Biotechnol Lett DOI 10.1007/s10529-015-1814-4
REVIEW
Plants and endophytes: equal partners in secondary metabolite production? Jutta Ludwig-Mu¨ller
Received: 30 January 2015 / Accepted: 12 March 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Well known plant production systems should be re-evaluated due to findings that the interesting metabolite might actually be produced by microbes intimately associated with the plant, socalled endophytes. Endophytes can be bacteria or fungi and they are characterized usually by the feature that they do not cause any harm to the host. Indeed, in some cases, such as mycorrhizal fungi or other growth promoting endophytes, they can be beneficial for the plant. Here some examples are reviewed where the host plant and/or endophyte metabolism can be induced by the other partner. Also, partial or complete biosynthesis pathways for plant secondary metabolites can be attributed to such endophytes. In other cases the host plant is able to metabolize substances from fungal origin. The question of the natural role of such metabolic changes for the endophyte will be briefly touched. Finally, the consequences for the use of plant cultures for secondary metabolite production is discussed. Keywords Antimicrobial activity Bioactive metabolites Biocontrol Endophyte In vitro conversion Secondary metabolites
J. Ludwig-Mu¨ller (&) Institute of Botany, Technische Universita¨t Dresden, 01062 Dresden, Germany e-mail: [email protected]
Introduction Plant secondary metabolite production relies on the increase of the plant material that is producing the metabolite of interest, alteration of secondary metabolite pathways, or the optimization of the production process of such plant materials (e.g. Chandra and Chandra 2011; Georgiev et al. 2012; Ludwig-Mu¨ller et al. 2014). In plants, the biosynthetic pathways and their regulation are complex. While developmental cues can regulate the synthesis of a range of secondary metabolites, some are only induced under certain circumstances, such as attacks by pathogens and/or insects. The synthesis of many metabolites is often the response to environmental signals, and these can be mimicked in culture to increase specific compounds. For example, caffeic acid could be increased by addition of the stress elicitor, jasmonic acid, but similarly was induced by an abiotic stress factor, for example O2 depletion (Nitzsche et al. 2004). The biosynthesis of taxol, however, could be induced by the bacterial toxin, coronatine, a mimic of the active JA-isoleucine conjugate (Katsir et al. 2008), which was a better elicitor than methyl jasmonate (Onrubia et al. 2011). Other examples for stressors that can induce secondary metabolite synthesis include elicitor molecules from pathogens, such as glucans, flagellin, harpins or systemin (Zhao et al. 2005). These and other findings allow the production of a specific metabolite of interest also in large scale.
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While some observations about the ability of plant endophytes to produce secondary plant metabolites were made about two decades ago (e.g. Strobel et al. 1993; Lee et al. 1996), only within recent years has it become clear that not only plants, but also their pathogens or endophytes can contribute to the patterns of their secondary metabolites. With pathogen/plant interactions the compounds cannot be easily produced because of the one-sided nature of the relationship that ultimately leads to the decay of the host plant. Endophytes, however, can live within their host plant for a long time. Since many endophytes produce secondary metabolites with interesting bioactive properties, their contribution to the wealth of secondary metabolites has attracted much attention. Therefore, the potential of endophytes to synthesize secondary metabolites and to use them in biotechnology has been recognized (e.g. Casella et al. 2013). There are already several recent reviews touching the ability of endophytes to produce plant metabolites (Aly et al. 2013; Kusari et al. 2013; Gandhi et al. 2015). Here some examples will be reviewed where partial or complete biosynthesis pathways can be attributed to such endophytes. The consequences for the use of plant cultures for secondary metabolite production will be discussed.
Definition of endophytes While pathogens adversely influence plant performance, endophytes might not always be obvious in their colonization of hosts (Schulz and Boyle 2005). Endophytes are generally considered as organisms (bacteria or fungi) that have a beneficial effect on their host during the interaction or only under certain circumstances, for example abiotic or biotic stress situations (e.g. Rodriguez et al. 2009). It is important to note that a given plant may contain several different endophyte species (Tan and Zou 2001). However, this cirumstance can also lead to the synthesis of novel metabolites in host and endophytes, because of their competing influences on each other (Kusari et al. 2012a). Based on their potential to induce defense responses (Arnold et al. 2003), some endophytes are also used for biocontrol: e.g. the fungal Acremonium or Heteroconium species (Joost 1995; Narisawa et al. 2000; Ja¨schke et al. 2010) or Bacillus subtilis that is already
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available as commercial product (Lahlali et al. 2013). In some cases, the effect can be attributed to the production of toxins as in the Acremonium zeae/maize interaction (Poling et al. 2008). Endophytes also contribute to plant defense mechanisms by preventing herbivory (Zhang et al. 2011) or reducing the offspring number of plant pests (Jaber and Vidal 2009). Secondary metabolites produced by the endophytes could alter the balance between beneficial interaction or defense, because endophytes can occasionally turn into pathogens (Schulz and Boyle 2005). On the one hand, plants can produce these compounds to alter growth of fungi within their tissue before and after colonization but, on the other hand, the fungi can produce secondary metabolites for their own benefit, for example to get rid of competitors in a host plant. The host plant can then modify either toxic compounds to deal with them if these are too harmful, or to use them in alternative processes (Fig. 1).
Endophytes produce bioactive compounds Mainly within the last decade endophytes have attracted greater attention due to their potential to synthesize a wide array of bioactive secondary metabolites (Tan and Zou 2001; Schulz et al. 2002; Strobel 2003; Prado et al. 2013). The endophytederived compounds belong to diverse structural groups such as terpenoids, steroids, xanthones, chinones, phenols, isocoumarins, benzopyranones, tetralones, cytochalasines, and enniatines (Schulz et al. 2002). They sometimes constitute variations of already known structures such as ergosterol, a fungal steroid, or the plant hormone indole-3-acetic acid (Lu et al. 2000). The biosynthesis of such compounds is important for the endophyte when it is in competition with other organisms surrounding the plant (Schulz et al. 1999). Therefore, in many cases these compounds possess antimicrobial activities. The protection of plants against pests can often be attributed to some of the compounds found in endophytes (e.g. Poling et al. 2008). To isolate endophytes from the close vicinity of their hosts was a successful strategy in the identification of (novel) organisms with high antimicrobial potential and in many cases new compounds could be isolated and characterized (e.g. Casella et al. 2013). However, there are also studies that report antimicrobial effects without further
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identification of the active principle in extracts of endophytes (e.g. Liu et al. 2001), so among these reports there is still a great potential for novel compounds. A screening for endophytes containing antimicrobial and herbicidal compounds showed that endophytes can also synthesize compounds directed against plants (Schulz et al. 1999). In addition, it was found in this study that the number of endophytes producing these herbicidal substances was three times higher than of soil microbes and twice that of plant pathogenic microorganisms. Insects were also proposed to influence endophytic fungi producing bioactive compounds and host plants (Kusari et al. 2013). Therefore, also insecticidal substances can be found among those produced by the endophytes. However, the scenario might become more complicated by observations that fungi contain their own symbionts. For example the plant pathogenic fungus, Rhizopus, does not synthesize the compound, rhizoxin, as previously attributed, but a symbiotic bacterium (Burkholderia) within the fungus (Partida-Martinez and Hertweck 2005). Also endophytic fungi can influence their metabolism in a complicated crosstalk if more tha one is present in a given host plant (Kusari et al. 2013).
Plant versus endophyte metabolism Plant/endophyte metabolism can interact on may levels (Fig. 1): (a) the endophyte induces host metabolism, (b) the host induces endophyte metabolism, (c) host and endophyte share parts of a specific pathway and contribute partially, (d) the host can metabolize products from the endophyte and vice versa (e) the endophyte can metabolize secondary compounds from the host. The two latter possibilities can concern only one, several or all enzymatic steps for biochemical transformation. Changes in host metabolism Some reports have shown that endophytes can induce host secondary metabolism but this aspect of the interaction is much less explored than the induction of endophyte metabolism, even though defense-related phenolic compounds in endophyte-infected roots were already described some time ago (Schulz et al. 1999). In a recent example in grasses (specifically in Lolium perenne) after infection with endophytes from the Clavicipitaceae family, an induction of phenols was observed (Qawasmeh et al. 2012). However, depending on the type of endophyte, metabolites could also be
Fig. 1 A simplified scheme showing possible interactions and roles for plant versus endophyte metabolite production and the contribution of the respective organisms to the other metabolic pathways. The induction of plant metabolism can be caused by environmental signals such as abiotic stress factors or pathogens/herbivores, but also by the endophytes themselves. In addition, developmental signals can induce secondary metabolite synthesis at defined stages, e.g. leaf colors or flowering, where pigments and volatiles are synthesized
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reduced or remain unchanged. Major compounds were glycosylated flavonoids and hydroxycinnamic acids; and chlorogenic acid was the major compound involved in an induction of the antioxidative activity of plant extracts after endophyte colonization (Qawasmeh et al. 2012). A different scenario is occurring when plants experience toxic compounds from the endophytes since some might have herbicidal activity (see ‘Endophytes produce bioactive compounds’). A Taxus species is able to glycosylate the endophyte-derived peptide leucinostatin A (Strobel and Hess 1997), whereas for other, non-host plants this compound may still be toxic. Such poisonous compounds may be used to determine the specificity of an interrelationship between fungus and plant. Influence of plant host on microbial metabolism As described above, endophytes can influence their host plant’s metabolism, but one can speculate that the established host range could also alter or influence the pattern of secondary metabolites in endophytic fungi. More so, host plants can influence the metabolite pattern in pathogenic fungi. For a complex of phytopathogenic Heterobasidium species, two pineinfecting and three non-pine infecting species showed different metabolite patterns which could be separated by principal component analysis after LC–MS analysis into five distinct groups, but also according to their hosts (Hansson et al. 2014). The main difference between the pine-infecting species and the non-pine infecting species was the production of the phytotoxic benzohydrofuran fomannoxin exclusively by the two pine infecting fungi (Hansson et al. 2014). However, due to the detection of this compound in another Heterobasidium sp. not infecting pine, the differences were thought to occur due to differential gene regulation. Synthesis of plant host compounds by endophytes The most fascinating topic is either biosynthesis or metabolism of host plant metabolites by endophytes, but only a few examples can be discussed here. In a recent review Kusari and colleagues (2013) pointed out that ‘‘The biosynthetic potential of endophytic fungi has gained impetus in recent times owing to the continual discovery of fungal endophytes capable of
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synthesizing plant compounds. However, the sustained production of the desired plant compounds has not yet been achieved using endophytes. It is thus imperative to investigate the diverse interactions that endophytes have with coexisting endophytes, host plants, insect pests, and other specific herbivores.’’ This implies that careful investigations on the nature of a given compound need to be done whether it is from plant origin, from the endophyte or can be produced by both. In the latter case the endophyte might contribute with the complete pathway, but another scenario might be that only parts of the biosynthesis derive from the endophyte. As a reason for the observed capability to synthesize these plant metabolites it was speculated that horizontal gene transfer is at least one possible scenario how the endophytes have gained their genes encoding biosynthetic enzymes. The most prominent example for (partial) plant metabolite synthesis so far might be the production of paclitaxel (Taxol), due to its medicinal use, by endophytes. Its production in cell cultures has been optimized to a large extent (Malik et al. 2011; Cusido et al. 2014). However, due to the large demand of the compound as anticancer drug, alternative sources to the plant material were sought (Heinig et al. 2013). The discovery of Taxol production in the endophytic fungus Taxomyces andreanae, which was found in Taxus brevifolia (Strobel et al. 1993), was followed by the isolation of other fungal endophytes from a variety of Taxus spp. (e.g. Strobel et al. 1996; Zhang et al. 2009). Such approaches have resulted in many reports on the identification on the microbial taxane production in a variety of species among them Alternaria, Aspergillus, Cladosporium, Fusarium, Monochaetia, Pestlotia, Pestalotiopsis, Pithomyces, Penicillium and Xylaria, which were isolated from yew and non-Taxus plants (cf Heinig et al. 2013). However, a reexamination of fungal and host plant taxane biosynthesis revealed that in this study no independent taxane biosynthesis in the endophytes was found (Heinig et al. 2013; see also Problems with single cultures). Other bioactive metabolites were found in an endophytic fungus, Pestalotiopsis microspora, isolated from another species belonging to the Taxaceae (Lee et al. 1996). The compound, torreyanic acid, has also anti-cancerogenic characteristics. Similar observations came from plant species from unrelated families, for example Berberidaceae, where an endophyte was able to produce podophyllotoxin (Eyberger
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et al. 2006); or Meliaceae, where an endophytic fungus, Fusarium sp., was reported to produce the compound, rohitukine, a precursor for another anticancer drug (Mohana Kumara et al. 2012). Of aryl tetralin lignans, podophyllotoxin, for example, is synthesized by Podophyllum but the production of this and other valuable compounds has not been achieved yet in a sustainable way (Puri et al. 2006). A novel endophytic fungus Trametes hirsuta that also produces these compounds has been envisioned as novel, promising biological system for the synthesis of podophyllotoxin (Puri et al. 2006). An endophyte related to a Chaetomium species was isolated from Hypericum perforatum that can produce hypericin and the putative biosynthetic precursor emodin in an in vitro culture system (Kusari et al. 2008). Kusari et al. (2012b) described the isolation of the novel endophytic fungus Eupenicillium parvum from the neem tree, Azadirachta indica, and found that the fungus also produced the insecticidal compounds, azadirachtin A and B. Of course, all these examples do not exclude the existence of other endophytes on the host plants mentioned. For example, Verma et al. (2009) report on the biodiversity of the microbial endophytes from Azadirachta indica. The examples mentioned above include fungi as endophytes but endophytic bacteria can also share compounds with their host. Alternanthera brasiliana (Amaranthaceae) stem extracts contain antimicrobial compounds from the oxylipin family (Trapp et al. 2015). Among these several that were also found in bacteria from the genus Bacillus isolated from Alternathera plants and the authors speculated that the host plant obtained the antimicrobial oxylipins from their bacterial endophytes (Trapp et al. 2015).
to the production of the corresponding aglycone in the medicinal host plant Cephalotaxus harringtonia via deglycosylation. The deglycosylated flavonoids turned out to display significant beneficial effects on the hyphal growth of germinated spores (Tian et al. 2014). These data proved that a close interaction between the two organisms took place on a biochemical level. Another example was detected for symbiotic fungi that alter host metabolism in a way that leaf-cutting ants preferred non-colonized plants or extracts from these. The individual leaf compounds responsible for this response were not identified, but thought to constitute low or non-volatile compounds (Estrada et al. 2013). In addition, there are many examples that endophytes can reduce the attractiveness of their host for herbivores. Another role of plant metabolite modification by endophytes is the detoxification of defense compounds of the host (Fig. 1). For example, maize plants synthesize the benzoxazinoids for their own defense against various pests. The toxic compounds are present in a glycosylated form, while deglycosylation leads to a non-toxic metabolite. Therefore, enzymatic deglycosylation will result in tolerant microorganisms, if they have the respective enzyme (Saunders and Kohn 2008). In this study it was also observed that fungi, which can degrade the toxic conpounds, can lead the way for colonization by other, not so tolerant endophytes. Alternative metabolic steps used to detoxify these plant compounds by other endophytic fungi include acylation, oxidation, reduction, hydrolysis, and nitration (Zikmundova´ et al. 2002).
Considerations for exploring the (biotechnological) potential of endophytes
Metabolism of plant host compounds by endophytes
Problems with single cultures
The fungal leaf endophyte, Paraconiothyrium variabile, is able to metabolize its host plant metabolome (Tian et al. 2014). However, the function of these biochemical reactions is not yet well understood. Therefore, the changes in the metabolome were monitored and these compounds, where the main changes were observed, were tested for beneficial effects on the fungus. A specific biotransformation of glycosylated flavonoids by the endophyte was observed (Tian et al. 2014) (Fig. 2). In all cases, this led
It is of interest to investigate endophytes better for bioprospecting, not only against plant pests (Schulz et al. 2002), but also against human pathogens such as Mycobacterium tuberculosis (Alvin et al. 2014) and as sources for novel drugs (Suryanarayanan et al. 2009). Microbes sometimes use different biosynthetic pathways and can synthesize cyclic peptides with high antimicrobial potential by a reaction so far not found in plants, namely nonribosomal peptide synthesis (e.g. Schwarzer et al. 2003). Generally, microbes are
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Biotechnol Lett Fig. 2 Some examples for in planta (right side) and in vitro (left side) bioconversion of metabolites by endophytes. Red boxes stand for a direct beneficial effect for the fungus, blue boxes indicate an effect mainly on the host plant. Black boxes indicate single enzymatic reactions, orange boxes depict complex pathways
desirable due to their short generation time, high biomass production due to high growth rates and good handling features in bioreactors. However, as pointed out above, the habitat of the host plant needs also be taken into account when novel compounds produced by endophytes should be isolated (Strobel 2003). When it is suspected that a certain pathway can be contributed by either plant or endophyte, then the contribution of each partner makes needs to be investigated. If only parts of the pathway derive from the endophyte then it is understandable that, as pointed out by Kusari et al. (2013), sustained production of the plant metabolite by the endophyte is not yet possible. However, for Taxol it was possible to get complete production from the endophytic fungus Taxomyces andreanae, isolated from the host tree Taxus brevifolia (Strobel et al. 1993), albeit not yet sufficient for biotechnological applications. This might be due to the observation that the fungal metabolites decrease to a large extent over a longer culture period (e.g. Gandhi et al. 2015). For example, the production of camptothecin was attenuated from the first to the seventh generation of subculturing of the camptothecin-producing endophytic fungus (Kusari et al. 2009) or even completely lost within two other fungal strains from the genus Aspergillus (Pu et al. 2013). However, this might be species dependent, because the same authors found that a Trichoderma species could still produce camptothecin even after the eighths generation with only minor reduction (Pu et al. 2013).
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Other possibilites to explain the lack of secondary metabolites in the endophytic cultures were brought up by Heinig et al. (2013). These authors claimed that they could not find unequivocally taxane synthesis in endophytic fungi alone. One criticism to previous publications was that PCR to identify fungal genes was performed with primers developed based on the plant sequences. They point out that ‘‘The presence of these genes would require the extensive horizontal gene transfer (HGT) between the yew trees and multiple endophytic fungi, representing a pathway with more than 20 steps.’’ (Heinig et al. 2013). Further experiments involving metabolite analysis and sequencing approaches did not support the presence of genes that could be capable of taxane synthesis in one strain. The take-home message is that more cautios investigations are necessary as to which partner is making the bioactive compounds, especially if these should be used for production purposes. Selection of plant material for endophyte isolation A rationale for eventually isolating endophytes from different plant species was proposed by Strobel (2003), who suggested that the plant species should be selected according to the environment where a specific benefit was observed. These conditions could be caused by abiotic or biotic stress factors and benefits conveyed by endophytes. For example, a plant from harsh aquatic environment that resists infection
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by an oomycete was selected by his group. In this plant, a novel antimicrobial agent was identified which was actually produced by a bacterial endophyte, Serratia marcescens, within the aquatic plant Rhyncholacis penicillata and helped in the protection against oomycetes (Strobel et al. 1999). Also, plants surrounded by other infected plants but themselves showing no symptoms could be a source to look for endophytes. By this approach an antimicrobial activity against the plant pathogenic fungus Colletotrichum musae was isolated (Tuntiwachwuttikul et al. 2008). Additional important points were raised by Yu et al. (2010) namely to look into areas with great biodiversity on the plant side, because these are most likely also to contain a large variety of endophytes. Also, the exploitation of traditional medicinal plants could lead to the identification of endophytes with a potential to produce bioactive compounds. Finally, the isolation of endophytes of plants from ancient land areas might be more promising, again possibly due to a longer evolutionary period and the potential to develop more different endophytic organisms (Yu et al. 2010). In vitro biotransformation by endophytes The metabolism of plant secondary compounds by endophytes can be explored by using plant endophytes for in vitro biotransformation of plant metabolites to valuable products (Fig. 2). The use of endophytes as biocatalysts might open up a new avenue for their biotechnological use. As biocatalysts, the endophytes can add stereospecificity to bioprocesses and thus reduce complicated purification steps of chemically-synthesized enantiomers (Borges et al. 2009). The biotransformation of a racemic mixture to stereoselective thioridazine sulfoxides was investigated (Borges et al. 2008). Prado et al. (2013) employed the endophytic fungus Paraconiothyrium variabile for an one-step enantioslective synthesis of (4S)-isosclerone from the natural precursor juglone. Other endophytes simply metabolize the core moiety to other, maybe even more bioactive compounds, or substances with novel properties. Curcuma longa contains the bioactive compound curcumin (Prana et al. 2010). Biotransformation was detected using vaious endophytic fungi isolated from the rhizome of the host plant, but the major compound was not indentifed in this work. The authors showed
that the metabolite production was dependent on the culture medium used. Oxidation and hydroxylation are simple enzymatic reactions to modify a structure. The stereoselective biooxidation at C4-flavans, specifically of (?)-catechin and (-)-epicatechin into dihydroflavan derivatives, was observed for the endophytic fungus Diaporthe sp. that was isolated originally from Camellia sinensis (Agusta et al. 2005; Shibuya et al. 2005). Later, Agusta et al. (2014) reported the conversion of the alkaloid berberine into its 7-N-oxide by cultures of the endophytic fungus Coelomycetes sp. Deacetylation, hydroxylation and epoxidation was described for the biotransformation of taxoids by endophytic fungi isolated originally from the inner bark of Taxus yunnanensis (Zhang et al. 1998). Volatiles are also on the list of compounds to be biotechnologically produced by endophytes (Abraha˜o et al. 2013). These compounds are important in the plant for either attracting or repelling mainly insects. For biotechnology they are of interest as flavor compounds and can be generated either by de novo synthesis or by metabolism, for example bio-oxidation of the respective terpenes (Bicas et al. 2009). Taken together, all these examples of manipulation of host metabolites in planta or in vitro (Fig. 2) indicate that the potential exploitation for endophytes in biotechnology is probably just at the beginning.
Conclusion and future prospects The observations pointed out above indicate that there will be many future challenges when appropriate cultivations systems need to be designed for secondary metabolite production. While microbial production systems depend often on the optimization of culture conditions, the production system might turn out to be problematic in cases where both partners contribute to the synthesis of the desired compound. Cultivation of endophytes without their host might result in the loss of the desired compound synthesis as pointed out above (see Problems with single cultures). Transgenic strategies to improve production in an endophyte grown without host plant can include genes from the same organism (homologous gene expression; e.g. Hwang et al. 2014), but more likely a suitable production
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host for the compound in question needs to be found (heterologous expression; e.g. Sakai et al. 2012). Therefore, such production systems could lead to a desired metabolite, if enough is known about the biosynthetic pathways. One challenge, therefore, remains the elucidation of the (control of) biosynthetic pathways by all participating partners. A controlled production with mature plants harboring the endophyte in question is very likely not possible due to sterility problems because of the microorganism(s) present. In addition, there is no way to know how many microbes are indeed present in a given plant species, and this hampers the controlled production of metabolites as well. Generating plant cultures harboring the endophyte might be possible, but whether the endophyte can be kept stable and productive in the plant over a long time is questionable, even though reports on the contamination of plant cultures with endophytes over a long period exist (Leifert and Cassells 2001). Elicitation of plant metabolite production by microbial culture filtrates (Verma et al. 2014) or elicitors generated from the microorganisms (Zhao et al. 2005) has been described in many cases (see also Introduction). However, in these cases the contribution to plant biosynthetic pathways is not by the living organisms. Alternatives could be the co-cultivation of sterile endophyte cultures together with sterile plant cultures, if the latter can be shown to have the ability of secondary metabolite production similar to the mother plant. Co-cultivation (or co-culture) of microorganisms, for example two fungal or two bacterial strains, has been exploited to generate novel compounds and to enhance the production of one desired product (Bertrand et al. 2014). To achieve larger scale production methods for co-fermentation can be used. This could also be a possible way to generate the desired metabolites from plant organ cultures, such as shoots or hairy roots, with defined endophytes during a cocultivation approach. Like for microbial co-cultivation careful monitoring of product formation would be required. Here another challenge emerges, namely the design of appropriate cultivation systems for larger scale production. Whether this might be a possibility to circumvent the problems with the contributions of the two partners—plant and endophyte - to a specific compound formation has to be elucidated in future work.
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Acknowledgments Work in the author’s laboratory on secondary metabolites is funded by the European Union, The German Ministry for Education and Science and the State of Saxony.
References Abraha˜o MRE, Molina G, Pastore GM (2013) Endophytes: recent developments in biotechnology and the potential for flavor production. Food Res Int 52:367–372 Agusta A, Maehara S, Ohashi K, Simanjuntak P, Shibuya H (2005) Stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. isolated from a tea plant. Chem Pharm Bull 53:1565–1569 Agusta A, Wulansari D, Praptiwi Nurkanto A, Fathoni A (2014) Biotransformation of protoberberine alkaloids by the endophytic fungus Coelomycetes AFKR-3 isolated from yellow moonsheed plant (Archangelisia flava (L.) Merr.). Proced Chem 13:38–43 Aly AH, Debbab A, Proksch P (2013) Fungal endophytes— secret producers of bioactive plant metabolites. Pharmazie 68:499–505 Arnold AE, Mejı´a LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, Herre EA (2003) Fungal endophytes limit pathogen damage in a tropical tree. Proc Natl Acad Sci USA 26:15649–15654 Bertrand S, Bohni N, Schnee S, Schumpp O, Gindro K, Wolfender J-L (2014) Metabolite induction via microorganism co-culture: a potential way to enhance chemical diversity for drug discovery. Biotechnol Adv 32: 1180–1204 Bicas JL, Dionisio AP, Pastore GM (2009) Bio-oxidation of terpenes: an approach for the flavor industry. Chem Rev 109:4518–4531 Borges KB, Borges WdS, Pupo MT, Bonato PS (2008) Stereoselective analysis of thioridazine-2-sulfoxide and thioridazine-5-sulfoxide: an investigation of rac- thioridazine biotransformation by some endophytic fungi. J Pharm Biomed Anal 46:945–952 Borges KB, Borges WdS, Dura´n-Patro´n R, Pupo MT, Bonato PS, Collado IG (2009) Stereoselective biotransformations using fungi as biocatalysts. Tetrahedron Asymmetry 20:385–397 Casella TM, Eparvier V, Mandavid H, Bendelac A, Odonne G, Dayan L, Duplais C, Espindola LS, Stien D (2013) Antimicrobial and cytotoxic secondary metabolites from tropical leaf endophytes: isolation of antibacterial agent pyrrocidine C from Lewia infectoria SNB-GTC2402. Phytochemistry 96:370–377 Chandra S, Chandra R (2011) Engineering secondary metabolite production in hairy roots. Phytochem Rev 10:371–395 Cusido RM, Onrubia M, Sabater-Jara AB, Moyano E, Bonfill M, Goossens A, Pedren˜o MA, Palazon J (2014) A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp. Biotechnol Adv 32:1157–1167 Estrada C, Wcislo WT, Van Bael SA (2013) Symbiotic fungi alter plant chemistry that discourages leaf-cutting ants. New Phytol 198:241–251
Biotechnol Lett Eyberger AL, Dondapati R, Porter JR (2006) Endophyte fungal isolates from Podophyllum peltatum produce podophyllotoxin. J Nat Prod 69:1121–1124 Gandhi SG, Mahajan V, Bedi YS (2015) Changing trends in biotechnology of secondary metabolism in medicinal and aromatic plants. Planta 241:303–317 Georgiev M, Agostini E, Ludwig-Mu¨ller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnology. Trends Biotechnol 30:528–537 ˚ , Karlsson M, Staerk D, Broberg Hansson D, Wubshet S, Olson A A (2014) Secondary metabolite comparison of the species within the Heterobasidion annosum s.l. complex. Phytochemistry 108:243–251 Hwang K-S, Kim HU, Charusanti P, Palsson BØ, Lee SY (2014) Systems biology and biotechnology of Streptomyces species for the production of secondary metabolites. Biotechnol Adv 32:255–268 Jaber LR, Vidal S (2009) Interactions between an endophytic fungus, aphids, and extrafloral nectaries: do endophytes induce extrafloral-mediated defences in Vicia faba? Funct Ecol 23:707–714 Ja¨schke D, Dugassa-Gobena D, Karlovsky P, Vidal S, Ludwig-Mu¨ller J (2010) Suppression of clubroot development in Arabidopsis thaliana by the endophytic fungus Acremonium alternatum. Plant Pathol 59: 100–111 Joost RE (1995) Acremonium in fescue and ryegrass: boon or bane? A review. J Anim Sci 73:881–888 Katsir L, Schilmiller AL, Staswick PE, He SY, Howe GA (2008) COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine. Proc Natl Acad Sci USA 105:7100–7105 Kusari S, Lamsho¨ft M, Zu¨hlke S, Spiteller M (2008) An endophytic fungus from Hypericum perforatum that produces hypericin. J Nat Prod 71:159–162 Kusari S, Zu¨hlke S, Spiteller M (2009) An endophytic fungus from Camptotheca acuminata that produces camptothecin and analogues. J Nat Prod 72:2–7 Kusari S, Hertweck C, Spiteller M (2012a) Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol 19:792–798 Kusari S, Verma VC, Lamshoeft M, Spiteller M (2012b) An endophytic fungus from Azadirachta indica A. Juss. that produces azadirachtin. World J Microbiol Biotechnol 28:1287–1294 Kusari S, Pandey SP, Spiteller M (2013) Untapped mutualistic paradigms linking host plant and endophytic fungal production of similar bioactive secondary metabolites. Phytochemistry 91:81–87 Lahlali R, Peng G, Gossen BD, McGregor L, Yu FQ, Hynes RK, Hwang SF, McDonald MR, Boyetchko SM (2013) Evidence that the biofungicide Serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance. Phytopathology 103:245–254 Lee JC, Strobel GA, Lobkovsky E, Clardy J (1996) Torreyanic acid: a selectively cytotoxic quinone dimer from the endophytic fungus Pestalotiopsis microspora. J Org Chem 61:3232–3233 Leifert C, Cassells AC (2001) Microbial hazards in plant tissue and cell cultures. In Vitro Cell Dev Biol Plant 37:133–138
Liu CH, Zou WX, Lu H, Tan RX (2001) Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi. J Biotechnol 88:277–282 Lu H, Zou WX, Meng JC, Hu J, Tan RX (2000) New bioactive metabolites produced by Colletotrichum sp., an endophytic fungus in Artemisia annua. Plant Sci 151:67–73 Ludwig-Mu¨ller J, Jahn L, Lippert A, Pu¨schel J, Walter A (2014) Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: strategies and applications. Biotechnol Adv 32:1168–1179 Malik S, Cusido´ RM, Mirjalili MH, Moyano E, Palazo´n J, Bonfill M (2011) Production of the anticancer drug taxol in Taxus baccata suspension cultures: a review. Proc Biochem 46:23–34 Mohana Kumara P, Zuehlke S, Priti V, Ramesha BT, Shweta S, Ravikanth G, Vasudeva R, Santhoshkumar TR, Spiteller M, Uma Shaanker R (2012) Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity. Antonie Van Leeuwenhoek 101:323–329 Narisawa K, Ohki T, Hashiba T (2000) Suppression of clubroot and Verticillium yellows in Chinese cabbage in the field by the root endophytic fungus, Heteroconium chaetospira. Plant Pathol 49:141–146 Nitzsche A, Tokalov SV, Gutzeit HO, Ludwig-Mu¨ller J (2004) Chemical and biological characterization of cinnamic acid derivatives from cell cultures of lavender (Lavandula officinalis) induced by stress and jasmonic acid. J Agric Food Chem 52:2915–2923 Onrubia M, Moyano E, Bonfill M, Cusido´ RM, Goossens A, Palazo´n J (2011) Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate. J Plant Physiol 170:211–219 Partida-Martinez LP, Hertweck C (2005) Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature 437:884–888 Poling SM, Wicklow DT, Rogers KD, Gloer JB (2008) Acremonium zeae, a protective endophyte of maize produces dihydroresorcylide and 7-hydroxydihydroresorcylides. J Agric Food Chem 56:3006–3009 Prado S, Buisson D, Ndoye I, Vallet M, Nay B (2013) One-step enantioselective synthesis of (4S)-isosclerone through biotransformation of juglone by an endophytic fungus. Tetrahedron Lett 54:1189–1191 Prana TK, Srikandace J, Sumitro E, Wulandari D (2010) The potency of endophytic fungi of turmeric (Curcuma longa L.) in biotransformation of curcumic compounds in various media. Res J Microbiol 5:1189–1198 Pu X, Qu X, Chen F, Bao J, Zhang G, Luo Y (2013) Camptothecin-producing endophytic fungus Trichoderma atroviride LY357: isolation, identification, and fermentation conditions optimization for camptothecin production. Appl Microbiol Biotechnol 97:9365–9375 Puri SC, Nazir A, Chawla R, Arora R, Riyaz-ul-Hasan S, Amnaa T, Ahmeda B, Verma V, Singh S, Sagar R, Sharma A, Kumar R, Sharma RK, Qazi GN (2006) The endophytic fungus Trametes hirsuta as a novel alternative source of podophyllotoxin and related aryl tetralin lignans. J Biotechnol 122:494–510
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Biotechnol Lett Qawasmeh A, Obied HK, Raman A, Wheatley W (2012) Influence of fungal endophyte infection on phenolic content and antioxidant activity in grasses: interaction between Lolium perenne and different strains of Neotyphodium lolii. J Agric Food Chem 60:3381–3388 Rodriguez RJ, White JF Jr, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330 Sakai K, Kinoshita H, Nihira T (2012) Heterologous expression system in Aspergillus oryzae for fungal biosynthetic gene clusters of secondary metabolites. Appl Microbiol Biotechnol 93:2011–2022 Saunders M, Kohn LM (2008) Host-synthesized secondary compounds influence the in vitro interactions between fungal endophytes of maize. Appl Env Microbiol 74:136–142 Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686 Schulz B, Rommert AK, Dammann U, Aust HJ, Strack D (1999) The endophyte host interaction: a balanced antagonism? Mycol Res 103:1275–1283 Schulz B, Boyle C, Draeger S, Rommert A-K, Krohn K (2002) Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol Res 106:996–1004 Schwarzer D, Finking R, Marahiel MA (2003) Nonribosomal peptides: from genes to products. Nat Prod Rep 20:275–287 Shibuya H, Agusta A, Ohashi K, Maehara S, Simanjuntak P (2005) Biooxidation of (?)- catechin and (-)- epicatechin into 3,4-dihydroxyflavan derivatives by the endophytic fungus Diaporthe sp. isolated from a tea plant. Chem Pharm Bull 53:866–867 Strobel GA (2003) Endophytes as sources of bioactive products. Microbes Infect 5:535–544 Strobel GA, Hess WM (1997) Glucosylation of the peptide leucinostatin A, produced by an endophytic fungus of European yew, may protect the host from leucinostatin toxicity. Chem Biol 4:529–536 Strobel G, Stierle A, Stierle D, Hess WM (1993) Taxomyces andreanae, a proposed new taxon for a bulbilliferous hyphomycete associated with Pacific yew (Taxus brevifolia). Mycotaxon 47:71–80 Strobel G, Yang X, Sears J, Kramer R, Sidhu RS, Hess WM (1996) Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiol 142:435–440 Strobel GA, Li JY, Sugawara F, Koshino H, Harper J, Hess WM (1999) Oocydin A, a chlorinated macrocyclic lactone with potent antioomycete activity from Serratia marcescens. Microbiol 145:3557–3564
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Suryanarayanan TS, Thirunavukkarasu N, Govindarajulu MB, Sasse F, Jansen R, Murali TS (2009) Fungal endophytes and bioprospecting. Fungal Biol Rev 23:9–19 Tan R, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep 18:448–459 Tian Y, Amand S, Buisson D, Kunz C, Hachette F, Dupont J, Nay B, Prado S (2014) The fungal leaf endophyte Paraconiothyrium variabile specifically metabolizes the hostplant metabolome for its own benefit. Phytochemistry 108:95–101 Trapp MA, Kai M, Mitho¨fer A, Rodrigues-Filho E (2015) Antibiotic oxylipins from Alternanthera brasiliana and its endophytic bacteria. Phytochemistry 110:72–82 Tuntiwachwuttikul P, Taechowisan T, Wanbanjob A, Thadaniti S, Taylor WC, Lansai A-D (2008) Secondary metabolites from Streptomyces sp. SUC1. Tetrahedron 64:7583–7586 Verma VC, Gond SK, Mishra A, Kumar A, Kharwar RN, Gange AC (2009) Endophytic actinomycetes from Azadirachta indica A. Juss.: isolation, diversity and anti-microbial activity. Microbial Ecol 57:749–756 Verma P, Khan SA, Mathur AK, Shanker K, Kalra A (2014) Fungal endophytes enhanced the growth and production kinetics of Vinca minor hairy roots and cell suspensions grown in bioreactor. Plant Cell Tiss Org Cult 118:257–268 Yu H, Zhang L, Li L, Zheng C, Guo L, Li W, Sun P, Qin L (2010) Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbial Res 165:437–449 Zhang J, Zhang L, Wang X, Qiu D, Sun D, Gu J, Fang Q (1998) Microbial transformation of 10-deacetyl-7-epitaxol and 1b-hydroxybaccatin I by fungi from the inner bark of Taxus yunnanensis. J Nat Prod 61:497–500 Zhang P, Zhou P-P, Yu L-J (2009) An endophytic taxol-producing fungus from Taxus media, Cladosporium cladosporioides MD2. Curr Microbiol 59:227–232 Zhang XX, Li CJ, Nan ZB, Matthew C (2011) Neotyphodium endophyte increases Achnatherum inebrians (drunken horse grass) resistance to herbivores and seed predators. Weed Res 52:70–78 Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333 Zikmundova´ M, Drandarov K, Bigler L, Hesse M, Werner C (2002) Biotransformation of 2-benzoxazolinone and 2-hydroxy-1,4-benzoxazin-3-one by endophytic fungi isolated from Aphelandra tetragona. Appl Env Microbiol 68:4863–4870
REVIEW
Plants and endophytes: equal partners in secondary metabolite production? Jutta Ludwig-Mu¨ller
Received: 30 January 2015 / Accepted: 12 March 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Well known plant production systems should be re-evaluated due to findings that the interesting metabolite might actually be produced by microbes intimately associated with the plant, socalled endophytes. Endophytes can be bacteria or fungi and they are characterized usually by the feature that they do not cause any harm to the host. Indeed, in some cases, such as mycorrhizal fungi or other growth promoting endophytes, they can be beneficial for the plant. Here some examples are reviewed where the host plant and/or endophyte metabolism can be induced by the other partner. Also, partial or complete biosynthesis pathways for plant secondary metabolites can be attributed to such endophytes. In other cases the host plant is able to metabolize substances from fungal origin. The question of the natural role of such metabolic changes for the endophyte will be briefly touched. Finally, the consequences for the use of plant cultures for secondary metabolite production is discussed. Keywords Antimicrobial activity Bioactive metabolites Biocontrol Endophyte In vitro conversion Secondary metabolites
J. Ludwig-Mu¨ller (&) Institute of Botany, Technische Universita¨t Dresden, 01062 Dresden, Germany e-mail: [email protected]
Introduction Plant secondary metabolite production relies on the increase of the plant material that is producing the metabolite of interest, alteration of secondary metabolite pathways, or the optimization of the production process of such plant materials (e.g. Chandra and Chandra 2011; Georgiev et al. 2012; Ludwig-Mu¨ller et al. 2014). In plants, the biosynthetic pathways and their regulation are complex. While developmental cues can regulate the synthesis of a range of secondary metabolites, some are only induced under certain circumstances, such as attacks by pathogens and/or insects. The synthesis of many metabolites is often the response to environmental signals, and these can be mimicked in culture to increase specific compounds. For example, caffeic acid could be increased by addition of the stress elicitor, jasmonic acid, but similarly was induced by an abiotic stress factor, for example O2 depletion (Nitzsche et al. 2004). The biosynthesis of taxol, however, could be induced by the bacterial toxin, coronatine, a mimic of the active JA-isoleucine conjugate (Katsir et al. 2008), which was a better elicitor than methyl jasmonate (Onrubia et al. 2011). Other examples for stressors that can induce secondary metabolite synthesis include elicitor molecules from pathogens, such as glucans, flagellin, harpins or systemin (Zhao et al. 2005). These and other findings allow the production of a specific metabolite of interest also in large scale.
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While some observations about the ability of plant endophytes to produce secondary plant metabolites were made about two decades ago (e.g. Strobel et al. 1993; Lee et al. 1996), only within recent years has it become clear that not only plants, but also their pathogens or endophytes can contribute to the patterns of their secondary metabolites. With pathogen/plant interactions the compounds cannot be easily produced because of the one-sided nature of the relationship that ultimately leads to the decay of the host plant. Endophytes, however, can live within their host plant for a long time. Since many endophytes produce secondary metabolites with interesting bioactive properties, their contribution to the wealth of secondary metabolites has attracted much attention. Therefore, the potential of endophytes to synthesize secondary metabolites and to use them in biotechnology has been recognized (e.g. Casella et al. 2013). There are already several recent reviews touching the ability of endophytes to produce plant metabolites (Aly et al. 2013; Kusari et al. 2013; Gandhi et al. 2015). Here some examples will be reviewed where partial or complete biosynthesis pathways can be attributed to such endophytes. The consequences for the use of plant cultures for secondary metabolite production will be discussed.
Definition of endophytes While pathogens adversely influence plant performance, endophytes might not always be obvious in their colonization of hosts (Schulz and Boyle 2005). Endophytes are generally considered as organisms (bacteria or fungi) that have a beneficial effect on their host during the interaction or only under certain circumstances, for example abiotic or biotic stress situations (e.g. Rodriguez et al. 2009). It is important to note that a given plant may contain several different endophyte species (Tan and Zou 2001). However, this cirumstance can also lead to the synthesis of novel metabolites in host and endophytes, because of their competing influences on each other (Kusari et al. 2012a). Based on their potential to induce defense responses (Arnold et al. 2003), some endophytes are also used for biocontrol: e.g. the fungal Acremonium or Heteroconium species (Joost 1995; Narisawa et al. 2000; Ja¨schke et al. 2010) or Bacillus subtilis that is already
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available as commercial product (Lahlali et al. 2013). In some cases, the effect can be attributed to the production of toxins as in the Acremonium zeae/maize interaction (Poling et al. 2008). Endophytes also contribute to plant defense mechanisms by preventing herbivory (Zhang et al. 2011) or reducing the offspring number of plant pests (Jaber and Vidal 2009). Secondary metabolites produced by the endophytes could alter the balance between beneficial interaction or defense, because endophytes can occasionally turn into pathogens (Schulz and Boyle 2005). On the one hand, plants can produce these compounds to alter growth of fungi within their tissue before and after colonization but, on the other hand, the fungi can produce secondary metabolites for their own benefit, for example to get rid of competitors in a host plant. The host plant can then modify either toxic compounds to deal with them if these are too harmful, or to use them in alternative processes (Fig. 1).
Endophytes produce bioactive compounds Mainly within the last decade endophytes have attracted greater attention due to their potential to synthesize a wide array of bioactive secondary metabolites (Tan and Zou 2001; Schulz et al. 2002; Strobel 2003; Prado et al. 2013). The endophytederived compounds belong to diverse structural groups such as terpenoids, steroids, xanthones, chinones, phenols, isocoumarins, benzopyranones, tetralones, cytochalasines, and enniatines (Schulz et al. 2002). They sometimes constitute variations of already known structures such as ergosterol, a fungal steroid, or the plant hormone indole-3-acetic acid (Lu et al. 2000). The biosynthesis of such compounds is important for the endophyte when it is in competition with other organisms surrounding the plant (Schulz et al. 1999). Therefore, in many cases these compounds possess antimicrobial activities. The protection of plants against pests can often be attributed to some of the compounds found in endophytes (e.g. Poling et al. 2008). To isolate endophytes from the close vicinity of their hosts was a successful strategy in the identification of (novel) organisms with high antimicrobial potential and in many cases new compounds could be isolated and characterized (e.g. Casella et al. 2013). However, there are also studies that report antimicrobial effects without further
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identification of the active principle in extracts of endophytes (e.g. Liu et al. 2001), so among these reports there is still a great potential for novel compounds. A screening for endophytes containing antimicrobial and herbicidal compounds showed that endophytes can also synthesize compounds directed against plants (Schulz et al. 1999). In addition, it was found in this study that the number of endophytes producing these herbicidal substances was three times higher than of soil microbes and twice that of plant pathogenic microorganisms. Insects were also proposed to influence endophytic fungi producing bioactive compounds and host plants (Kusari et al. 2013). Therefore, also insecticidal substances can be found among those produced by the endophytes. However, the scenario might become more complicated by observations that fungi contain their own symbionts. For example the plant pathogenic fungus, Rhizopus, does not synthesize the compound, rhizoxin, as previously attributed, but a symbiotic bacterium (Burkholderia) within the fungus (Partida-Martinez and Hertweck 2005). Also endophytic fungi can influence their metabolism in a complicated crosstalk if more tha one is present in a given host plant (Kusari et al. 2013).
Plant versus endophyte metabolism Plant/endophyte metabolism can interact on may levels (Fig. 1): (a) the endophyte induces host metabolism, (b) the host induces endophyte metabolism, (c) host and endophyte share parts of a specific pathway and contribute partially, (d) the host can metabolize products from the endophyte and vice versa (e) the endophyte can metabolize secondary compounds from the host. The two latter possibilities can concern only one, several or all enzymatic steps for biochemical transformation. Changes in host metabolism Some reports have shown that endophytes can induce host secondary metabolism but this aspect of the interaction is much less explored than the induction of endophyte metabolism, even though defense-related phenolic compounds in endophyte-infected roots were already described some time ago (Schulz et al. 1999). In a recent example in grasses (specifically in Lolium perenne) after infection with endophytes from the Clavicipitaceae family, an induction of phenols was observed (Qawasmeh et al. 2012). However, depending on the type of endophyte, metabolites could also be
Fig. 1 A simplified scheme showing possible interactions and roles for plant versus endophyte metabolite production and the contribution of the respective organisms to the other metabolic pathways. The induction of plant metabolism can be caused by environmental signals such as abiotic stress factors or pathogens/herbivores, but also by the endophytes themselves. In addition, developmental signals can induce secondary metabolite synthesis at defined stages, e.g. leaf colors or flowering, where pigments and volatiles are synthesized
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reduced or remain unchanged. Major compounds were glycosylated flavonoids and hydroxycinnamic acids; and chlorogenic acid was the major compound involved in an induction of the antioxidative activity of plant extracts after endophyte colonization (Qawasmeh et al. 2012). A different scenario is occurring when plants experience toxic compounds from the endophytes since some might have herbicidal activity (see ‘Endophytes produce bioactive compounds’). A Taxus species is able to glycosylate the endophyte-derived peptide leucinostatin A (Strobel and Hess 1997), whereas for other, non-host plants this compound may still be toxic. Such poisonous compounds may be used to determine the specificity of an interrelationship between fungus and plant. Influence of plant host on microbial metabolism As described above, endophytes can influence their host plant’s metabolism, but one can speculate that the established host range could also alter or influence the pattern of secondary metabolites in endophytic fungi. More so, host plants can influence the metabolite pattern in pathogenic fungi. For a complex of phytopathogenic Heterobasidium species, two pineinfecting and three non-pine infecting species showed different metabolite patterns which could be separated by principal component analysis after LC–MS analysis into five distinct groups, but also according to their hosts (Hansson et al. 2014). The main difference between the pine-infecting species and the non-pine infecting species was the production of the phytotoxic benzohydrofuran fomannoxin exclusively by the two pine infecting fungi (Hansson et al. 2014). However, due to the detection of this compound in another Heterobasidium sp. not infecting pine, the differences were thought to occur due to differential gene regulation. Synthesis of plant host compounds by endophytes The most fascinating topic is either biosynthesis or metabolism of host plant metabolites by endophytes, but only a few examples can be discussed here. In a recent review Kusari and colleagues (2013) pointed out that ‘‘The biosynthetic potential of endophytic fungi has gained impetus in recent times owing to the continual discovery of fungal endophytes capable of
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synthesizing plant compounds. However, the sustained production of the desired plant compounds has not yet been achieved using endophytes. It is thus imperative to investigate the diverse interactions that endophytes have with coexisting endophytes, host plants, insect pests, and other specific herbivores.’’ This implies that careful investigations on the nature of a given compound need to be done whether it is from plant origin, from the endophyte or can be produced by both. In the latter case the endophyte might contribute with the complete pathway, but another scenario might be that only parts of the biosynthesis derive from the endophyte. As a reason for the observed capability to synthesize these plant metabolites it was speculated that horizontal gene transfer is at least one possible scenario how the endophytes have gained their genes encoding biosynthetic enzymes. The most prominent example for (partial) plant metabolite synthesis so far might be the production of paclitaxel (Taxol), due to its medicinal use, by endophytes. Its production in cell cultures has been optimized to a large extent (Malik et al. 2011; Cusido et al. 2014). However, due to the large demand of the compound as anticancer drug, alternative sources to the plant material were sought (Heinig et al. 2013). The discovery of Taxol production in the endophytic fungus Taxomyces andreanae, which was found in Taxus brevifolia (Strobel et al. 1993), was followed by the isolation of other fungal endophytes from a variety of Taxus spp. (e.g. Strobel et al. 1996; Zhang et al. 2009). Such approaches have resulted in many reports on the identification on the microbial taxane production in a variety of species among them Alternaria, Aspergillus, Cladosporium, Fusarium, Monochaetia, Pestlotia, Pestalotiopsis, Pithomyces, Penicillium and Xylaria, which were isolated from yew and non-Taxus plants (cf Heinig et al. 2013). However, a reexamination of fungal and host plant taxane biosynthesis revealed that in this study no independent taxane biosynthesis in the endophytes was found (Heinig et al. 2013; see also Problems with single cultures). Other bioactive metabolites were found in an endophytic fungus, Pestalotiopsis microspora, isolated from another species belonging to the Taxaceae (Lee et al. 1996). The compound, torreyanic acid, has also anti-cancerogenic characteristics. Similar observations came from plant species from unrelated families, for example Berberidaceae, where an endophyte was able to produce podophyllotoxin (Eyberger
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et al. 2006); or Meliaceae, where an endophytic fungus, Fusarium sp., was reported to produce the compound, rohitukine, a precursor for another anticancer drug (Mohana Kumara et al. 2012). Of aryl tetralin lignans, podophyllotoxin, for example, is synthesized by Podophyllum but the production of this and other valuable compounds has not been achieved yet in a sustainable way (Puri et al. 2006). A novel endophytic fungus Trametes hirsuta that also produces these compounds has been envisioned as novel, promising biological system for the synthesis of podophyllotoxin (Puri et al. 2006). An endophyte related to a Chaetomium species was isolated from Hypericum perforatum that can produce hypericin and the putative biosynthetic precursor emodin in an in vitro culture system (Kusari et al. 2008). Kusari et al. (2012b) described the isolation of the novel endophytic fungus Eupenicillium parvum from the neem tree, Azadirachta indica, and found that the fungus also produced the insecticidal compounds, azadirachtin A and B. Of course, all these examples do not exclude the existence of other endophytes on the host plants mentioned. For example, Verma et al. (2009) report on the biodiversity of the microbial endophytes from Azadirachta indica. The examples mentioned above include fungi as endophytes but endophytic bacteria can also share compounds with their host. Alternanthera brasiliana (Amaranthaceae) stem extracts contain antimicrobial compounds from the oxylipin family (Trapp et al. 2015). Among these several that were also found in bacteria from the genus Bacillus isolated from Alternathera plants and the authors speculated that the host plant obtained the antimicrobial oxylipins from their bacterial endophytes (Trapp et al. 2015).
to the production of the corresponding aglycone in the medicinal host plant Cephalotaxus harringtonia via deglycosylation. The deglycosylated flavonoids turned out to display significant beneficial effects on the hyphal growth of germinated spores (Tian et al. 2014). These data proved that a close interaction between the two organisms took place on a biochemical level. Another example was detected for symbiotic fungi that alter host metabolism in a way that leaf-cutting ants preferred non-colonized plants or extracts from these. The individual leaf compounds responsible for this response were not identified, but thought to constitute low or non-volatile compounds (Estrada et al. 2013). In addition, there are many examples that endophytes can reduce the attractiveness of their host for herbivores. Another role of plant metabolite modification by endophytes is the detoxification of defense compounds of the host (Fig. 1). For example, maize plants synthesize the benzoxazinoids for their own defense against various pests. The toxic compounds are present in a glycosylated form, while deglycosylation leads to a non-toxic metabolite. Therefore, enzymatic deglycosylation will result in tolerant microorganisms, if they have the respective enzyme (Saunders and Kohn 2008). In this study it was also observed that fungi, which can degrade the toxic conpounds, can lead the way for colonization by other, not so tolerant endophytes. Alternative metabolic steps used to detoxify these plant compounds by other endophytic fungi include acylation, oxidation, reduction, hydrolysis, and nitration (Zikmundova´ et al. 2002).
Considerations for exploring the (biotechnological) potential of endophytes
Metabolism of plant host compounds by endophytes
Problems with single cultures
The fungal leaf endophyte, Paraconiothyrium variabile, is able to metabolize its host plant metabolome (Tian et al. 2014). However, the function of these biochemical reactions is not yet well understood. Therefore, the changes in the metabolome were monitored and these compounds, where the main changes were observed, were tested for beneficial effects on the fungus. A specific biotransformation of glycosylated flavonoids by the endophyte was observed (Tian et al. 2014) (Fig. 2). In all cases, this led
It is of interest to investigate endophytes better for bioprospecting, not only against plant pests (Schulz et al. 2002), but also against human pathogens such as Mycobacterium tuberculosis (Alvin et al. 2014) and as sources for novel drugs (Suryanarayanan et al. 2009). Microbes sometimes use different biosynthetic pathways and can synthesize cyclic peptides with high antimicrobial potential by a reaction so far not found in plants, namely nonribosomal peptide synthesis (e.g. Schwarzer et al. 2003). Generally, microbes are
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Biotechnol Lett Fig. 2 Some examples for in planta (right side) and in vitro (left side) bioconversion of metabolites by endophytes. Red boxes stand for a direct beneficial effect for the fungus, blue boxes indicate an effect mainly on the host plant. Black boxes indicate single enzymatic reactions, orange boxes depict complex pathways
desirable due to their short generation time, high biomass production due to high growth rates and good handling features in bioreactors. However, as pointed out above, the habitat of the host plant needs also be taken into account when novel compounds produced by endophytes should be isolated (Strobel 2003). When it is suspected that a certain pathway can be contributed by either plant or endophyte, then the contribution of each partner makes needs to be investigated. If only parts of the pathway derive from the endophyte then it is understandable that, as pointed out by Kusari et al. (2013), sustained production of the plant metabolite by the endophyte is not yet possible. However, for Taxol it was possible to get complete production from the endophytic fungus Taxomyces andreanae, isolated from the host tree Taxus brevifolia (Strobel et al. 1993), albeit not yet sufficient for biotechnological applications. This might be due to the observation that the fungal metabolites decrease to a large extent over a longer culture period (e.g. Gandhi et al. 2015). For example, the production of camptothecin was attenuated from the first to the seventh generation of subculturing of the camptothecin-producing endophytic fungus (Kusari et al. 2009) or even completely lost within two other fungal strains from the genus Aspergillus (Pu et al. 2013). However, this might be species dependent, because the same authors found that a Trichoderma species could still produce camptothecin even after the eighths generation with only minor reduction (Pu et al. 2013).
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Other possibilites to explain the lack of secondary metabolites in the endophytic cultures were brought up by Heinig et al. (2013). These authors claimed that they could not find unequivocally taxane synthesis in endophytic fungi alone. One criticism to previous publications was that PCR to identify fungal genes was performed with primers developed based on the plant sequences. They point out that ‘‘The presence of these genes would require the extensive horizontal gene transfer (HGT) between the yew trees and multiple endophytic fungi, representing a pathway with more than 20 steps.’’ (Heinig et al. 2013). Further experiments involving metabolite analysis and sequencing approaches did not support the presence of genes that could be capable of taxane synthesis in one strain. The take-home message is that more cautios investigations are necessary as to which partner is making the bioactive compounds, especially if these should be used for production purposes. Selection of plant material for endophyte isolation A rationale for eventually isolating endophytes from different plant species was proposed by Strobel (2003), who suggested that the plant species should be selected according to the environment where a specific benefit was observed. These conditions could be caused by abiotic or biotic stress factors and benefits conveyed by endophytes. For example, a plant from harsh aquatic environment that resists infection
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by an oomycete was selected by his group. In this plant, a novel antimicrobial agent was identified which was actually produced by a bacterial endophyte, Serratia marcescens, within the aquatic plant Rhyncholacis penicillata and helped in the protection against oomycetes (Strobel et al. 1999). Also, plants surrounded by other infected plants but themselves showing no symptoms could be a source to look for endophytes. By this approach an antimicrobial activity against the plant pathogenic fungus Colletotrichum musae was isolated (Tuntiwachwuttikul et al. 2008). Additional important points were raised by Yu et al. (2010) namely to look into areas with great biodiversity on the plant side, because these are most likely also to contain a large variety of endophytes. Also, the exploitation of traditional medicinal plants could lead to the identification of endophytes with a potential to produce bioactive compounds. Finally, the isolation of endophytes of plants from ancient land areas might be more promising, again possibly due to a longer evolutionary period and the potential to develop more different endophytic organisms (Yu et al. 2010). In vitro biotransformation by endophytes The metabolism of plant secondary compounds by endophytes can be explored by using plant endophytes for in vitro biotransformation of plant metabolites to valuable products (Fig. 2). The use of endophytes as biocatalysts might open up a new avenue for their biotechnological use. As biocatalysts, the endophytes can add stereospecificity to bioprocesses and thus reduce complicated purification steps of chemically-synthesized enantiomers (Borges et al. 2009). The biotransformation of a racemic mixture to stereoselective thioridazine sulfoxides was investigated (Borges et al. 2008). Prado et al. (2013) employed the endophytic fungus Paraconiothyrium variabile for an one-step enantioslective synthesis of (4S)-isosclerone from the natural precursor juglone. Other endophytes simply metabolize the core moiety to other, maybe even more bioactive compounds, or substances with novel properties. Curcuma longa contains the bioactive compound curcumin (Prana et al. 2010). Biotransformation was detected using vaious endophytic fungi isolated from the rhizome of the host plant, but the major compound was not indentifed in this work. The authors showed
that the metabolite production was dependent on the culture medium used. Oxidation and hydroxylation are simple enzymatic reactions to modify a structure. The stereoselective biooxidation at C4-flavans, specifically of (?)-catechin and (-)-epicatechin into dihydroflavan derivatives, was observed for the endophytic fungus Diaporthe sp. that was isolated originally from Camellia sinensis (Agusta et al. 2005; Shibuya et al. 2005). Later, Agusta et al. (2014) reported the conversion of the alkaloid berberine into its 7-N-oxide by cultures of the endophytic fungus Coelomycetes sp. Deacetylation, hydroxylation and epoxidation was described for the biotransformation of taxoids by endophytic fungi isolated originally from the inner bark of Taxus yunnanensis (Zhang et al. 1998). Volatiles are also on the list of compounds to be biotechnologically produced by endophytes (Abraha˜o et al. 2013). These compounds are important in the plant for either attracting or repelling mainly insects. For biotechnology they are of interest as flavor compounds and can be generated either by de novo synthesis or by metabolism, for example bio-oxidation of the respective terpenes (Bicas et al. 2009). Taken together, all these examples of manipulation of host metabolites in planta or in vitro (Fig. 2) indicate that the potential exploitation for endophytes in biotechnology is probably just at the beginning.
Conclusion and future prospects The observations pointed out above indicate that there will be many future challenges when appropriate cultivations systems need to be designed for secondary metabolite production. While microbial production systems depend often on the optimization of culture conditions, the production system might turn out to be problematic in cases where both partners contribute to the synthesis of the desired compound. Cultivation of endophytes without their host might result in the loss of the desired compound synthesis as pointed out above (see Problems with single cultures). Transgenic strategies to improve production in an endophyte grown without host plant can include genes from the same organism (homologous gene expression; e.g. Hwang et al. 2014), but more likely a suitable production
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host for the compound in question needs to be found (heterologous expression; e.g. Sakai et al. 2012). Therefore, such production systems could lead to a desired metabolite, if enough is known about the biosynthetic pathways. One challenge, therefore, remains the elucidation of the (control of) biosynthetic pathways by all participating partners. A controlled production with mature plants harboring the endophyte in question is very likely not possible due to sterility problems because of the microorganism(s) present. In addition, there is no way to know how many microbes are indeed present in a given plant species, and this hampers the controlled production of metabolites as well. Generating plant cultures harboring the endophyte might be possible, but whether the endophyte can be kept stable and productive in the plant over a long time is questionable, even though reports on the contamination of plant cultures with endophytes over a long period exist (Leifert and Cassells 2001). Elicitation of plant metabolite production by microbial culture filtrates (Verma et al. 2014) or elicitors generated from the microorganisms (Zhao et al. 2005) has been described in many cases (see also Introduction). However, in these cases the contribution to plant biosynthetic pathways is not by the living organisms. Alternatives could be the co-cultivation of sterile endophyte cultures together with sterile plant cultures, if the latter can be shown to have the ability of secondary metabolite production similar to the mother plant. Co-cultivation (or co-culture) of microorganisms, for example two fungal or two bacterial strains, has been exploited to generate novel compounds and to enhance the production of one desired product (Bertrand et al. 2014). To achieve larger scale production methods for co-fermentation can be used. This could also be a possible way to generate the desired metabolites from plant organ cultures, such as shoots or hairy roots, with defined endophytes during a cocultivation approach. Like for microbial co-cultivation careful monitoring of product formation would be required. Here another challenge emerges, namely the design of appropriate cultivation systems for larger scale production. Whether this might be a possibility to circumvent the problems with the contributions of the two partners—plant and endophyte - to a specific compound formation has to be elucidated in future work.
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Acknowledgments Work in the author’s laboratory on secondary metabolites is funded by the European Union, The German Ministry for Education and Science and the State of Saxony.
References Abraha˜o MRE, Molina G, Pastore GM (2013) Endophytes: recent developments in biotechnology and the potential for flavor production. Food Res Int 52:367–372 Agusta A, Maehara S, Ohashi K, Simanjuntak P, Shibuya H (2005) Stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. isolated from a tea plant. Chem Pharm Bull 53:1565–1569 Agusta A, Wulansari D, Praptiwi Nurkanto A, Fathoni A (2014) Biotransformation of protoberberine alkaloids by the endophytic fungus Coelomycetes AFKR-3 isolated from yellow moonsheed plant (Archangelisia flava (L.) Merr.). Proced Chem 13:38–43 Aly AH, Debbab A, Proksch P (2013) Fungal endophytes— secret producers of bioactive plant metabolites. Pharmazie 68:499–505 Arnold AE, Mejı´a LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, Herre EA (2003) Fungal endophytes limit pathogen damage in a tropical tree. Proc Natl Acad Sci USA 26:15649–15654 Bertrand S, Bohni N, Schnee S, Schumpp O, Gindro K, Wolfender J-L (2014) Metabolite induction via microorganism co-culture: a potential way to enhance chemical diversity for drug discovery. Biotechnol Adv 32: 1180–1204 Bicas JL, Dionisio AP, Pastore GM (2009) Bio-oxidation of terpenes: an approach for the flavor industry. Chem Rev 109:4518–4531 Borges KB, Borges WdS, Pupo MT, Bonato PS (2008) Stereoselective analysis of thioridazine-2-sulfoxide and thioridazine-5-sulfoxide: an investigation of rac- thioridazine biotransformation by some endophytic fungi. J Pharm Biomed Anal 46:945–952 Borges KB, Borges WdS, Dura´n-Patro´n R, Pupo MT, Bonato PS, Collado IG (2009) Stereoselective biotransformations using fungi as biocatalysts. Tetrahedron Asymmetry 20:385–397 Casella TM, Eparvier V, Mandavid H, Bendelac A, Odonne G, Dayan L, Duplais C, Espindola LS, Stien D (2013) Antimicrobial and cytotoxic secondary metabolites from tropical leaf endophytes: isolation of antibacterial agent pyrrocidine C from Lewia infectoria SNB-GTC2402. Phytochemistry 96:370–377 Chandra S, Chandra R (2011) Engineering secondary metabolite production in hairy roots. Phytochem Rev 10:371–395 Cusido RM, Onrubia M, Sabater-Jara AB, Moyano E, Bonfill M, Goossens A, Pedren˜o MA, Palazon J (2014) A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp. Biotechnol Adv 32:1157–1167 Estrada C, Wcislo WT, Van Bael SA (2013) Symbiotic fungi alter plant chemistry that discourages leaf-cutting ants. New Phytol 198:241–251
Biotechnol Lett Eyberger AL, Dondapati R, Porter JR (2006) Endophyte fungal isolates from Podophyllum peltatum produce podophyllotoxin. J Nat Prod 69:1121–1124 Gandhi SG, Mahajan V, Bedi YS (2015) Changing trends in biotechnology of secondary metabolism in medicinal and aromatic plants. Planta 241:303–317 Georgiev M, Agostini E, Ludwig-Mu¨ller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnology. Trends Biotechnol 30:528–537 ˚ , Karlsson M, Staerk D, Broberg Hansson D, Wubshet S, Olson A A (2014) Secondary metabolite comparison of the species within the Heterobasidion annosum s.l. complex. Phytochemistry 108:243–251 Hwang K-S, Kim HU, Charusanti P, Palsson BØ, Lee SY (2014) Systems biology and biotechnology of Streptomyces species for the production of secondary metabolites. Biotechnol Adv 32:255–268 Jaber LR, Vidal S (2009) Interactions between an endophytic fungus, aphids, and extrafloral nectaries: do endophytes induce extrafloral-mediated defences in Vicia faba? Funct Ecol 23:707–714 Ja¨schke D, Dugassa-Gobena D, Karlovsky P, Vidal S, Ludwig-Mu¨ller J (2010) Suppression of clubroot development in Arabidopsis thaliana by the endophytic fungus Acremonium alternatum. Plant Pathol 59: 100–111 Joost RE (1995) Acremonium in fescue and ryegrass: boon or bane? A review. J Anim Sci 73:881–888 Katsir L, Schilmiller AL, Staswick PE, He SY, Howe GA (2008) COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine. Proc Natl Acad Sci USA 105:7100–7105 Kusari S, Lamsho¨ft M, Zu¨hlke S, Spiteller M (2008) An endophytic fungus from Hypericum perforatum that produces hypericin. J Nat Prod 71:159–162 Kusari S, Zu¨hlke S, Spiteller M (2009) An endophytic fungus from Camptotheca acuminata that produces camptothecin and analogues. J Nat Prod 72:2–7 Kusari S, Hertweck C, Spiteller M (2012a) Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol 19:792–798 Kusari S, Verma VC, Lamshoeft M, Spiteller M (2012b) An endophytic fungus from Azadirachta indica A. Juss. that produces azadirachtin. World J Microbiol Biotechnol 28:1287–1294 Kusari S, Pandey SP, Spiteller M (2013) Untapped mutualistic paradigms linking host plant and endophytic fungal production of similar bioactive secondary metabolites. Phytochemistry 91:81–87 Lahlali R, Peng G, Gossen BD, McGregor L, Yu FQ, Hynes RK, Hwang SF, McDonald MR, Boyetchko SM (2013) Evidence that the biofungicide Serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance. Phytopathology 103:245–254 Lee JC, Strobel GA, Lobkovsky E, Clardy J (1996) Torreyanic acid: a selectively cytotoxic quinone dimer from the endophytic fungus Pestalotiopsis microspora. J Org Chem 61:3232–3233 Leifert C, Cassells AC (2001) Microbial hazards in plant tissue and cell cultures. In Vitro Cell Dev Biol Plant 37:133–138
Liu CH, Zou WX, Lu H, Tan RX (2001) Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi. J Biotechnol 88:277–282 Lu H, Zou WX, Meng JC, Hu J, Tan RX (2000) New bioactive metabolites produced by Colletotrichum sp., an endophytic fungus in Artemisia annua. Plant Sci 151:67–73 Ludwig-Mu¨ller J, Jahn L, Lippert A, Pu¨schel J, Walter A (2014) Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: strategies and applications. Biotechnol Adv 32:1168–1179 Malik S, Cusido´ RM, Mirjalili MH, Moyano E, Palazo´n J, Bonfill M (2011) Production of the anticancer drug taxol in Taxus baccata suspension cultures: a review. Proc Biochem 46:23–34 Mohana Kumara P, Zuehlke S, Priti V, Ramesha BT, Shweta S, Ravikanth G, Vasudeva R, Santhoshkumar TR, Spiteller M, Uma Shaanker R (2012) Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity. Antonie Van Leeuwenhoek 101:323–329 Narisawa K, Ohki T, Hashiba T (2000) Suppression of clubroot and Verticillium yellows in Chinese cabbage in the field by the root endophytic fungus, Heteroconium chaetospira. Plant Pathol 49:141–146 Nitzsche A, Tokalov SV, Gutzeit HO, Ludwig-Mu¨ller J (2004) Chemical and biological characterization of cinnamic acid derivatives from cell cultures of lavender (Lavandula officinalis) induced by stress and jasmonic acid. J Agric Food Chem 52:2915–2923 Onrubia M, Moyano E, Bonfill M, Cusido´ RM, Goossens A, Palazo´n J (2011) Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate. J Plant Physiol 170:211–219 Partida-Martinez LP, Hertweck C (2005) Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature 437:884–888 Poling SM, Wicklow DT, Rogers KD, Gloer JB (2008) Acremonium zeae, a protective endophyte of maize produces dihydroresorcylide and 7-hydroxydihydroresorcylides. J Agric Food Chem 56:3006–3009 Prado S, Buisson D, Ndoye I, Vallet M, Nay B (2013) One-step enantioselective synthesis of (4S)-isosclerone through biotransformation of juglone by an endophytic fungus. Tetrahedron Lett 54:1189–1191 Prana TK, Srikandace J, Sumitro E, Wulandari D (2010) The potency of endophytic fungi of turmeric (Curcuma longa L.) in biotransformation of curcumic compounds in various media. Res J Microbiol 5:1189–1198 Pu X, Qu X, Chen F, Bao J, Zhang G, Luo Y (2013) Camptothecin-producing endophytic fungus Trichoderma atroviride LY357: isolation, identification, and fermentation conditions optimization for camptothecin production. Appl Microbiol Biotechnol 97:9365–9375 Puri SC, Nazir A, Chawla R, Arora R, Riyaz-ul-Hasan S, Amnaa T, Ahmeda B, Verma V, Singh S, Sagar R, Sharma A, Kumar R, Sharma RK, Qazi GN (2006) The endophytic fungus Trametes hirsuta as a novel alternative source of podophyllotoxin and related aryl tetralin lignans. J Biotechnol 122:494–510
123
Biotechnol Lett Qawasmeh A, Obied HK, Raman A, Wheatley W (2012) Influence of fungal endophyte infection on phenolic content and antioxidant activity in grasses: interaction between Lolium perenne and different strains of Neotyphodium lolii. J Agric Food Chem 60:3381–3388 Rodriguez RJ, White JF Jr, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330 Sakai K, Kinoshita H, Nihira T (2012) Heterologous expression system in Aspergillus oryzae for fungal biosynthetic gene clusters of secondary metabolites. Appl Microbiol Biotechnol 93:2011–2022 Saunders M, Kohn LM (2008) Host-synthesized secondary compounds influence the in vitro interactions between fungal endophytes of maize. Appl Env Microbiol 74:136–142 Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686 Schulz B, Rommert AK, Dammann U, Aust HJ, Strack D (1999) The endophyte host interaction: a balanced antagonism? Mycol Res 103:1275–1283 Schulz B, Boyle C, Draeger S, Rommert A-K, Krohn K (2002) Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol Res 106:996–1004 Schwarzer D, Finking R, Marahiel MA (2003) Nonribosomal peptides: from genes to products. Nat Prod Rep 20:275–287 Shibuya H, Agusta A, Ohashi K, Maehara S, Simanjuntak P (2005) Biooxidation of (?)- catechin and (-)- epicatechin into 3,4-dihydroxyflavan derivatives by the endophytic fungus Diaporthe sp. isolated from a tea plant. Chem Pharm Bull 53:866–867 Strobel GA (2003) Endophytes as sources of bioactive products. Microbes Infect 5:535–544 Strobel GA, Hess WM (1997) Glucosylation of the peptide leucinostatin A, produced by an endophytic fungus of European yew, may protect the host from leucinostatin toxicity. Chem Biol 4:529–536 Strobel G, Stierle A, Stierle D, Hess WM (1993) Taxomyces andreanae, a proposed new taxon for a bulbilliferous hyphomycete associated with Pacific yew (Taxus brevifolia). Mycotaxon 47:71–80 Strobel G, Yang X, Sears J, Kramer R, Sidhu RS, Hess WM (1996) Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiol 142:435–440 Strobel GA, Li JY, Sugawara F, Koshino H, Harper J, Hess WM (1999) Oocydin A, a chlorinated macrocyclic lactone with potent antioomycete activity from Serratia marcescens. Microbiol 145:3557–3564
123
Suryanarayanan TS, Thirunavukkarasu N, Govindarajulu MB, Sasse F, Jansen R, Murali TS (2009) Fungal endophytes and bioprospecting. Fungal Biol Rev 23:9–19 Tan R, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep 18:448–459 Tian Y, Amand S, Buisson D, Kunz C, Hachette F, Dupont J, Nay B, Prado S (2014) The fungal leaf endophyte Paraconiothyrium variabile specifically metabolizes the hostplant metabolome for its own benefit. Phytochemistry 108:95–101 Trapp MA, Kai M, Mitho¨fer A, Rodrigues-Filho E (2015) Antibiotic oxylipins from Alternanthera brasiliana and its endophytic bacteria. Phytochemistry 110:72–82 Tuntiwachwuttikul P, Taechowisan T, Wanbanjob A, Thadaniti S, Taylor WC, Lansai A-D (2008) Secondary metabolites from Streptomyces sp. SUC1. Tetrahedron 64:7583–7586 Verma VC, Gond SK, Mishra A, Kumar A, Kharwar RN, Gange AC (2009) Endophytic actinomycetes from Azadirachta indica A. Juss.: isolation, diversity and anti-microbial activity. Microbial Ecol 57:749–756 Verma P, Khan SA, Mathur AK, Shanker K, Kalra A (2014) Fungal endophytes enhanced the growth and production kinetics of Vinca minor hairy roots and cell suspensions grown in bioreactor. Plant Cell Tiss Org Cult 118:257–268 Yu H, Zhang L, Li L, Zheng C, Guo L, Li W, Sun P, Qin L (2010) Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbial Res 165:437–449 Zhang J, Zhang L, Wang X, Qiu D, Sun D, Gu J, Fang Q (1998) Microbial transformation of 10-deacetyl-7-epitaxol and 1b-hydroxybaccatin I by fungi from the inner bark of Taxus yunnanensis. J Nat Prod 61:497–500 Zhang P, Zhou P-P, Yu L-J (2009) An endophytic taxol-producing fungus from Taxus media, Cladosporium cladosporioides MD2. Curr Microbiol 59:227–232 Zhang XX, Li CJ, Nan ZB, Matthew C (2011) Neotyphodium endophyte increases Achnatherum inebrians (drunken horse grass) resistance to herbivores and seed predators. Weed Res 52:70–78 Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333 Zikmundova´ M, Drandarov K, Bigler L, Hesse M, Werner C (2002) Biotransformation of 2-benzoxazolinone and 2-hydroxy-1,4-benzoxazin-3-one by endophytic fungi isolated from Aphelandra tetragona. Appl Env Microbiol 68:4863–4870
