1. Fungal Endophytes and their medical value in
association with Cannabis sativa
By: Victoria Nedley
December 2013.
© The Samuel Roberts Noble Foundation
Abstract
Cannabis sativa is an indispensable gift from the earth, withmultiple uses and
a majority of its importance recognized in its medicinal qualities.This paper
investigates the role fungal endophytes, extrapolated from recent findings found in
every plant species,play in the medical values attributed to C. sativa. Mysteries are
enquired and investigated,and parallelsare drawn from what is known about fungal
endophytes. The fungal endophytes found in C.sativa should be accredited with the
vast expansion of unique secondary metabolitesfound in C. sativa; as well as be
recognized for their beneficiary actionswhich have probably allowed C. sativa such a
long stay as a species.

2. INTRODUCTION
Empirical evidence suggests that symbiosis may be one of the most important vectors to
speciation, giving random genetic mutation a backseat role in the evolution of new species.
Because of this reason, paying particular attention to interactions between species, and the
points at which these interactions become dependent – from cospeciation to host specificity
and eventually altogether so dependent that they are in fact inseparable —is of utmost
importance in understanding the ebb and flow of life formation. Take the example of Lichens,
two organisms that [most likely because of their size] are still fully recognizable in their own
right; but coexist as one organism together. Or a more extreme example would be that of
organelles inside cells; once an ancient protist – now a version of its old self, so closely involved
with another that it has the opportunity to reduce. So much so that it uses the metabolism of
the other for certain things but still holds fast to the important aspects of itself which led it to
being there in the first place…i.e. chloroplasts.
The steps in between the initial dependency and the evident dissolution into one
another are of particular interest. One such interaction is that of the fungal endophytes and
their particular plant hosts. Still distinguishedas separate entities, these endophytes are
woven within and throughout host plant tissues and organs; both inter and intracellularly. The
symbiosis is markedby the trade-off recognizedof both species. The endophyte provides
protection via stress reduction, secondary metabolite production, and increasednutrient and
water uptake; while the plant provides a viable source of energy. Metabolite production is
predominantly the issue of discussion. Because of the closeness of their relationship, it has
been proven that endophytes often produce similar if not exactly the same metabolites as their
plant partners [Stierle et al, 1993; Tan and Zou, 2001].
Upwards of thirty fungal endophytes have been isolatedfrom the plant tissues of C.
sativa [Kusari, 2012]; but their attribution to the medicinal qualities we praise C. sativa for
have not been recognized. I present evidence for the acknowledgement of the role fungal
endophytes in C. sativa play in the multiple medical uses of C. sativa. From this, the fungal
endophytes found in C. sativa, especially those found in the apical and lateral buds, should be
investigatedfor specific medical value. If specific action cannot be completely inferred, due to
synergistic effects – the importance of the crude C. sativa should be exaggerated.
FUNGAL ENDOPHYTES; A MODEST INTRODUCTION
“An endophyte is a bacterial (including actinomycete) or fungal microorganism,
which spends the whole or part of its life cycle colonizing inter and or intra-cellularly inside the
healthy tissues of the host plant, typically causing no apparent symptoms of disease.” [Tan and
Zou, 2001].
Fungal Endophytes [FE’s] have been found in all plant species that have been
examinedfor them [Saikkonen, 1998]; therefore it may be safe to say that fungal endophytes
are present in all plant species. With this in mind, the silent admiration upon a tree or a flower
holds with it a certain complexity unseen by the eye. As the microbial world goes, not only are
they smearedwith organisms on the outside, but interwoven throughout by microbes on the
inside; a parallel to the human biome, which has just begun to be so accurately studied and

3. commentedupon. Symbiotic speciation and the serial endosymbiosis theory also bring further
wattage to this light. Many ‘domesticated parasites’ become so dependent on their host that
they reduce themselves to organelles using the hosts mechanisms for metabolism and are even
passed down to progeny via vertical transmission [Moran, 2008; Saikkonen 1998]. Identifiable,
isolatable FE’s are not yet reduced but many are found to be transmittedmaternally by hyphae
in the seeds [Saikkonen, 1998]. One step closer to complete dissolution.
FE’s offer multiple benefits to the host plant in exchange for two of the most important
things necessary for survival, food and shelter. Those benefits were first realizedin the
somewhat miraculous observation of grasses and their uncanny tolerance to drought [Carroll,
1988]. In return for harboring and feeding these endophytes, plant hosts are rewarded with a
variety of leg-ups. Including but not limitedto: increasedtolerance to stress, protection and
defense against disease andpredation, and increasednutrient and water uptake
[Higginbotham, 2013]. FE’s have even been noted to further extend a plant hosts allelopathic
abilities andto produce phytohormones, allowing for faster healthier growth; which further
increases fitness for both species [Saikkonen, 1998].
Probably the most fascinating aspect of fungal endophytes, and the central mechanism
behind all of their benefits, is their vast production of complex secondary metabolites. Even
more so, the fact that many of the chemical constituents we value plant species for are at the
same time synthesized identically by their endophytic counterpart. [Stierle et al, 1993; Tan and
Zou, 2001; Schulz, 2002; Shweta, 2009]. Owen and Hundley [2004] go as far as to say they are
“the chemical synthesizers inside plants”. It is my goal to scientifically persuade the argument
that at the very least, some of the chemical metabolites in Cannabis sativa are produced either
solely or simultaneously by their fungal endophytes.
MYSTERIES OF C. SATIVA
(featuring possible clues and answers)
A thorough review of the natural chemical constituents of C. sativa, done by Turner et
al., reveals there to be 421 known metabolites as of 1980. Today, according to a new study
done in 2005 by ElSohly and Slade, 12 new metabolites have been identified; and novel
compounds are being discovered and reported constantly [Turner, 1980].
While fungi enjoy a vast spectrum of lifespans, they are arguably always shorter than
their plant host counterpart [Kendrick, 2000]. While the plant host completes one lifecycle, the
FE could produce new generations daily. The options for mutation, resistance, etc… are much
higher in a species of such quick turnaround; and their responses to pathogen mutation,
resistance, etc… are also much quicker and more relevant. Saikkonen gives a great example of
this: you are an elderly woodland plant, and you are 100 years old – ask yourself how you are
to keepup with the everyday evolution of a threatening pathogen. Your genotype will not
change drastically enough –via random mutation- in one lifetime to implement new synthesis of
new compounds to combat new challenges. The answer to this is: endophytes – the tree
harbors them and uses them in a sense to keepup with the evolutionary arms race going on
around and within it. Because they will have hundreds of generations under their belt,
changing in response to threats posed to the plant and itself, they are like a developing

4. weapon. The novel compounds from C. sativa being discoveredand reported, are a result of
endophytic fungi responding to changes in threats to itself and its host plant. Whether these
compounds are medicinally important isn’t exactly relevant here, just the fact that they are
traceable, while the genotype of the host plant remains generally the same.
One class of medicinally valuable chemicals derivedfrom C. sativa, are the
cannabinoids. Cannabinoids are quite mysterious due to the fact that although they belong to
a relatively ubiquitous chemical class – the terpenophenolics, they are not found anywhere else
in nature. Not even in the closely relatedHumulus genus. There are upwards of 150 unique
cannabinoids found in C. sativa. [Turner, 1980] From what is known about FE’s, it seems more
than obvious that they are playing a strong role here. Whether the mechanism of action is
present in the production of the cannabinoids themselves or in the sym biotic alteration of the
pre-existing plant metabolites or in the induction of transcription by the FE in the plant host
[Soliman, 2013], their role here cannot be denied. Producing such a rich diversity of complex
and unique compounds is not often a feature of plant metabolism; whereas fungi are known
specifically for producing many intricate distinctive compounds [Tan and Zou, 2001], with their
vast sexual reproduction strategies and short life spans [Kendrick, 2000; Heaton et al. 2012].
Another facet of C.sativa that offers us something to consider, is the fact that the
absence or presence of compounds can vary via geological location [Turner et al, 1980]. This is
not the same as say American ginseng and Chinese ginseng growing in different geological
areas and producing very similar but not exactly the same active constituent. What this is
saying is that compounds will physically not be present in C.sativa growing in Bangladesh,
that are present in C. sativa growing in South America. Whereas American andChinese
ginseng are recognizedare two different species, C. sativa is recognizedas one species with
multiple strains. Part of this must be due to the endophytes that are available in these different
regions. As it is commonly known, the center equator tropics has a much higher species
diversity then is observed elsewhere in the world. It would not be surprising to find out that
there are more isolatable compounds in the tropical variations of C.sativa than in the Middle
Eastern strains. Because there exists higher species diversity, there are more species of
endophytes to infect host plants and produce novel compounds. FE’s probably contribute to
the vast number of strains that are available, with altering ratios of cannabinoids.
EXAMPLES OF ENDO/HOST PRODUCTION OF IDENTICAL AND NOVEL METABOLITES
Although the investigative journey to understanding FEs and their metabolism is newly
underway, there exists a paltry amount of published information regarding the chemical
metabolites produced by FE’s. Again, not unexpectedly, many of the chemical compounds we
have grown to associate with plants are also, if not solely or indirectly via induction, produced
by their FE(s). This information should be taken into consideration and investigation should
be implementedto isolate C.sativa FEs in search of production of compounds initially
attributed to the plant itself.
Taxus brevofolia, the Pacific Yew, is in danger of extinction due to the popularity of a
diterpenoid, found in the inner bark of the tree, which has shown promise in the reduction of
tumors. The chemical is synthesizedinto a medication known as paclitaxel used to treat
various forms of cancer, such as breast cancer, ovarian cancer, and lung cancer [Stierle, 1993].

5. Stierle andher group discovered that taxol was being produced also by one of its fungal
endophytes, Taxomyces andreanae . This is significant, the pacific yew is a slow growing tree
and alternate sources for exploitation are in dire need. Stierle andher group discovered this
information 20 years ago, it is astonishing that immediate attention was not directed towards
endophytes at this time. Tan and Zou propose the exciting concept of “upscale fermentation” of
the fungus to allow for a reproducible sink of taxol. Imagine the possibilities here.
In 2009 another group of scientists, [Shewta et al. 2009], discovered that strains of the
endophyte Fusarium solani of the host plant Apodytes dimidiata were producing camptothecin
and derivatives of camptothecin, which is a biologically active alkaloid, observed to be anti -
tumor with a novel mechanism of action [Cordell et al. 2001].
Tan and Zou present an exhaustive list in which they isolate multiple endophytes from
a variety of plant hosts. They find an assortment of novel compounds, and some that are
biologically active. These compounds, like their host plants, can be: anti-viral, anti-fungal,
anti-bacterial, anti-… One in particular that caught my attention was an unidentifiedfungal
endophyte species that was found to produce compounds that are anti -bacterial against
Methicillin-Resistant Staphylococcus aureus, as well as Vancomycin-Resistant Enterococcus
faecium [Tan and Zou, 2001]. This is further proof that because of their shortenedlifespan and
ability to produce complex unique compounds, they can keepup evolutionarily at the same
pace as other microbes that we classify as pathogens.
To be sure, the world exists in a balance – and everything is a double edged sword.
Sometimes it has been found that these isolatedendophytes don’t produce sizeable amounts of
the desired product, or that without the activation of their host species stress response they do
not produce the compound at all. Hopefully, these are not complete set-backs. More will be
said on these and other problems in the exploitation section.
IMPORTANCE OF THE CRUDE
If there is any silver lining to be taken from the information presented, one is the
importance of the crude. It has been proven that plant derived medicines are more effective in
the crude form [Cordell et al. 2001]. Specifically concentrating on C.sativa, noted before
upwards of 30 FEs have been isolated from C.sativa and a majority of those are present in the
apical and lateral buds [Kusari, 2013]. The apical and lateral buds are the medicinally applied
portions of the plant; and in Turners review of natural constituents of C.sativa he does specify
where those constituents are concentrated. A careful investigation of concentration of
metabolites alignedwith concentration of fungal endophyte species could reveal predictions of
which species to concentrate on to look for specific me tabolic products.
Simple deductions can be made to predict the relevance of C.sativa’s FEs and the
medicinal values associated. When curing and drying the apical and lateral buds, fungal
hyphae are present. If burning does not affect the conditions of the cannabinoids, then it
doesn’t rule out the existence of cannabinoids and/or other alkaloids and metabolites
produced by fungal compounds in the crude extract. The synergistic effects should not be
ignored, and could in fact be, in part, a major part of the healing properties. The fact that
C.sativa has such a wide range of effects – from nausea to glaucoma [Borgelt, 2013] – is only

6. back up to the argument that multiple constituents are responsible and synergistically related
to the medical values associatedwith C. sativa.
EXPLOITATION OF NOVEL AND KNOWN COMPOUNDS
Biomass is a key point to keepin mind, as Tan and Zou 2001 projected – a reproducible
sink of fermentedfungi producing novel and known biologically active compounds is not
entirely out of scientific generation andactualization. Each year new strains could be collected
from the environment, which are actively participating in the arms race. As we face future
resistant pathogens, this could be a sustainable and viable source of medical compounds in
which to combat said pathogens. Because they are so small, the fact that they don’t produce
high concentrations of the desired product can be counteracted by their small size. Insteadof
clearing fields and forests for medically valuable plant resources, several large buildings will be
employed solely for fermentation andisolation of valuable metabolites. An alternate source, to
lessen the pressure we have put on plants as medicenes. Sometimes one must resort to
monetary value to open the eyes of others. And, if that be the case, there are billions to be
made here; although my attitude should be expressed, better said by Ehrenfeld, 1998:
“Assigning value to that which we do not own and whose purpose we do not understand…is the
ultimate in presumptuous folly.” [Cordell et al. 2001].
There is much to be done in this respect, and many discoveries to be made. Not only
will this lead to a more hopeful future for the medical world, but it many answer questions as
deep as the formation of complex life. Surely one cannot go astray with this direction.
CONCLUSION
There is a developing history in the transformation of genetic material between host
organisms and symbionts, parasites, pathogens and the like. All interactions are important,
here the Gaia theory comes to mind [Moran, 2008; Margulis, 2008]. Alternate sources of
medically valuable metabolites are to be found upon further investigation in the fungal
endophyte world. Their ability to synthesize complex compounds at the small size and lifespan,
make them an ideal tool to combat pathogens. The multitude of uses of C. sativa employed
medically should be attributed, at least in part, to its myriad fungal endophytes; and the
importance of the endophyte containing crude should be inflated. We know that C. sativa is a
hardy plant, which can grow in multiple areas of the world. We know that C. sativa produces
unique compounds. We know that C. sativa has been around for thousands of years. What do
fungal endophytes have to do with all the great things we know about C. sativa?
For other stimulating information, findout about fungal endophytes and their use as
bio-pesticides [Backman and Sikora, 2008; Gautam, 2012; Mejia et al, 2008; Qadri et al. 2013;
Vega, 2008]

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