endohpytesas sources of bioactive products by adelaide17madette

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									                                                         Microbes and Infection 5 (2003) 535–544


                               Endophytes as sources of bioactive products
                                                                Gary A. Strobel *
                                   Department of Plant Sciences, Montana State University, Bozeman, MT 59717, USA


   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 [1]. 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 [3]. By definition, these
lives each year than any other group of infectious agents [1].                 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 [4]. 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 [2]. 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: uplgs@montana.edu (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 [5]. 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 [3]. 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 [3]. 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 [6]. 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 [7]. 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 [8]. 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 [9]. 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 [10].                                                                  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 [3]. 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 [12] (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+) [8]. 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 [11].                                                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 [13].
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 [8]. 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 [8]. 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 [14].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 [8]. 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. [15]. 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 [16]. 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 [21]. In addition, at
most commonly isolated endophytic species is Pestalotiopsis              least three endophytes of T. wallichiana produce taxol, in-
microspora [17]. Organisms virtually identical to the taxo-              cluding Sporormia minima and Trichothecium sp. [22]. 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 [23]. In addition, several
pines, loquats, guavas, mangoes and a large number of orna-              fungal endophytes of this plant (filbert) produce taxol in
mental plants [16]. Generally, the commonly held view is                 culture [23]. 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 [20]. 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 [15]. 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 [15]. 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 [17]. 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 [18]. 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 [19]. 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. [20] and factors controlling its productions were                during the processes of cell division [24]. 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 [25]. 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 [11]. 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 [26].                  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 [27]. 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 [28]. 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 [28]. 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 [29]. 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
[30]. 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
                                                                              [35] (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 [36]. The fact that complete organic synthesis of a bioac-
[30]. 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 [37] (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 [38]. 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 [17]. 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-[12]-cytochalasins [38].
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 [33] (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) [34]. 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) [39]. 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 [40]. 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 [40]. 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 [41]. 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 [42]. 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. Thus, if a microbial source for a medicinally impor-            report. This report, in part, was modified from another review
tant substance can be found, then its supply is better guaran-           published in Critical Reviews in Biotechnology (2002).
teed than if its sole source is from one or more obscure, rare,
or difficult to cultivate higher plants. Quite commonly, local
native peoples have endowed themselves with the knowledge                References
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