Brazilian Cerrado and the Atlantic Forest [16], and can represent an inexhaustible source of microorganisms.

A search using the words “endophytes” and “Brazil” in the PubMed database resulted in 67 papers that performed isolation and bioprospecting of endophytes from Brazilian biomes during 2012 – 2017. From the analyzed articles, the data collected were the name of the plant from which the endophytes were isolated, the endophytes isolated from each plant, the methods used to identify the endophytes, the biotechnological potential of the endophytes, and the isolated secondary metabolites. Endophytes were grouped at kingdom and family levels based on Mycobank [MycoBank (http://www.mycobank.org/) classification and list of prokaryotic names with standing nomenclature (http://www.bacterio.net/)].

[Table 1] lists the scientific names of the 54 plant species of which the endophytes were isolated in Brazil from 2012 to 2017. The plants studied belong to 30 families and the frequency of each family is shown in [Fig. 1]. The most representative plant families are Fabaceae, Myrtaceae, and Asteraceae, representing more than 25% of the studies ([Fig. 1]). The Fabaceae family includes several important agricultural and food plants, and Asteraceae members provide products such as cooking oils, sunflower seeds, and sweetening agents. The Myrtaceae family also provides many products, including timber, essential oils, and horticultural plants (http://tolweb.org). Interestingly, the most representative families have obvious significance in the agriculture and food industries; in contrast, fewer studies have been conducted on the biodiversity of endophytes of medicinal plants. Biomes and the approximate location where the collections were made were estimated using “Google Maps” (https://www.google.com.br/maps) and compared with the biome map proposed by Myers et al. [16] ([Fig. 2]). The highest number of collections was carried out in the Atlantic Forest biome, mainly in the states of São Paulo, Minas Gerais, and Paraná, with the largest species cataloged in these states. In recent years, no studies on endophytic biodiversity have been conducted in Pampa ([Fig. 2]).

Table 1 Taxonomic classification of plants containing endophytes and the collection sites. The scientific names of the plants were searched in the NCBI Taxonomy database to note the family in which the plant is classified.

Plant	Family	City and State	Reference
Alibertia macrophylla	Rubiaceae	São Paulo, São Paulo	[71]
Alternanthera brasiliana	Amaranthaceae	São Paulo, São Paulo	[72]
Ananas comosus	Bromeliaceae	São Paulo, São Paulo	[73]
Aspidosperma tomentosum	Apocynaceae	Rio de Janeiro, Rio de Janeiro	[74]
Avicennia nitida	Verbenaceae	Cananéia, São Paulo	[75]
Avicennia schaueriana	Verbenaceae	Bertioga, São Paulo	[39]
Baccharis trimera	Asteraceae	Ouro Branco, Minas Gerais	[76]
Bauhinia forficate	Fabaceae	Recife, Pernambuco	[77]
Bauhinia guianensis	Fabaceae	Manus, Amazonas	[78]
Borreria verticillata	Rubiaceae	Recife, Pernambuco	[56]
Citrus sinensis	Rutaceae	Piracicaba, São Paulo	[79]
Coffea Arabica	Rubiaceae	Viçosa, Minas Gerais	[80]
Eichhornia azurea	Pontederiaceae	Porto Rico, Paraná	[81]
Eichhornia crassipes	Pontederiaceae	Porto Rico, Paraná	[81]
Eucalyptus benthamii	Myrtaceae	São Paulo, São Paulo	[82]
Eucalyptus grandis	Myrtaceae	Belo Oriente, Minas Gerais	[83]
Eucalyptus urophylla	Myrtaceae	Belo Oriente, Minas Gerais	[83]
Eugenia bimarginata	Myrtaceae	Belo Orizonte, Minas Gerais	[29]
Fragaria chiloensis	Rosaceae	Lavras, Minas Gerais	[84]
Glycine max	Fabaceae	Viçosa, Minas Gerais	[85]
Hadrolaelia jongheana	Orchidaceae	Serra do Brigadeiro, Minas Gerais	[71]
Hoffmannseggella caulescens	Orchidaceae	Serra do Brigadeiro, Minas Gerais	[71]
Hoffmannseggella cinnabarina	Orchidaceae	Serra do Brigadeiro, Minas Gerais	[71]
Hyptis suaveolens	Lamiaceae	Miranda, Mato Grosso do Sul	[50]
Laguncularia racemose	Combretaceae	Cananéia, São Paulo	[60]
Lippia sidoides	Verbenaceae	São Cristóvão, Sergipe	[86]
Luehea divaricate	Malvaceae	Maringá, Paraná	[87]
Lychnophora ericoides	Asteraceae	Furnas, Minas Gerais	[35]
Maytenus ilicifolia	Celastraceae	Curitiba, Parana	[20]
Melia azedarach	Meliaceae	São Carlos, São Paulo	[66]
Musa spp	Musacea	Manacapuru, Amazonas	[39]
Myrcia guianensis	Myrtaceae	Santarém, Bahia	[88]
Opuntia ficus-indica	Cactaceae	Itaıba, Pernambuco	[77]
Oryza glumaepatula	Poaceae	Seropédica, Rio de Janeiro	[52]
Paullinia cupana	Sapindaceae	Manus, Amazonas	[53]
Phaseolus vulgaris	Fabaceae	Viçosa, Minas Gerais	[89]
Piper hispidum	Piperaceae	Maringa, Paraná	[38]
Rhizophora mangle	Rhizophoraceae	Cananéia, São Paulo	[75]
Ricinus communis	Euphorbiaceae	Curitiba, Paraná	[90]
Saccharum officinarum	Poaceae	Seropédica, Rio de Janeiro	[91]
Schinus terebinthifolius	Anacardiaceae	Curitiba, Paraná	[20]
Senna spectabilis	Fabaceae	Araraquara, São Paulo	[2]
Smallanthus sonchifolius	Asteraceae	Ribeirão Preto, Sâo Paulo	[92]
Solanum cernuum	Solanaceae	Belo Horizonte, Minas Gerais	[32]
Spondias mombin	Anacardiaceae	Redenção, Pará	[74]
Strychnos toxifera	Loganiaceae	Manaus, Amazonas	[61]
Theobroma cacao	Malvaceae	Brasılia, Distrito Federal	[40]
Trichilia elegans	Meliaceae	Maringá, Paraná	[93]
Vellozia gigantean	Velloziaceae	Tocantins	[94]
Vernonia polyanthes	Asteraceae	Ouro Preto, Minas Gerais	[68]
Vigna unguiculata	Fabaceae	Juazeiro, Bahia	[95]
Vitis labrusca	Vitaceae	Salesópolis, São Paulo	[96]
Vochysia divergens	Vochysiaceae	Miranda, Mato Grosso do Sul	[27]
Zea mays	Poaceae	Anchieta, Espirito Santos	[50]

Fig. 1 Families of plants from which endophytes have been isolated. The size of the wedges is proportional to the number of genera that correspond to each family.

Fig. 2 Collection sites of plants harboring endophytes in different Brazilian biomes. Site locations of collected plants were estimated with “Google Maps” (https://www.google.com.br/maps) based on the information provided in the literature. The circles and their colors indicate where and the number of studies performed.

Diversity of Endophytes in Brazil

Taxonomic classification

Table 1S, Supporting Information, contains a taxonomic identification of the microorganisms isolated as endophytes from the plants listed in [Table 1]. Of the 54 species of plants studied, 307 species (belonging to 300 genera) were reported (Table 1S, Supporting Information), 51 and 49% of the isolated genera belong, respectively, to the kingdoms bacteria and fungi ([Fig. 3]).

Fig. 3 The frequency of kingdom and phylum of endophytes isolated in Brazil between the years 2012 and 2017. Bars represent kingdom, and colors represents phylum inside each kingdom.

Among the bacteria kingdom, more than 50% of genera belong to the phylum Proteobacteria, followed by Actinobacteria and Firmicutes ([Fig. 3]). As reported previously [17], 99% of fungal genera isolated as endophytes belong to the Ascomycota and Basidiomycota phylum, with the dominance of Ascomycota isolates (~ 85%) ([Fig. 3]).

[Fig. 4] represents the number of occurrences of different endophytic genera in the studies in Brazil from 2012 to 2017. Diaporthe was the fungal genus reported in the largest number of studies, present as an endophyte in 48% of the analyzed articles ([Fig. 4]). Among the bacteria, Bacillus was the most frequent genus identified in 77% of the articles ([Fig. 4]). These data agree with several studies on the biodiversity of endophytes [18], [19]. Possibly the Diaporthe and Bacillus species have developed effective strategies to escape plant defenses, or even produce metabolites that may be useful for host development or defense against plant pathogens [20], [21], [22]. Despite the high diversity of endophytes of Brazilian plants (Table 1S, Supporting Information), of the 300 genera reported as endophytes, 101 bacteria and 83 fungal genera were reported as endophytes in only one publication ([Fig. 4] and Table S1, Supporting Information), suggesting that the endophytic community in Brazil remains little explored.

Fig. 4 Visualization of occurrence of bacterial and fungal genera isolated as endophytes in 67 papers published during 2012 – 2017 (number of occurrences against itself with small artificial jitter added to the points so that they do not completely overlap). Blue circles represent bacteria genera, and red circles represent fungi.

Problems in DNA sequence analysis

The identification of endophytes at the species level is performed based on taxonomy, ecology, and applied reasons, such as the discovery of new products based on genomic analysis. Raja et al. [23] reported that 28% of the articles published in the Journal of Natural Products did not have any identification for fungal strains producing active molecules, and 31% of strain identification was based only on morphological aspects. Because correct species identification is a key step in ensuring reproducibility for biotechnology purposes and may reveal important information about its possible biochemical properties, a correct and robust method for species recognition should be applied in biodiversity studies and bioprospecting of endophytes.

For many years, microbiologists have used morphology as the sole criterion for species identification. However, morphological characteristics do not always present good performance in the identification of the species, and the absence of phenotypic information, such as the lack of sporulation in laboratory conditions, increase the difficulties of identification, even at the genus level [24]. Thus, molecular approaches become a reliable alternative for the identification of endophytes.

To date, the ITS and 16S rRNA regions remain the first choice for identification of fungi and bacteria, respectively. In the analyzed articles, 95.5% used molecular markers to identify species and the majority, 93.5%, used only one ribosomal marker (Table 1S, Supporting Information). However, to identify cryptic species in some genera, such as Diaporthe [25], Fusarium [26], Microbispora [27], and Streptomyces [28], ribosomal markers are not informative enough. In addition to this problem, most of the analyzed papers performed the identification of the strains based on similarity, comparing the DNA sequences with the GenBank database using the BLAST tool. However, it is already known that BLAST search-based identifications should be carried out with caution since in GenBank, there are misidentified sequences and entries with other annotation problems [23]. An interesting alternative that can minimize these problems is to use the “sequence from type” filter in the blast searches, or to conduct searches in the RefSeq Targeted Loci project (http://www.ncbi.nlm.nih.gov/refseq/targetedloci/), which maintains curated sets of full-length sequences of type material for ribosomal RNAs [25]. Therefore, in order to obtain a correct identification of the species, this should be performed through a phylogenetic analysis using an evolutionary framework with homologous sequences of all type strain of each genus [23]. In contrast to similarity-based identification, phylogeny reconstructs the tree-like pattern that describes the evolutionary relationships between species with a predictive value [25].

In order to evaluate the accuracy of the species identification of endophytes isolated in Brazil, we have reanalyzed the sequences published in 18 articles describing strains with biotechnological potential ([Table 2]). First, the sequences were compared to the sequences available in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/) using the Blast tool, and selecting the option “blast only in type strains sequences”. The value of 95% similarity was used as discriminatory for identification at the genus level. Sequences of all type strains of each genus of fungi and bacteria were obtained from MycoBank (http://www.mycobank.org/) and from the list of prokaryotic names with standing in nomenclature (http://www.bacterio.net/), respectively. The species identification of each strain was based on Bayesian phylogenic analysis according to Savi et al. [27].

Table 2 Comparative analysis between the identification of endophytes performed in 18 published articles with the identification carried out in this study based on Bayesian phylogenetic analysis.

Host plant	Strain	GenBank code	Identification reported in the literature	Phylogenetic identification performed in this study	Incongruence	Reference
– = No difference in the identification. All figures noted in this table are available as Supporting Information
Eugenia bimarginata	UFMGCB2032	KF681521	Mycosphaerella sp.	Phaeophleospora sp. (Fig. 17S)	Mycosphaerella was divided into several genera, and the isolate belongs to the genus Phaeophleospora	[29]
Baccharis trimera	UFMGCB4425	KJ404206	Alternaria sp.	Alternaria sp. Sect. Alternata (Fig. 3S)	With phylogenetic analysis, the isolate is identified at the section level	[76]
	UFMGCB4428	KJ404203	Chaetomium sp.	Another genus than Chaetomium (Fig. 7S)	Low sequence quality
	UFMGCB4580	KJ404213	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
	UFMGCB4453	KJ404204	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
	UFMGCB4451	KJ404215	Phomopsis sp.	Diaporthe sp. (Fig. 25S)	–
	UFMGCB4570	KJ404212	Epicoccum sp.	Epicoccum sp. (Fig 10S)	–
	UFMGCB4468	KJ404201	Guignardia sp.	Phyllosticta sp. (Fig. 19S)	–
	UFMGCB4429	KJ404217	Nigrospora sp.	Nigrospora sp. (Fig. 15S)	–
	UFMGCB4436	KJ404209	Nigrospora sp.	Nigrospora sp. (Fig. 15S)	–
	UFMGCB4571	KJ404211	Podospora sp.	Podospora sp. (Fig. 20S)	–
	UFMGCB4498	KJ404214	Preussia sp.	Preussia sp. (Fig. 21S)	–
	UFMGCB4528	KJ404216	Preussia sp.	Preussia sp. (Fig. 21S)	–
	UFMGCB4510	KJ404202	Preussia africana	Preussia africana (Fig. 21S)	–
	UFMGCB4423	KJ404196	Preussia pseudominima	Preussia minima (Fig. 21S)	–
	UFMGCB4592	KJ404199	Preussia sp.	Preussia sp. (Fig. 21S)	–
Lychnophora ericoides	RLe7	KF057056	Streptomyces albospinus	Streptomyces sp. (Fig. 24S)	Housekeeping gene sequences are required for species identification	[35]
Hyptis suaveolens	F7	KF554491	Aspergillus terreus	Aspergillus sp. Sect. terrei (Fig. 4S)	With phylogenetic analysis, the isolate is identified only at the section level	[36]
Piper hispidum		JF766997	Phlebia sp.	Phlebia sp. (Fig. 18S)	–	[49]
Mikania glomerata		KT962838	Diaporthe citri	Diaporthe sp. (Fig. 9S)	Based on phylogeny, it is not possible to identify the isolate as D. citri	[37]
Piper hispidum		JF766998	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–	[58]
		JF767007	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
Lippia sidoides		KU639597	Lactococcus lactis	Lactococcus lactis (Fig. 11S)	–	[86]
Lychnophora ericoides	RLe9	KF057070	Streptomyces sp.	Streptomyces sp. (Fig. 24S)	–	[33]
	RLe8	KF057057	Streptomyces sp.	Streptomyces sp. (Fig. 24S)	–
	RLe6	KF057069	Streptomyces cattleya	Streptomyces sp. (Fig. 24S)	Housekeeping gene sequences are required for species identification
	RLe4	KF057055	Streptomyces cattleya	Streptomyces sp. (Fig. 24S)	Housekeeping gene sequences are required for species identification
	RLe1	KF057065	Streptomyces cattleya	Streptomyces sp. (Fig. 24S)	Housekeeping gene sequences are required for species identification
	RLe11	KF057067	Streptomyces cattleya	Streptomyces sp. (Fig. 24S)	Housekeeping gene sequences are required for species identification
	RLe13	KF057064	St. angustmyceticus	Streptomyces sp. (Fig. 24S)	Housekeeping gene sequences are required for species identification
	RLe10	KF057058	Kitasatospora cystarginea	Kitasatospora or Streptomyces (Fig. 24S)	Housekeeping gene sequences are required to differentiate Kitasatospora from the genus Streptomyces
	RLe03	KF057068	Streptomyces mobaraensis	Streptomyces sp. (Fig. 24S)	Housekeeping gene sequences are required for species identification
	RLe12	KF057053	Streptomyces albospinus	Streptomyces sp. (Fig. 24S)	Housekeeping gene sequences are required for species identification
Schinus terebinthifolius	LGMF626	KM510497	Alternaria sp. Sect. Alternata	Alternaria sp. Sect. Alternata (Fig. 3S)	–	[48]
	LGMF692	KM510498	Alternaria sp. Sect. Alternata	Alternaria sp. Sect. Alternata (Fig. 3S)	–
	LGMF713	KM510499	Bjerkandera sp.	Bjerkandera centroamericana (Fig. 6S)	B. centroamericana was described after the publications of the cited paper [48]
	LGMF627	KM510503	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
	LGMF653	KM510508	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
	LGMF655	KM510505	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
	LGMF657	KM510509	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
	LGMF694	KM510507	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
	LGMF701	KM510512	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
	LGMF698	KM510513	Penicillium sp.	Penicillium sp. section Citrina (Fig. 16S)	With ITS phylogenetic analysis, the isolate is identified at the section level
Piper hispidum		JF766989	Lasiodiplodia theobromae	Lasiodiplodia sp. (Fig. 12S)	With ITS phylogenetic analysis, the isolate is not identified at the species level	[38]
		JF766998	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
		JF767000	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–
		JF767007	Bipolaris sp.	Bipolaris sp. (Fig. 27S)	–
		JF766997	Phlebia sp.	Phlebia floridensis (Fig. 18S)	With ITS phylogenetic analysis, the isolate was identified at the species level
		JF767001	Bipolaris sp.	Bipolaris sp. (Fig. 27S)	–
		JF766996	Colletotrichum sp.	C. gloesporioides complex (Fig. 8S)	With ITS phylogenetic analysis, the isolate is identified at the complex level
		JF767002	C. gloesporioides	C. gloesporioides complex (Fig. 8S)	With ITS phylogenetic analysis, the isolate is identified at the complex level
		JF766993	Bipolaris sp.	Bipolaris sp. (Fig. 27S)	–
		JF766992	Bipolaris sp.	Bipolaris sp. (Fig. 27S)	–
		JF766991	Alternaria sp.	Alternaria sp. Sect. Alternata (Fig. 3S)	With ITS phylogenetic analysis, the isolate is identified at the section level
		JF766990	Alternaria sp.	Alternaria sp. Sect. Alternata (Fig. 3S)	With ITS phylogenetic analysis, the isolate is identified at the section level
		JF767006	Colletotrichum sp.	C. boninense complex (Fig. 8S)	With ITS phylogenetic analysis, the isolate is identified at the complex level
		JF767005	Bipolaris sp.	Bipolaris sp. (Fig. 27S)	–
		JF767004	Colletotrichum sp.	C. gloesporioides complex (Fig. 8S)	With ITS phylogenetic analysis, the isolate is identified at the complex level
		JF766988	Phyllosticta capitalensis	Phyllosticta sp. (Fig. 19S)	P. paracapitalensis was described after the publication of this paper [38], and using only the ITS sequence, it is not possible to differentiate this species from P. capitalensis
Sabicea cinerea	SNB-GSS10	KF164383	Diaporthe sp.	Diaporthe sp. (Fig. 25S)	–	[57]
Baccharis trimera	PS6	KM925013	Bacillus amyloliquefaciens	Bacillus sp. section Subtilis (Fig. 5S)	The isolate did not cluster with any type strain of Bacillus section Subtilis and may represent a new species	[76]
	M10	KM925011	Bacillus amyloliquefaciens	Bacillus sp. section Subtilis (Fig. 5S)	The isolate did not cluster with any type strain of Bacillus section Subtilis and may represent a new species
	M28	KM925012	Bacillus subtilis	Bacillus sp. section Subtilis (Fig. 5S)	The isolate is in the same branch as B. subtilis and B. tequilensis, therefore, using only 16S rRNA, it was not possible to identify the strain at the species level
Musa spp	ALB629	JQ435867	Bacillus subtilis	Bacillus sp. section Subtilis (Fig. 5S)	The isolate did not cluster with any type strain of Bacillus section Subtilis and may represent a new species	[40]
Vochysia divergens		KY458125	Actinomadura sp.	Actinomadura sp. (Fig. 1S)	–	[54]
		KY421547	Actinomadura sp.	Actinomadura sp. (Fig. 1S)	–
		KY411896	Aeromicrobium ponti	Aeromicrobium ponti (Fig. 2S)	–
		KY411900	Microbispora sp.	Microbispora sp. (Fig. 13S)	–
		KY411898	Microbispora sp.	Microbispora sp. (Fig. 13S)	–
		KY423496	Micrococcus sp.	Micrococcus sp. (Fig. 14S)	–
		KY458126	Sphaerisporangium sp.	Sphaerisporangium sp. (Fig. 22S)	–
		KY423333	S. thermocarboxydus	Streptomyces sp. (Fig. 24S)	Housekeeping gene sequences are required for species identification
		KY421546	Williamsia serinedens	Williamsia serinedens (Fig. 23S)	–
			Microbacterium sp.	Microbacterium sp. (Fig. 26S)	–

We performed 27 new phylogenetic analyzes of 78 isolates belonging to 26 genera ([Table 2] and Figs. 1S–27S, Supporting Information) in order to verify if the published identifications are correct or if there are misidentified species. Based on these analyses, we noticed that two isolates were erroneously identified at the genus level in the previous articles: Pereira et al. [29] identified the strain UFMGCB2032 (Genbank code KF681521), isolated from Eugenia bimarginata, as Mycosphaerella sp. based on phylogenetic analysis. The authors used only ITS sequences from a few type species of the Mycosphaerella genus and other GenBank sequences. However, Crous [30], using morphological and molecular approaches, demonstrated that Mycosphaerella is polyphyletic and represents a complex of genera and species, containing about 10 000 species names, and has been split into more than 23 genera based on phylogenetic analysis and asexual morphs [30], [31]. Based on these data and the previously listed article, we found that the isolate reported by Pereira shows high similarity to sequences of the genus Phaeophleospora, and in our new phylogenetic analysis (Fig. 19S, Supporting Information), the strain UFMGCB2032 is clustered to Phaeophleospora gregaria, Phaeophleospora scytalidii, and Phaeophleospora eugeniicola, confirming the identification of this strain as Phaeophleospora sp. The second misidentification at the genus level was performed by Vieira et al. [32] for the isolate UFMGCB4428 (GeneBank code KJ404203) (Fig. 8S, Supporting Information). The authors identified strain UFMGCB4428 as Chaetomium sp. based on 90% similarity in BLAST analysis. However, despite the poor sequence quality (represented by several indeterminate bases, “N”, present in the sequence), the first 190 bp of the ITS sequence do not have similarity to the ITS sequences of the Chaetomium species, which may suggest a mixture of DNA sequences.

Isolate RLe10 (GenBank code KF057058) was identified as Kitasatospora cystarginea by Conti et al. [33]. However, several reports have shown that the partial sequences of 16S rRNA did not have enough information to differentiate Kitasotospora species from Streptomyces [28], [34], as observed in our analysis (Fig. 26S, Supporting Information). A multilocus approach is required for the identification of species in these cryptic genera.

The remaining 75 isolates were correctly identified at the genus level, however, 17 isolates belonging to the genera Aspergillus, Phyllosticta, Colletotrichum, Lasiodiplodia, Streptomyces, and Bacillus were misidentified at the species level ([Table 2]) [33], [35], [36], [37], [38], [39], [40].

The difference in the identification of two isolates belonging to species of Phyllosticta and Lasidioplodia genera ([Table 2]) published by Orlandelli et al. [38] was due to the description of new species in the genera Phyllosticta and Lasiodiplodia after their publication. In these cases, the isolates clustered with more than one species (Figs. 21S and 13S, Supporting Information), making identification at the species level impossible. As an example, the species Phyllosticta paracapitalensis, recently described, is not differentiated from Phyllosticta capitalensis using only ITS sequences [41], requiring a multilocus sequence for species identification.

Silva et al. [36], Souza et al. [39], and Falcão et al. [40] erroneously identified several isolates at the species level in the genera Aspergillus and Bacillus. In our phylogenetic analyses, these isolates were not identified at the species level, but as belonging to Aspergillus section terrei and Bacillus section subtilis (Figs. 4S and 5S, Supporting Information). The dentification of species within these sections is not possible using only ITS sequences [42], [43].

Chagas et al. [35] and Conti et al. [33] identified seven isolates belonging to the genus Streptomyces based on the 16S rRNA partial sequence (~ 400 bp). The authors performed the phylogenic analysis using only a few species of the more than 500 species belonging to the genus Streptomyces. In addition to the low number of species used for phylogenetic analyses, in some cases, the isolate presented 100% similarity with more than one species, such as the strain RLe13. In these cases, the authors identified the strain as belonging to the species based on the similarity to the sequence of species deposited in the CBS database, even without phylogenetic support. Several authors have reported the low discriminatory power of ribosomal markers and have suggested a minimum of four loci to identify species within the genus Streptomyces [28], [44]. This same difficulty is observed for fungi, such as the Diaporthe genus, in which few species are identified using only ribosomal markers, such as the ITS sequences ([Table 2] and Figs. 10S and 27S, Supporting Information) [25]. In these cases, a multilocus sequences analysis, using protein-coding genes, is recommended [25], [27], [41], [44]. Among the protein-coding markers used to identify fungal species, the translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), and actin (act), GAPDH and subunits of RNA polymerase (RPB1 and RPB2) have been commonly used to infer phylogenetic relationships and species identification [25], [44]. For bacteria, the use of housekeeping genes has been confirmed as highly reproducible, low cost, and with the same efficiency as DNA-DNA hybridization for species identification. The most common multilocus analysis consists of sequences of gyrB (DNA gyrase, beta subunit), rpoD (RNA polymerase, σ factor), recA (recombinase A), and trpB (tryptophan synthase, beta subunit) genes [27], [28], [44], [45].

Biological activity of endophytes found in Brazil

Research on natural products is still the most effective way to discover new compounds, and less than 10% of the worldʼs biodiversity has been evaluated for its biological potential [46]. Endophytes have great importance in the production of compounds with a unique structure, which may result from several biological interactions [47], however, the challenge for drug discovery is how to access this chemical potential. Most of the studies on endophytic biodiversity published in Brazil (2012 – 2017) present some bioprospecting studies, such as evaluation of antibiotic, antioxidant, antiparasitic, or cytotoxicity activities, or the use of endophytes to promote plant growth or reactive dye discoloration [40], [48], [49], [50], [51], [52], [53], [54] ([Table 3]). As a result of the success obtained in terms of discovering active metabolite-producing endophytes, a large number of compounds [2], [55], [56], [57] or known compounds with unreported biological properties [33], [36], [58], [59], [60] have been reported.

Table 3 Taxa of endophytes isolated in Brazil, and biological activities and bioactive compounds produced by the endophytes.

Endophytic	Biological activities	Compound	Reference
The bolds indicate new compounds.
Streptomyces albospinus	Crude extract showed antifungal activity, however, none of the isolated compounds showed activity against the fungi Coniochaeta sp., used as an antimicrobial marker	(2 R *,4 S *)-2-((1′ S *)-hydroxy-4′-methylpentyl)-4-(hydroxymethyl)butanolide (3 R *,4 S *,5 R *,6 S *)-tetrahydro-4-hydroxy-3,5,6-trimethyl-2-pyranone 1-O-(phenylacetyl)glycerol (S)-4-benzyl-3-oxo-3,4-dihydro-1Hpyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (S)-4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde cyclo(L-Tyr-L-Pro)	[35]
Aspergillus terreu	All compounds displayed antioxidant, cytotoxic, and antiparasitic activities (Schistosoma mansoni and Leishmania amazonensis); compound butyrolactone I showed activity against Escherichia coli	Terrain Butyrolactone I Butyrolactone V	[36]
Diaporthe sp.	Cytotoxic activity	2,4,6-Tri-O-methyl-1,3,5-tri-O-acetylglucose 2,3,4,6-Tetra-O-methyl-1,5-di-O-acetyl-glucose 2,4-Di-O-methyl-1,3,5,6-tetra-O-acetyl-glucose	[58]
Mycosphaerella sp.	Antifungal activity	2-Amino-3,4-dihydroxy-2 – 25-(hydroxymethyl)-14-oxo-6,12-eicosenoic acid Myriocin	[29]
Streptomyces cattleya	Nocardamine showed cytotoxic and antiparasitic activities	Salicylamide Nocardamine Propioveratrone Acetoveratrone	[33]
Streptomyces albospinus	Nocardamine showed cytotoxic and antiparasitic activities	Nocardamine Dehydroxynocardamine Physostigmine	[33]
Streptomyces sp.	2,3-Dihydro-2,2-dimethyl-4(1H)-quinazolinone showed cytotoxic activity against several tumor cell lines; deferoxamine showed antiparasitic activity against Tripanosoma cruzi	3-Hydroxybenzamide 4-Hydroxy-3-methoxybenzamide 3-Hydroxy-4methoxybenzamide Benzamide trans-4-Hydroxyscytalone cis-4-Hydroxyscytalone 2-Phenylacetamine Veratramide 2,3-Dihydro-2,2-dimethyl-4(1H)-quinazolinone Deferoxamine	[33]
Alternaria sp. Sect. Alternata	The compounds were present as one fraction with antibacterial activity, however, none of them were evaluated as pure form	E-2-Hexyl-cinnamaldehyde 3-Benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione 3-Isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione E-2-Hexyl-cinnamaldehyde 3-Isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione 3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione	[48]
Cochliobolus sativus	Antileishmanial activity	Cochlioquinone A Isocochlioquinone A Anhydrocochlioquinone A	[68]
Diaporthe sp.	Mycoepoxydiene and eremofortin F showed cytotoxic activity	Mycoepoxydiene Altiloxin A Enamidin Eremofortin F	[57]
Mycosphaerella sp.	Antifungal activity	(2S,3R,4R)-(E)-2-Amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoeicos-6,12-dienoic acid (2S,3R,4R)-(E)-2-Amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoeicos-6-enoic acid	[29]
Microbispora sp.	Compound 1-Vinyl-β-carboline-3-carboxylic acid displayed antibacterial and cytotoxicity activities	1-Vinyl-β-carboline-3-carboxylic acid JBIR-133 Kitasetaline Methyl 1-(propionic acid)-β-carboline-3-carboxylic acid Indole-3-carbaldehyde Indole-3-acetic acid Indole-3-carboxylic acid	[97]
Phomopsis sp.	cytochalasins J, H and alternariol showed antioxidant activity, and cytochalasins H showed antifungal activity	2-Hydroxy-alternariol Cytochalasins J Cytochalasins H 5′-Epialtenuene Monomethyl ether Alternariol Cytosporone C	[2]
Papulaspora immersa Nigrospora sphaerica	Cytotoxic activity	(22E,24R)-8,14-Epoxyergosta-4,22-diene-3,6-dione Aphidicolin	[65]
Mycoleptodiscus indicus	All compounds were weakly active when tested in antileishmanial and cytotoxicity assays	Mycoleptones A Mycoleptones B Mycoleptones C Austdiol Eugenitin 6-Methoxyeugenin 9 – 22 Hydroxyeugenin	[56]
Lewia infectoria	Excluding Pyrenocin A and Novae Zelandin A, the others compounds showed antimicrobial activity.	Acremonisol A Semicochliodinol A Cochliodinol Griseofulvin Pyrenocin A Novae zelandin A Alterperyleno	[55]
Cocultivation Alternariatenuissima/ Nigrospora sphaerica	Antifungal activity	Stemphyperylenol Altertoxin I Alterperylenol Alternariol Alternariol monomethyl ether	[35]
Phomopsis longicolla	Antibacterial activity	3-Nitropropionic acid	[59]
Mycoleptodiscus indicus	Increases the production of glucoamylases by Aspergilus niveus	Eugenitin	[63]
Gliocladium sp	Citotoxicity	Cyclo-(glycyl-Ltyrosyl)-4,4-dimethylallyl ether	[61]
Aeromicrobium ponti	Only the diketopiperazines did not show antibacterial activity	1-Acetyl-βcarboline Indole-3-carbaldehyde tryptophol 3-(Hydroxyacetyl)indole Brevianamide F Cyclo-(L-Pro-L-Phe) Cyclo(L-Pro-L-Tyr) Cyclo-(L-Pro-LLeu) Cyclo-(L-Val-L-Phe)	[54]

It is well known that the culture conditions can drastically influence the profile of the metabolites produced by a specific strain. To evaluate the influence of culture conditions on the antibacterial activity of endophytic isolates of the medicinal plant Schinus terebinthifolius, Tonial et al. [48] explored the production of metabolites using 4 N (Nitrogen) and 3 C (Carbon) sources, different temperatures, pHs, and incubation time. Interestingly, independent of the species analyzed, galactose was the most effective source of carbon to produce active metabolites, acidification provided the best results in terms of activity against Candida albicans, while optimal temperature and nitrogen source varied depending on the strain.

In 2012, Koolen et al. [61] reported for the first time the isolation of cyclo-(glycyl-Ltyrosyl)-4,4-dimethylallyl ether, a diketopiperazine alkaloid, from Gliocladium sp. The compound showed high bactericidal activity against Micrococcus luteus (43.4 µM). Diketopiperazine alkaloids are known to possess a broad spectrum of actions exhibiting antibacterial, antifungal, and cytotoxic activities [62].

Andrioli et al. [63] explored the potential of the eugenitin compound, isolated from the endophytic strain Mycoleptodiscus indicus, to increase the production of the enzyme glucoamylase by Aspergillus niveus. Eugenitin increased the activity of A. niveus glucoamylase twofold, improving the production of glucose and ethanol using starch as a carbon source. The authors explored an unusual biological application to fungal metabolites [64], and their data highlight the importance of understanding the communication between endophytes in the activation of genes related to the production of metabolites [3]. In the same aspect, Chagas et al. [35] evaluated the interaction between endophytes of Smallanthus sonchifolius, aiming to understand the chemical communication between Alternaria tenuissima and Nigrospora sphaerica. A. tenuissima produced polyketides with antifungal activity in response to N. sphaerica. In addition, the effect of this relationship on the host was also evaluated, and even at concentrations higher than those required for antifungal activity, the compounds did not present phytotoxic activity to the host.

A screening program for the discovery of compounds produced by endophytic fungi from plants belonging to the Asteraceae family resulted in the isolation of two compounds, the steroid (22E,24R)-8,14-epoxyergosta-4,22-diene-3,6-dione (a) and the diterpene aphidicolin (b), with strong cytotoxicity against HL-60 cells [65]. Compounds (a) and (b) have been reported previously in the literature, however, the mechanism of action in HL-60 cells has not been elucidated. Using molecular targets, the authors suggested that compound (a) could influence the G2/M transition of the mitotic cells cycle, while compound (b) showed apoptotic activity. Since leukemia represents a common type of cancer among adults, and in the United States in 2012 more than 40 000 new cases were reported, finding new drugs and understanding how they act against leukemia cells are extremely important discoveries.

Pereira et al. [29] worked with 400 endophytic fungi isolated from different Brazilian ecosystems in order to select active strains against of Cryptococcus neoformans and Cryptococcus gattii. Strain Mycosphaerella sp. UFMGCB 2032, isolated from Eugenia bimarginata, showed remarkable antifungal activity, with MIC values of 31.2 and 7.8 µg/mL against Cryptococcus neoformans and C. gattii, respectively. After several chromatography techniques, two compounds were isolated and identified as responsible for antifungal activity, (2S,3R,4R)-(E)-2-amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoeicos-6,12-dienoic acid and (2S,3R,4R)-(E)-2-amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoeicos-6-enoic acid, against C. neoformans and C. gattii, with MICs of 1.3 – 2.50 µg/mL and 0.5 µg/mL, respectively. These compounds may represent an option to treat infections caused by Cryptococcus species.

The focus of many studies on natural products is to find compounds with antimicrobial or cytotoxic activities, while the antiparasitic potential remains little explored. Leishmaniasis is an endemic disease in Brazil [66] and is one of the most neglected diseases in the world, affecting poor people in developing countries [67]. In order to find new compounds for the treatment of Leishmaniasis, do Nascimento et al. [68] analyzed 16 endophytic fungi of Vernonia palyanthes. Using a bioassay-guided fractionation of the extract produced by the endophyte Cochliobolus sativus, the compounds cochlioquinone A, isocochlioquinone A, and anhydrocochlioquinone A were identified as responsible for antileishmanial activity, with EC₅₀ values of 1.7, 4.1, and 50.5 µg/mL, respectively.

Seven new compounds have been reported from endophytes found in Brazil in recent years ([Table 3]). The compounds belong to four chemical classes, butanolide (γ-lactone, δ-lactone), glyceride (monoacylglycerol) [35], alternariol [2], and azaphilone (mycoleptones) [56] ([Table 3]). Their chemical structures are listed in [Fig. 5]. The a – d compounds ([Fig. 5]) were isolated in a large amount from the crude extract of endophytes but showed no activity in the biological evaluations [2], [35]. The absence of activity under laboratory conditions may not necessarily reflect the role of these metabolites in nature. Knowledge of biological functions in the interaction of microbes with the environment can provide insight into how these molecules are used by the microorganism [69]. The mycoleptones ([Fig. 5 e – g]) are azophilones with an unusual methylene bridge and were produced by the endophytic strain M. indicus isolated from the medicinal plant Borreria verticillata. Azaphilones are known for their range of biological activity, such as antimicrobial, nematicidal, and anti-inflammatory [70]. Andrioli et al. [56] demonstrated that the new mycoleptones are non-selective compounds with antileishmanial and cytotoxic activities.

Fig. 5 Chemical structures of the new compounds isolated from endophytes in Brazil in the years 2012 – 2017, reported by Chagas et al. [35] (a – c); Chapla et al. [2] (d), and Andrioli et al. [56] (e – g). Chemical structures were obtained using the software Chemdraw (https://chemistry.com.pk/software/chemdraw-free/).

Secondary metabolites produced by endophytes, in general, are less toxic to eukaryotes than to prokaryotes, since endophytes should not harm the host plant [17]. This is especially true if we compare the number of compounds with antibacterial activity (16) with those showing antifungal activity (8) ([Table 3]). However, the need for new compounds for the treatment of neglected diseases present in Brazil, such as Leishmaniasis, has led to the development of programs to find metabolites with antiparasitic activity. These programs resulted in the isolation of 17 compounds with antiparasitic activity, the equivalent number of compounds with antibacterial activity ([Table 3]). The host tolerance to these compounds may be the result of similar molecules produced by the plants, or even the secretion of metabolites that inactivate the toxic metabolites produced by endophytes [6].

Conclusions

Brazil represents one of the largest biodiversities in the world and most of the biological sources remain underexplored. Between the years 2012 – 2017, more than 300 genera of fungi and bacteria were identified as endophytes of 54 plant species, with Diaporthe and Bacillus being the most isolated genera. The prevalence of these genera as endophytes may be related to the escape of the host immune response or the production of secondary metabolites that encompass advances in plant resistance to insects and pathogens. Endophytes found in Brazil have been linked as a source of bioactive molecules, some of them with a new molecular structure. Biotechnological advances contribute to enhancing the importance of Brazilian diversity, and new species and bioactive compounds are waiting to be reported.

Please note: this article was changed according to the following erratum: Some lines in [Table 2] were misrepresented and corrected.

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