Microbes and Infection 5 (2003) 535–544 www.elsevier.com/locate/micinf Review Endophytes as sources of bioactive products Gary A. Strobel * Department of Plant Sciences, Montana State University, Bozeman, MT 59717, USA Abstract An increase in the number of people in the world having health problems caused by various cancers, drug-resistant bacteria, parasitic protozoans, and fungi is a cause for alarm. An intensive search for newer and more effective agents to deal with these disease problems is now under way and endophytes are a novel source of potentially useful medicinal compounds. © 2003 Éditions scientiﬁques et médicales Elsevier SAS. All rights reserved. Keywords: Antibiotic; Plants; Infectious disease; Cancer; Ethnobotany 1. Introduction century, many of the 0.1 million fungi that have been de- scribed were those associated with various higher organisms The advent of the development of drug resistance in hu- as either parasites or saprophytes on dead and dying biologi- man pathogenic bacteria among such microbes as Staphylo- cal materials. Thus, the question, where are the remaining coccus spp., Mycobacterium tuberculosis, Streptococcus 0.9 million fungi? Microorganisms seem to occupy virtually spp. and others has prompted a search for more and better every living and non-living niche on earth. This includes antibiotics . Together with this is an increasing need for those in the thermal vents, in deep rock sediments, and in more and better antimycotics, especially as the human popu- desert as well as marine environments. For the purposes of lation is developing more fungal infections as a result of the this discussion, this review concentrates on those microor- AIDS epidemic and the increased numbers of patients with ganisms, mostly fungi, that reside in plants. organ transplants, whose immune systems are weakened. In addition, the world’s arsenal is not large for the treatment of In the past few decades, plant scientists have begun to parasitic protozoan infections, e.g. malaria, leshmaniasis, realize that plants may serve as a reservoir of untold numbers trypanosomiasis, and ﬁlariasis, which probably claim more of organisms known as endophytes . By deﬁnition, these lives each year than any other group of infectious agents . microorganisms (mostly fungi and bacteria) live in the inter- It now appears that an enormous, relatively untapped source cellular spaces of plant tissues. Some of these endophytes of microbial diversity is represented by the microbial endo- may be producing bioactive substances that may be involved phytes. They produce antimicrobial agents and seem to have in a host-endophyte relationship. As a direct result of the role unique genetic and biological systems that may have appli- that these secondary metabolites may play in nature, they cations outside the host plant in which they normally reside. may ultimately be shown to have applicability in medicine. A The forests of the world are sources of these microbial worldwide scientiﬁc effort to isolate endophytes and study endophytes (fungi and bacteria). This review discusses the their natural products is now under way . While there are rationale and the methods used, as well as some results myriads of epiphytic microorganisms associated with plants, obtained by those isolating and studying endophytes for their the endophytic ones now seem to be attracting more atten- medicinal potential. tion. This may be the case, since closer biological associa- tions may have developed between these organisms in their 2. Endophytes respective hosts than the epiphytes or soil-related organisms. Hence, the result of this may be the production of a greater It has been estimated that there may be as many as 1 number and diversity of classes of biologically derived mol- million different fungal species on our planet . In the past ecules, possessing a range of biological activities. In fact, a * Corresponding author. Tel.: +1-406-994-5148; fax: +1-406-994-7600. recent comprehensive study has indicated that 51% of bio- E-mail address: firstname.lastname@example.org (G.A. Strobel). logically active substances isolated from endophytic fungi © 2003 Éditions scientiﬁques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 7 3 - X 536 G.A. Strobel / Microbes and Infection 5 (2003) 535–544 were previously unknown . This compares with only 38% isolated. It is also obvious that the earlier and numerous of novel substances from soil microﬂora. works of Petrini, Fisher and Carroll have led the way in One of the least studied biochemical-chemical systems in showing how widespread, diverse and interesting these plant- nature is the relationship between microorganisms and their associated microorganisms are in nature [3,5]. While literally plant hosts. For instance, it appears that all higher plants are hundreds of reports have appeared in the world’s literature on hosts to one or more endophytic microbes. These microbes many new endophytic microorganisms, it appears that little is include the fungi, bacteria and actinomycetes, which prima- being done to understand the complex chemical and bio- rily reside in the tissues beneath the epidermal cell layers, chemical mechanisms that govern the biology of the endo- and the host tissues are transiently symptomless . It is well phytic processes. In fact, it is becoming increasingly clear understood that endophytic infections are at least inconspicu- that host speciﬁcity is a bona ﬁde phenomenon in endophyte- ous and as a result, the host tissues are transiently symptom- higher plant relationships . Such plant speciﬁcity implies less, and the colonization of the tissues is internal to the that complex biochemical interactions are occurring between surface of the plant . The exact physical relationship of the the host and its associated microorganism. In turn, knowl- endophyte to the plant has, in most cases remained obscure, edge of such interactions can provide guidance as to which because it is extremely difﬁcult to ﬁnd, by electron micros- endophytes might be selected in the search for novel medici- copy, an endophyte within plant tissues. Conceivably, the nal natural products. microbes live within the intercellular spaces of the tissues There are approximately 300,000 different plant species and it also seems likely that the penetration of living cells on our planet. And of the several hundred of these that we may occur, but it is not easy to observe under natural condi- have examined, each one has a complement of endophytic tions. microbes. Those plants growing in unique environmental These endophytic relationships may have begun to evolve settings, having ethnobotanical uses, having extreme age or from the time that higher plants ﬁrst appeared on the earth, interesting endemic locations generally produce novel endo- hundreds of millions of years ago. Evidence of plant- phytic microorganisms. Novel taxonomy of an endophyte or associated microbes has been discovered in the fossilized the acquisition of one that is only rarely seen, generally offers tissues of stems and leaves . As a result of these long-held a prospect for also ﬁnding novel bioactive natural products. associations, it is possible to imagine that some of these Therefore, we strive to obtain one or more of these circum- endophytic microbes may have devised genetic systems al- stances. lowing for the transfer of information between themselves Overall, our rationale for studying endophytic microbes as and the higher plant and vice versa . Obviously, this would potential sources of new medicinals is related to the fact that permit a more rapid and reliable mechanism for the endo- this is a relatively unexplored area of biochemical diversity. phyte to deal with everchanging environmental conditions Furthermore, our search is driven by the fact that the contri- and perhaps allow for more compatibility with the plant host. bution of the endophyte to the plant maybe to provide protec- For these reasons, it may have been the case that plant- tion to it by virtue of antimicrobial compounds that it pro- associated microorganisms evolved biochemical pathways duces. Some of these compounds may be of interest resulting in the production of plant growth hormones. Each medicinally, since they possess antifungal, antibacterial, an- of the ﬁve classes of these substances (auxins, abscisins, timalarial, and a host of other biological activities. Finally, of ethylene, gibberellins, and kinetins) is, in fact, known from a major concern to the medical community is the latent toxicity list of a range of representative plant-associated fungi and of any prospective drug to the higher organisms such as bacteria . In addition, independent evolution of the endo- animal and human tissues. It would appear that since the phytic microbes may have allowed them to better adapt to a plant is also a eukaryotic system, in which the endophyte plant host and perhaps develop to a point where they could exists, the antibiotics made by the endophyte may have contribute to the relationship by carrying out such functions reduced cell toxicity; otherwise, death of the host tissue may as protection from pathogens, insects, and grazing animals. occur. Thus, the plant itself has naturally served as a selection Thus, eventually various types of relationships may have system for microbes having bioactive molecules with re- formed, leading to symbiosis and ultimately to host speciﬁc- duced toxicity toward higher organisms. ity . With some exceptions, this brief review gives speciﬁc A relatively recent text presents an outstanding review of examples of our experiences in studying endophytes isolated the biology of endophytes along with some aspects of their from various higher plants obtained from a wide range of isolation, description, taxonomy, and uses in agriculture and forests around the world. It also discusses the results of some forestry. It also describes the threats that some endophyte- other laboratories studying these interesting organisms and plant associations have in animal production . On the their associated natural products. other hand, in the same text, there is a comprehensive review on the uses and importance of the mycorrhizal fungi to plant 3. Isolation of endophytes growth. Thus, while mycorrhizal fungi show the highest degree of plant compatibility, they exhibit relatively low host Endophytes, by deﬁnition, live in close association with speciﬁcity, and useful products from them have not been living plant tissues. Thus, in a discussion of this type, it is G.A. Strobel / Microbes and Infection 5 (2003) 535–544 537 critical to understand the methods and choices used to com- monly isolate endophytic microorganisms. In order to ac- quire endophytes, we ﬁrst select a plant species that may be of interest because of its unique biology, age, endemism, ethnobotanical history, and/or environmental setting. We also have learned that perennial plants growing in tropical or semitropical areas of the world are hosts to a greater diversity of endophytes than those growing in drier or colder areas of the world having less plant diversity. This is a critical step in the entire process of obtaining and studying endophytes, because it represents a biological rationale in plant selection. As such, it has the tendency to eliminate work involved in doing a completely random search of all ﬂora in any given area. Thus, we try to establish a speciﬁc rationale for the discovery of endophytes for each plant selected for study. As Fig. 1. The structure of taxol, the world’s ﬁrst billion dollar antitumor agent. an example, a plant is selected from a harsh aquatic environ- ment in which it is being constantly wounded by passing organisms in higher plants will aid in the development of a rocks and other debris. Nevertheless, the plant still resists drug discovery program involving these organisms. A search infection by common oomyceteous fungi (water molds that for a rare, and thus, expensive product such as taxol may be are phytopathogenic) that would normally be expected to facilitated by examining the endophytic microorganisms of cause disease, since many portals of entry have been pro- certain plants for their ability to make this drug. Taxol, a duced in the host plant. Thus, is it possible that highly functionalized diterpenoid, is found in each of the endophytes/epiphytes associated with the aquatic plant may world’s yew (Taxus) species  (Fig. 1). In electrospray be protecting it from attack by these pathogenic fungi? This mass spectroscopy, taxol usually gives two peaks, one at situation was studied in the case of oocydin A (a novel molecular weight 854 (M + H+), and the other at molecular antifungal agent) that is produced by a strain of Serratia weight 876 (M + Na+) . This compound is the world’s ﬁrst marcescens on an aquatic plant, Rhyncholacis penicillata, billion dollar anticancer drug, and it is used to treat a number growing in the river systems draining the Auyan Tepui of of other human tissue-proliferating diseases as well. Its cost southwest Venezuela . makes it unavailable to many people worldwide. Therefore, Once the plant is selected for study, it is identiﬁed, and a alternative sources are needed, since organic synthesis, while small portion is harvested. Furthermore, it is exactly located having been accomplished, is not economically feasible . geographically with a global positioning device. Usually, Given the fact that endophytes are virtually universally small stem pieces are cut from the plant and placed in plastic present in all of the world’s higher plants, it was reasoned that bags after any excess moisture is removed. Every attempt is yew trees conceivably might support certain endophytic mi- made to store the materials at 4 °C until isolation procedures croorganisms that also make taxol . Thus, if a microbial can be instituted. Plant materials are thoroughly surface source of the drug were available it could eliminate the need treated with 70% ethanol until they dry under a laminar ﬂow to harvest and extract the slow-growing and relatively rare hood. Then, with a sterile knife blade, outer tissues are yew trees. The price for the drug would also be reduced, since removed from the samples and the inner tissues are carefully taxol could be produced via fermentation in much the same excised and placed on water agar plates. After several days of way that penicillin is fermented. It was also speculated that incubation, hyphal tips of the fungi are removed and trans- the ability of any endophyte to make taxol may have arisen ferred to potato dextrose agar plates. In some occasions, from the exchange of genetic material from the yew tree to bacterial forms also emerge, including Streptomyces spp. one or more microorganisms living in close association with The endophytes are encouraged to sporulate on speciﬁc plant it . By the early 1990s, however, no endophytic fungi had materials and are eventually identiﬁed. Aspects of the biol- been isolated or were even known from any of the world’s ogy and biochemistry of the endophytes are then examined. representative yew species. After several years of effort, a Some speciﬁc examples are given below, which show ap- novel taxol-producing endophytic fungus, Taxomyces an- proaches, not only in discovering novel bioactive substances dreanae, was discovered in Taxus brevifolia .The most produced by endophytes, but also the aspects of their rela- critical line of evidence for the presence of taxol in the tionship to the higher plant host as well. culture ﬂuids of this fungus, among others, was the electro- spray mass spectrum of the putative taxol isolated from 4. Speciﬁc endophytes T. andreanae as well as C-14 labelling studies, which irrefut- 4.1. Microorganisms producing taxol ably showed the presence of fungal-derived taxol in the culture . This early work set the stage for a more compre- A background understanding that involves some speciﬁc hensive examination of the ability of other Taxus species and examples and rationale of the presence of endophytic micro- other plants to yield endophytes producing taxol. 538 G.A. Strobel / Microbes and Infection 5 (2003) 535–544 One of the most commonly found endophytes of the endophytes of yews. Thus, it may be that taxol had its origins world’s yews is Pestalotiopsis spp. . Generally represen- in certain fungi and ultimately, if there is lateral gene transfer, tative of this fungal genus, it is among the most commonly it may have been in the direction of the microbe to the higher isolated endophytic fungi of tropical plants . In fact, it is plant. rare not to isolate one of these species from any tropical Recently, in addition to our laboratory, others have begun plant. It appears that this fungal genus could be considered to make observations on taxol production by endophytes, the “Escherichia coli” of the rainforest because it is omni- including the discovery of taxol production by Tubercularia present. However, its role in the plant, and in the ecosystem sp. isolated from Southern Chinese yew (Taxus mairei) in the in general, is only beginning to be understood. One of the Fujian province of Southeastern China . In addition, at most commonly isolated endophytic species is Pestalotiopsis least three endophytes of T. wallichiana produce taxol, in- microspora . Organisms virtually identical to the taxo- cluding Sporormia minima and Trichothecium sp. . Us- nomic description of P. microspora are numerous, and they ing high-performance liquid chromatography and electro- have usually been isolated as leaf and stem pathogens of spray mass spectroscopy, taxol has been discovered in economically important tropical plants, such as the palms, Corylus avellana cv. Gasaway . In addition, several pines, loquats, guavas, mangoes and a large number of orna- fungal endophytes of this plant (ﬁlbert) produce taxol in mental plants . Generally, the commonly held view is culture . It is important to note, however, that taxol that this fungus is a relatively weak plant pathogen, but at production by all endophytes in culture is in the range of times acts in a more aggressive manner, resulting in major submicrograms to micrograms per liter. Also, commonly, the plant loss. The widely held view that this is a relatively fungi will attenuate taxol production in culture, with some obscure genus of interest only to tropical pathologists should possibility for recovery, if certain activator compounds are undoubtedly be revised. It seems that this fungus and its close added to the medium . Efforts are being made to deter- relatives are not as important as plant pathogens since they mine the feasibility of making microbial taxol a commercial play some role as endophytic fungi living in symbiotic rela- possibility. tionships to plants in each of the world’s temperate and It is conceivable that taxol is made by a number of higher tropical rainforests. In a global perspective, given the wide plants in the world as well as their associated endophytes. distribution of this genus, it probably represents one of the Conclusive evidence, however, for the existence of taxol largest biomasses of any plant-associated endophytic fungus from any biological source must be based on sound spectro- in the world. scopic evidence, since the monoclonal antibodies produced An examination of the endophytes of Taxus wallichiana by Hawaii Biotech (immunological method), while having yielded P. microspora, and a preliminary monoclonal anti- high molecular recognition speciﬁcity, are not totally speciﬁ- body test indicated that it may produce taxol . After cally reactive with taxol. preparative thin-layer chromatography, a compound was iso- As new uses for taxol in medical applications have been lated and shown by spectroscopic techniques to be taxol. proven, the interest in ﬁnding other organisms that make this Labeled taxol was produced by this organism from several valuable compound has risen. One rationale to help in this C-14 precursors that had been administered to it . Fur- new search is based on the observations that certain microbe- thermore, several other P. microspora isolates were obtained plant systems seemed to have evolved in which the microbe from bald cypress in South Carolina and were also shown to (endophyte) is aiding the plant as well as itself in its survival. produce taxol . This was the ﬁrst indication that endo- Thus, plants in extremely moist conditions (aquatic plants), phytes residing in plants other than Taxus spp. were produc- or ones growing in the world’s rainforests (at a more or less ing taxol. Therefore, a speciﬁc search was conducted for constant 90-100% relative humidity) are prone to attack by a taxol-producing endophytes in continents not known for any- certain group of extremely pathogenic fungi, and defense indigenous Taxus spp. This included an examination of the mechanisms are necessary for survival. Such disease de- prospects that taxol-producing endophytes exist in South fenses may be offered by the endophyte normally associated America and Australia. From the extremely rare, and previ- with the plant. One of the main groups of microorganisms ously thought to be extinct, Wollemi pine (Wollemia nobilis), causing diseases in plants is the oomycetes (water molds). Pestalotiopsis guepini was isolated, which was shown to This group of fungi is exempliﬁed by such plant pathogens as produce taxol . Also, quite surprisingly, a rubiaceous Phytophthora, Pythium and Aphanomyces. Each of these plant—Maguireothamnus speciosus—yielded a novel fun- pathogenic fungi is extremely sensitive to taxol. In fact, these gus, Seimatoantlerium tepuiense, that produces taxol. This fungi are killed in an identical manner by taxol as certain endemic plant grows on the tops of the tepuis in the sensitive human cells, such as those originating from breast Venzuelan-Guyana in southwestern Venezuela . Further- and ovarian cancer cell lines [24,25]. The mode of action of more, fungal taxol production also has been noted in Perico- taxol is to preclude tubulin molecules from depolymerizing nia sp.  and factors controlling its productions were during the processes of cell division . Tubulin molecules determined. Simply stated, it appeared that fungi more com- in taxol-sensitive fungi, such as the oomycetes are affected in monly produced taxol than higher plants, and the distribution the same manner as human cancer cells . In fact, the of those fungi making taxol is worldwide and not conﬁned to tubulin gene in Pythium sp. is virtually identical to the tubu- G.A. Strobel / Microbes and Infection 5 (2003) 535–544 539 lin gene in humans. Thus, it seems that taxol, in nature, serves duced novel peptide antibiotics, designated munumbicins A, the yew tree (Taxus spp.) from which it originates commer- B, C, and D, in honor of Reggie Munumbi Miller. These cially, by warding off plant pathogens, especially in the antibiotics possess wide-spectrum activity against many hu- oomycete group. Simply stated—taxol is a fungicide! It is man as well as plant pathogenic fungi and bacteria, and a almost impossible to ﬁnd Taxus spp. that show any infections Plasmodium sp. This endophyte was fermented, and the caused by any of these oomycetes. Therefore, it seems rea- broth was extracted with an organic solvent and the contents sonable that taxol, related taxanes, and some other com- of the residue were puriﬁed by bioassay-guided high- pounds existing in nature, kill the oomycetes in a manner performance liquid chromatography. The residue primarily similar to the killing of actively dividing human cancer cells. contained four functionalized peptides with molecular A search for these compounds using the oomycetes as a weights of 1269.6, 1298.5, 1312.5 and 1326.5. Numerous screening tool seems like a reasonable approach. The proce- other related compounds, possessing bioactivity, with differ- dure is rapid and effective and represents a noteworthy union ing masses and lower quantities were also present in the of plant pathology to modern medicine. To date, each com- culture broth extract. With few exceptions, the peptide por- pound that has been isolated from endophytic fungi, or other tion of each component contained only the common amino plant-associated microbes, that is effective against any of the acids threonine, aspartic acid (asparagine), glutamic acid oomycetes, is also effective against certain cancer cell lines (glutamine), valine, and proline, in varying ratios. The mu- (Strobel, 2001, unpublished). For instance, this association numbicins possessed widely differing biological activities, has held true in the discovery of oocydin A, a chlorinated depending upon the target organism. For instance, munum- macrocyclic lactone from S. marcescens that has potent bicin B had a minimal inhibitory concentration of 2.5 µg/ml activity against the oomycetes and also kills certain cancer against a methicillin-resistant strain of Staphylococcus au- cell lines . Thus, it appears that the approach of carefully reus, whereas munumbicin A was not active against this developing a rationale for plant selection for endophyte iso- organism. In general, the munumbicins demonstrated activ- lation has merit. ity against Gram-positive bacteria such as Bacillus anthra- cis, and multi-drug-resistant M. tuberculosis. However, the 4.2. Endophytes producing antibiotics most impressive biological activity of any of the munum- A search for speciﬁc endophytes that may produce antibi- bicins was that of munumbicin D (see Fig. 2) against the otics can have its origins in ethnobotany, which utilizes the malarial parasite Plasmodium falciparum, having an IC50 of medicinal lore of native people. In the Northern Territory of 4.5 ± 0.07 ng/ml, which is better than that of chloroquine, the Australia, various Aboriginal groups use the ground-up mass world’s gold standard. 1 of snakevine (Kennedia nigriscans) to promote the healing More recently other plants from various locations have of skin wounds and infections. The snakevine, known as yielded other endophytic streptomycete isolates including “mangerrporlo” in Dalabon and Mayali, is harvested as a NRRl 30566 from the fern leafed grevillea (Grevillea pte- fresh stem piece, placed on some hot coals for a short time ridifolia) also of the Northern Territory of Australia. This (10 min), mashed into a pulp, and then applied as a sticky microbe produces novel wide-spectrum antibiotics termed paste to a cut, wound, or infection. Because of the native uses kakadumycins, and they are chemically related to echinomy- of this plant, it was selected as a source of endophytic cin (Castillo et al., 2002, unpublished). In addition, there are microorganisms with the idea that some of the healing prop- now over 20 other endophytic actinomycetes, in hand, which erties of the snakevine may, in fact, be produced as a result of we have collected from plants all over the world that we are the products of one or more endophytes. In fact, one of the characterizing chemically and biologically. endophytes isolated from this medicinal plant was a Strepto- Cryptosporiopsis cf. quercina is the imperfect stage of myces sp. This is of particular interest because, as a culture, it Pezicula cinnamomea, a fungus commonly associated with was extremely bioactive against a number of test microor- hardwood species in Europe. This fungus and related species ganisms. Interest in this endophyte was further piqued be- cause actinomycetes have not been reported to be endophytic 1 I take special note in this section on the munumbicins, to acknowledge on dicotyledonous plants. However, recently a Streptomyces the help and guidance of my late friend, Aboriginal guide, and Arnhem land sp. was reported on an annual plant–Lolium perenne . council member Mr. Reggie Munumbi Miller, of Manyallaluk village, near This lolium endophyte produces a weak antibiotic, desig- Katherine, Australia. He and his assistants graciously identiﬁed trees and nated methylalbonoursin, which is a diketopiperazine, con- shrubs of ethnobotanical interest in and around his village for scientiﬁc examination and sampling purposes. His reason for providing permission densed from leucine and phenylalanine. Thus, although for sample gathering was a part of his goal to encourage greater understan- streptomycetes are the source of over 70% of the world’s ding between the European peoples and the Aboriginal communities of antibiotics, their source is most frequently from soil. Australia. He strongly desired that young people of Manyallaluk village On the other hand, it now appears that some Streptomyces should be proud of their heritage and culture. He also wished to educate spp. have taken up residence in plants. As a result, it appears others on the value and importance of Aboriginal culture, which is widely considered to be the oldest in the world. I had sent him representative SEM that this may be an entirely untapped source of novel phar- photos of the endophytes of the plants of Manyallaluk. One of these endo- maceuticals. The streptomycete obtained from the snakevine phytes was S. munumbi. These novel antibiotics, described above, were plant was designated NRRL 30562 . This microbe pro- presented to the world in his honor and memory. 540 G.A. Strobel / Microbes and Infection 5 (2003) 535–544 Fig. 2. A 14-d-old culture of Streptomyces NRRL-30562 growing on a Petri plate of potato dextrose agar. This microbe represents the ﬁrst (in the 1990s) endophytic streptomycete to have been isolated from any dicotyledonous plant. In this case, the snakevine—Kennedia nigricans—growing on Aboriginal land in the Northern Territory of Australia, was the source of this microbe. The plant itself is used by Aborigines to heal open wounds. Further examination of it revealed that it is a host to an endophytic streptomycete that produces a family of extremely potent novel peptide antibiotics known as the munumbicins. Photo courtesy of Gary Strobel and Uvi Castillo, Montana State University. occur as endophytes in many parts of the world. It was isolated as an endophyte from Tripterigeum wilfordii, a me- dicinal plant native to Eurasia . On Petri plates, C. quercina demonstrated excellent antifungal activity against some important human fungal pathogens—Candida albi- cans and Trichophyton spp. Since infections caused by fungi are a growing health problem, especially among AIDS pa- tients, and those who are otherwise immunocompromised, new antimycotics are needed to combat this problem. A unique peptide antimycotic, termed cryptocandin, was iso- lated and characterized from C. quercina . This com- pound contains a number of peculiar hydroxylated amino acids and a novel amino acid—3-hydroxy-4-hydroxy methyl proline (Fig. 3). The bioactive compound is related to the known antimycotics, the echinocandins and the pneumocan- dins . Cryptocandin is also active against a number of plant pathogenic fungi, including Sclerotinia sclerotiorum and Botrytis cinerea. Cryptocandin is currently being tested and developed by several companies for use against a number of fungi causing diseases of skin and nails. The ecomycins are produced by Pseudomonas viridiﬂava Fig. 3. The structure of cryptocandin (antifungal agent) with each of the . P. viridiﬂava is a member of a group of plant-associated amino acid residues indicated. G.A. Strobel / Microbes and Infection 5 (2003) 535–544 541 Fig. 5. Jesterone from P. jesteri, a newly described Pestalotiopsis species. Fig. 4. The structure of ambuic acid, an antifungal metabolite common to Pestalotiopsis microspora. pestalotiopsis, namely Pestalotiopsis jesteri, produces jest- erone and hydroxy-jesterone that possess antifungal activity ﬂuorescent bacteria that are either weakly pathogenic or  (Fig. 5). Jesterone, subsequently, has been prepared by saprophytic. It is generally associated with the leaves of organic synthesis with complete retention of biological activ- many grass species and is located on and within the tissues ity . The fact that complete organic synthesis of a bioac- . The ecomycins represent a family of novel lipopeptides tive molecule from the endophytic fungus P. microspora has and have molecular weights of 1153 and 1181. Besides been successfully completed has important ramiﬁcations. common amino acids such as alanine, serine, threonine, and A surprise was the realization that endophytes produce glycine, some unusual amino acids are also involved in the substances that can inﬂuence the immune system of animals. structure of the ecomycins, including homoserine and Subglutinols A and B are immunosuppressive compounds b-hydroxyaspartic acid. The ecomycins are active against produced by Fusarium subglutinans, an endophyte of T. wil- such pathogenic fungi as Cryptococcus neoformans and fordii  (Fig. 6). The compounds both have IC50 values of C. albicans. Another group of antifungal compounds is the 0.1 µM in the mixed lymphocyte reaction assay. In the same pseudomycins, produced by a plant-associated pseudo- assay, cyclosporin is roughly as potent as the subglutinols. monad [31,32]. These compounds are being examined more thoroughly as 4.3. Endophytes with other important biological activities immunosuppressive agents. Their role in the endophyte and its relationship to the plant are unknown. As mentioned earlier, P. microspora as an endophyte and The alkaloids are also commonly found in endophytic as a producer of taxol, is commonly isolated from rainforest fungi. Such fungal genera as xylaria, phoma, hypoxylon, and plants. Since it is so commonly found, other aspects of its chalara are representative producers of a relatively large biology should be examined. One extremely interesting fea- group of substances known as the cytochalasins, of which ture of P. microspora and other species of this genus is their over 20 are now known . Many of these compounds wide genetic and biological diversity. For instance, from one possess antitumor and antibiotic activities, but because of small cypress limb, 21 isolates of P. microspora were ob- their cellular toxicity they have not been developed into tained . Only two seemed to be identical in all cultural pharmaceuticals. Three novel cytochalasins have recently and biological respects. Of these isolates, only nine produced been reported from Rhinocladiella sp. as an endophyte on taxol. In another study, the methylene chloride extracts of T. wilfordii. These compounds have antitumor activity and 15 isolates of P. microspora, obtained from at least four have been identiﬁed as 22-oxa--cytochalasins . continents, were examined by thin-layer chromatography Thus, it is not uncommon to ﬁnd the cytochalasins in endo- and no two chromatograms were identical (Strobel, 2001 phytic fungi, and workers in this ﬁeld need to be alert to the unpublished). Enormous variability must exist in this organ- fact that redundancy in discovery does occur. Chemical re- ism, arising by mutation, genetic crossing, or by as yet dundancy usually occurs with certain groups of organisms in unsubstantiated mechanisms, such as genetic exchange with its hosts. It appears that P. microspora is a microbial factory of bioactive secondary metabolites. For this reason, some investigators have examined Pestalotiopsis spp. for novel compounds that may have potential use for medicine. Although biochemical diversity does exist in this endo- phytic fungus, it seems that there may be some secondary metabolites that various isolates of this fungus hold in com- mon. One such secondary metabolite is ambuic acid, an antifungal agent, which has been recently described from several isolates of P. microspora found in many of the world’s rainforests  (Fig. 4). Torreyanic acid, a selec- tively cytotoxic quinone dimer (anticancer agent and anti- biotic), was isolated from a P. microspora originally obtained as an endophyte associated with the endangered tree, Torreya taxifolia (Florida torreya) . A newly described species of Fig. 6. Subglutinol A, an immunosuppressant from Fusarium subglutinans. 542 G.A. Strobel / Microbes and Infection 5 (2003) 535–544 which previous studies have already established the chemical human telomeres, and this points to the possibility that P. identity of major biologically active compounds. For in- microspora may serve as a means to make artiﬁcial human stance, as with the cytochalasins, they are commonly associ- chromosomes, a totally unexpected result. ated with the xylariaceaous fungi. 4.4. Endophytes producing volatile antibiotics 6. Prospects for the discovery of other important compounds from endophytes Muscodor albus is a newly described endophytic fungus obtained from small limbs of Cinnamomum zeylanicum (cin- A comprehensive study on the endophytes of any indi- namon tree) . This xylariaceaous (non-spore producing) vidual rainforest higher plant species has not been done, fungus effectively inhibits and kills certain other fungi and much less a study on any individual plant in its entirety, from bacteria by virtue of a mixture of volatile compounds that it its complete root system to its stems, petioles, leaves and produces . The majority of these compounds have been ﬂowers. The prospects of ﬁnding endophytes (fungi and identiﬁed by gas chromatography/mass spectrometry and bacteria) that are speciﬁc to any given higher plant or even then ultimately made into an artiﬁcial mixture that mimicked occurring only in a local region in a forest seem great, given the antibiotic effects of the volatile compounds produced by the paucity of work in this area. Generally, it seems that novel the fungus . Each of the ﬁve classes of volatile com- taxa of these microbes are the most likely source of novel pounds produced by the fungus had some inhibitory effect bioactive molecules such as antifungal agents, antibiotics, against the test fungi and bacteria, but none was lethal. immunosuppressants, and other molecules of interest and However, collectively they acted synergistically to cause importance. The most difﬁcult problem, however, is to prop- death in a broad range of plant and human pathogenic fungi erly choose those among thousands of plant species on the and bacteria. The most effective class of inhibitory com- planet to be the most fruitful for study. It seems that endemic pounds was the ester, of which isoamyl acetate was the most plants growing in moist, warm, and geologically isolated biologically active. The ecological implications and potential climates are among the ﬁrst choices for study. For instance, practical beneﬁts of the “mycofumigation” effects of M. the top of Mt. Waialeale in the island of Kawai, in the albus are very promising in both human and agricultural Hawaiian Islands, receives one of the world’s greatest applications. amounts of rainfall at nearly 450 in. per annum. It would appear that microbial competition in such an area would be ﬁerce given the abundance of both water and plants. As such, 5. Surprising results of molecular biological studies on the number and diversity of natural products produced by P. microspora microbes surviving in such an area would be enormous. Rationale such as this can serve to direct a concerted study on Of compelling interest is an explanation as to how the the endophytes existing in various suitable environments. For genes for taxol production may have been acquired by P. mi- instance, this forested area of Hawaii is also home to a crospora [8,15]. Although the complete answer to this ques- plethora of native plants ranging from the common Metrosi- tion is not at hand, some other relevant genetic studies have deros spp. (Ohi’a lehua) to the native Hawaiian tree been done on this organism. P. microspora Ne 32, is one of fern—Cibotium spp. Recent sampling of this area has re- the most easily genetically transformable fungi that has been vealed endophytes in all plants studied. The likelihood of studied to date. In vivo addition of telomeric repeats to ﬁnding novel bioactive molecules from these endophytic foreign DNA generates extrachromosomal DNAs in this fun- microorganisms also seems quite possible. gus . Repeats of the telomeric sequence 5’-TTAGGG-3’ were appended to non-telomeric transforming DNA termini. The new DNAs, carrying foreign genes and the telomeric 7. Endophytes and ethnobotany repeats, replicated independently of the chromosome and expressed the information carried by the foreign genes. The Other areas of the world holding fascinating plants and addition of telomeric repeats to foreign DNA is unusual associated endophytes are the tepuis of Venezuela, the rain- among fungi. This ﬁnding may have important implications forests of Central America, and the monsoonal areas of in the biology of P. microspora Ne 32, since it explains at Australia. In addition, the golden triangle of Thailand, and least one mechanism through which new DNA can be cap- the highlands and coastal areas of Papua New Guinea, the tured by this organism and eventually expressed and repli- entire country of Madagascar, and the upper Amazon regions cated. Such a mechanism also points to an explanation of are other areas with great biodiversity . In each of these how the enormous biochemical variation may have arisen in areas, novel endophytic fungal taxa have been discovered as this fungus. Also, this initial work represents a framework to well as a series of new bioactive compounds. Again, each of aid in the understanding of the ways this fungus may adapt these areas of the world has abundant rainfall, wide plant itself to the environment of its plant hosts, and suggests that species diversity, and many endemic plants. The search for the uptake of plant DNA into its own genome may occur. In novel endophytes and their associated secondary products addition, the telomeric repeats have the same sequence as should also be directed towards plants that commonly serve G.A. Strobel / Microbes and Infection 5 (2003) 535–544 543 native populations for medicinal purposes. It is conceivable Acknowledgements that these plants have microbes that mimic the chemistry of their respective host plants and make the same bioactive The author expresses appreciation to the National Science natural product(s) or derivatives that are more bioactive than Foundation, the USDA, Novozymes Biotech, The BARD those of their respective host. This is exempliﬁed with the Foundation of Israel, the R&D Board of the State of Montana case of taxol from yews and also taxol being produced by a and the Montana Agricultural Experiment Station, in provid- series of endophytes from yews as well as other plant ing ﬁnancial support for some of the work reviewed in this sources. 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