On Cannabis Endophytes: An answer to a question

Herb Notes

Originally published in Plant Healer Quarterly, Vol. IX, Edition IV (Winter 2019/2020)

During my 2019 class at the Confluence on the endophytes of medicinal plants, someone asked if Cannabis has endophytes, and whether I would speak on the topic. They were curious, for instance, whether the endophytes in Cannabis influence cannabinoid production. Other than answering, “Yes, all plants have endophytes”, I didn’t have anything else to share. I decided to look into it, and here are the results (and the topic of one of my classes at this year’s Confluence).

Endophytes 101

Let’s start with a primer on endophytes for folks who missed last year’s class and accompanying article series. (Skip ahead if you’re already an endophyte maven.) Endophytes are bacteria, archaea, fungi, and protists that live within plants (hence, endo “inside” and phyte “plant”).  They live in between or inside of plant cells. This is in contrast to epiphytes, minute critters living on the surface of plants. All of these constitute the microbiome of a plant, just as we animals have microbiomes.

Fungal endophytes first shacked up with plants at least 400 million years ago based on the fossil record, and an ancient association between endophytes and plants implies that they’ve influenced one another’s evolution. For example, the selection for beneficial changes in the genes of a pine tree and its descendants may not happen quickly enough to survive the introduction of a new parasite to the neighborhood. But the resident endophytes of a particular tree — providing a deep well of genetic richness — may rapidly help the tree fend off attack. That tree and its endophytes then survive to pass on their genes. (Endophytes are often passed vertically from parent to progeny.)

Along these lines, endophytes can benefit the host plant in several ways. For instance, some endophytes produce metabolites that deter grazing by insects or animals, or fend off infection by pathogenic microbes. Other endophytes enhance the growth or development of a plant. Still others suppress growth of nearby competitive plant species. On the flip side, endophytes get a home and nourishment from the plant. Lest you think it’s all sunshine and roses, some endophytes are like your Uncle Larry who moved in “temporarily” and hasn’t left the couch since. Meaning, some endophytes appear to be parasitic, taking up space and energy in the plant without providing anything in return.

Cohabitation of endophytes and plants is relevant to us as herbalists.  More specifically, some of what we call “plant medicine” is actually plant and endophyte medicine, or even largely endophyte medicine.  Why is this?  Because endophytes influence the secondary metabolites (the “medicine”) present in a plant.  Some endophytes do this by stimulating the plant to increase synthesis of a particular plant metabolite. This is the case regarding the anti-inflammatory alkylamides of Echinacea (1, 2). Others endophytes induce production of a plant metabolite that otherwise wouldn’t be present. trigger the plant to produce a chemical that wouldn’t be there in the absence of the endophyte (e.g. resveratrol in Doug fir) (3). Weirdly enough, some endophyte and plant pairs make the same chemical, as with the synthesis of paclitaxel by yew trees and a yew tree endophyte (3). Finally, some endophytes generate compounds that the plant itself doesn’t make. For instance, endophytes in guduchi make inhibitors of xanthine oxidase, which is involved in uric acid production (4).  Incidentally, guduchi is traditionally indicated for gout, a form of arthritis due to uric acid precipitation within joints.

On to Cannabis

Cannabis is getting a lot of attention for properties beyond its psychoactive effects. Cannabis is chemically complex, with over 400 secondary metabolites identified so far. These include cannabinoids, terpenoids, flavonoids, and lignans that display all sorts of activities in lab-based research and, in some cases, in clinical studies. The most famous of these, the cannabinoids, are a chemical class initially identified in Cannabis. Chemically related molecules in this “phytocannabinoid” class are found elsewhere in the plant world, and even in a fungus (5). We, as humans, even have our own “endocannabinoids” that are involved in a range of physiological functions.

Yes, Cannabis has endophytes

Finally, the meat of the article! Cannabis endophytes have been found in the leaves, petioles, twigs, buds and roots (6, 10, 12), with multiple genera of fungi (8-11) and bacteria (7, 11,12) being isolated from the plant.  Who is observed and in what numbers — microbially speaking — varies based on plant cultivar, growth region, growth stage, and part (6, 7). Some of the endophytes are known people pathogens such as Aspergillus and Pseudomonas that may be problematic for Cannabis users who are immunocompromised. More on this later…

Endophytes and cannabinoid production

Well, do any of them influence cannabinoid production? Like endophyte-mediated enhancement of alkylamide production in Echinacea, are there endophytes in Cannabis that ramp up, say, CBG production by the plant? Or themselves make a cannabinoid, like the yew tree endophytes that make paclitaxel?

And……..not.  At least, as of this writing, no influence of endophytes on cannabinoid production has been reported in research found in Medline, Ovid, or Google Scholar databases. Plenty of researchers speculate that endophytes do, indeed, influence cannabinoid production, but so far it’s been crickets in terms of published data.  I’ll go out on a limb and say that more than one lab is probably looking at this question, since everyone and their Uncle Larry seem to be using weed-themed products. (CBD deodorant, oh my!) To truly nail how a plant’s endophytes influence secondary metabolite production, a plant has to be grown under sterile conditions from a seed that has been cleared of both endophytes and surface microbes. Either multiple of these plants are generated from such seeds or the initial endophyte-free plant generated is cloned into multiple plants. Then the comparison is made between endophyte-free plants and those inoculated with their native endophytes. (An aside: plants originating from different seeds will have genetic variability that may translate into variability in cannabinoid levels, endophytes be damned.) There may be technical challenges doing these sorts of experiments with Cannabis that are beyond my reckoning. Dunno. (2021 update….still nothing.)

Endophytes and Cannabis growth

I was disappointed not to have anything juicy to report back to the cannabinoid-curious audience member regarding the influence of Cannabis endophytes on production. As a consolation prize, I searched for reports on the influence of Cannabis endophytes on growth of the plant. Certain endophytes in medicinal and food plants have been found to improve growth of their host plants. As yet, little has been published on this with respect to Cannabis and its endogenous endophytes. As discussed below, what may have been Cannabis endophytes influenced aspects of hemp growth. Hemp is a form Cannabis with minimal levels of the psychoactive cannabinoid THC. Also, endophytes from Cannabis can improve the growth of other plant species. Here are the deets:

(i) A group from China reported production of thicker hemp fibers following inoculation of hemp plants with several species of endophyte (13). Inoculation with one of the endophytes also increased the spacing in between branches and stems of the hemp plants (the “internode”)(13), which results in leggier plants. Leggier plants would presumably mean longer hemp fibers. However, I could only read the abstract and parts of the data tables. The rest was in Chinese, so I couldn’t read the methods or other details. I don’t know, for instance, whether the endophytes had been isolated from Cannabis plants or were from other plant sources. The word “re-inoculation” in the title suggests the former. And the fungal genera such as Fusarium and Chaetomium listed in data table are genera found in Cannabis. (I’ve yet to find a translation of the Chinese article, and, given that this article is simply a long winded answer to a question at a conference presentation, I’ve not gone as far as bribing someone to translate it.)

(ii) Endophytes isolated from Cannabis cultivars and grown in culture have been found to produce metabolites that can increase the growth of other species of plant. For example, endophytes from wild Cannabis increased the growth of rapeseed plants along with increasing their resistance to various types of stressors (14).  Similarly, inoculation of rice plants with an endophytic fungus isolated from Cannabis improved growth and chlorophyll production (15). The fungus did this by stimulating production of plant hormones and via producing several plant hormones itself (15).

(iii) A proprietary commercial blend of endophytes (“Mammoth P™”) isolated from who knows where (the manufacturer knows) increased the size of Cannabis plants, including bud yields (16). Buds contain the highest concentration of cannabinoids in the plant, so this would theoretically mean a higher yield of cannabinoids per plant. (This doesn’t answer the question of whether the endogenous endophytes of Cannabis regulate cannabinoid synthesis but is a step in that direction.)

Endophytes and disease resistance

Continuing down the consolation prize path entailed a review of what’s known regarding Cannabis endophytes and disease resistance. Just like our resident microbes help protect us from incoming pathogenic microbes, Cannabis endophytes may protect the plant from pathogens.  The battle of the bugs, as it were.

Anyone who’s grown Cannabis knows that a wide variety of pathogenic microbes like to establish residence in and on the plant. Particularly notorious pathogens include the fungi Botrytis cinerea and Trichothecium roseum and even a protist, Phythium (17, 18). The famous botanist Lyster Dewey said in the early 1900s that hemp has no enemies; but it turns out that it does, particularly in a green house setting.

Studies done in a dish have shown that endophytes isolated from Cannabis can inhibit or kill both Botrytis and Trichothecium (19) along with a passel of other plant pathogens (10, 20). This inhibition was due to metabolites spewed out by the endophytes. An even sneakier way that Cannabis endophytes can hogtie disease-causing microbes is by inactivating the virulence factors that make them pathogenic (21). This inactivation is called quorum quenching. In a bit more detail, endophytic bacteria isolated from Cannabis disrupted the cell to cell communication of a bacterial test strain (21). This cell to cell communication is called quorum sensing, and is used by bacteria (single celled organisms) to coordinate with each other and act more like a multicellular organism. Quorum sensing for many types of bacteria contributes to their pathogenicity.

Its yet to be seen how these experiments-in-a-dish apply to an actual plant, but the data are compelling. Keep in mind that if these results do carry over to actual endophyte-pathogen interactions in Cannabis, it’s not that the resident endophytes are being nice to the plant, they’re simply protecting their home and meal ticket.

Is the presence of endophytes in Cannabis a risk factor for us?

By definition, endophytes don’t cause disease in their host plant. But, as mentioned earlier, some Cannabis endophytes are pathogenic in people. Immunocompromised people who smoke Cannabis have developed dangerous lung infections (aspergillosis) with Aspergillus, a common endophyte in the plant (22, 23). An immunocompetent person also developed aspergillosis in association with smoking Cannabis (23). Other fungal endophytes in Cannabis produce a variety of mycotoxins (22, 23).

To be clear, the issue of human pathogens as plant endophytes issue isn’t limited to Cannabis. Many food crops harbor microbes that are pathogenic to people (24).

Remaining questions

So many questions remain regarding the relationship between one of the earliest cultivated plants and its endophytes. I’m mostly interested in the “gee whiz” aspects, not being a grower or user of Cannabis. But the questions have practical relevance.

(i) Do Cannabis endophytes impact the synthesis of secondary metabolites such as cannabinoids, terpenoids, flavonoids and such in Cannabis? The main question that kicked off this whole exercise. Presumably the answer is “of course they do”, as with the situation in other plants.  Just waiting on the studies looking at this….

(ii) Are there endophytes in Cannabis capable of synthesizing cannabinoids or precursor molecules, and if so, are they able to do this in culture outside of the plant?  You can bet that some pharmaceutical and/or nutraceutical companies are screening for these.

(iii) How much is the regional and strain variation in Cannabis chemistry due to their particular collection of endophytes? In other words, do region, cultivar, and plant microbiome independently influence chemistry (unlikely) or is one of these factors more dominant? The presence and relative levels of plant secondary metabolites in general vary based on geographical region and cultivar. Endophyte populations within a plant species also vary based on region and cultivar.

(iv) Can Cannabis endophytes isolated from the wild and inoculated into cultivated strains improve plant health/growth/yield? The idea is a biological fertilizer rather than a chemical one. This is already being done with exogenous endophytes, such as the commercial product mentioned earlier. But it would be interesting and useful if wild Cannabis endophytes could impart some benefit to their tame and prone-to-disease counterparts.

So there you have it: a treatise on Cannabis endophytes. I’ll continue to follow the research literature, so please keep your eyes out for a follow up to this article.

References and further reading

  1. Maggini, V, et al (2017) Plant-endophytes interaction influences the secondary metabolism in Echinacea purpurea (L.) Moench: an in vitro model. Scientific Rep. 7:16924.
  2. Gualandi, RJ Jr. (2010) Fungal endophytes enhance growth and production of natural products in Echinacea purpurea (Moench.). ” Master’s Thesis, University of Tennessee.
  3. Owen, NL & N Hundley (2004) Endophytes — The chemical synthesizers inside plants. Science Progress. 87(2):79-99.
  4. Kapoor, N & S Saxena. (2018) Endophytic fungi of Tinospora cordifolia with anti-gout properties. 3 Biotech. 8(6):264.
  5. Hanus, LO, et al (2016) Phytocannabinoids: A unified critical inventory. Nat Prod Rep. 33:1357-92.
  6. Winston, ME, et al (2014) Understanding Cultivar-Specificity and Soil Determinants of the Cannabis Microbiome. PLoS ONE 9(6): e99641.
  7. Backer, R, et al (2019) Closing the yield gap for Cannabis: A meta-analysis of factors determining Cannabis yield.  Front Plant Sci. 10:495.
  8. Zubek, S, et al (2012) Fungal root endophyte associations of medicinal plants. Nova Hedwigia. 94(4):525-40.
  9. Kusari, P, et al (2013) Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens. Fungal Diversity. 60:137-51.
  10. Guatam, AK, et al (2013) Isolation of endophytic fungi from Cannabis sativa and study their antifungal potential. Arch Phytopathol Plant Protection. 46(6):627-35.
  11. Scott, M, et al (2018) Endophytes of industrial hemp (Cannabis sativa L.) cultivars: identification of culturable bacteria and fungi in leaves, petioles and seeds. Can J Microbiol. 64(10:664-80.
  12. Kusari, P, et al (2014) Quorum quenching is an antivirulence strategy employed by endophytic bacteria. Applied Microbiol Biotechnol. DOI 10.1007/s00253-014-5807-3.
  13. Jin, X, et al (2014) Effects of Endophytic Fungi Re-inoculation on Physiological and Agronomic Characters of Hemp (Cannabis sativa). Plant Diversity. 36(1):65-69.
  14. Azfal, I, et al (2015) Selective isolation and characterization of agriculturally beneficial endophytic bacteria from wild hemp using canola. Pak J Bot. 47(5):1999-2008.
  15. Lubna, SA, et al (2019) Growth-promoting bioactivities of Bipolaris sp. CSL-1 isolated from Cannabis sativa suggest a distinctive role in modifying host plant phenotypic plasticity and functions. Acta Physiologiae Plant. 41:65
  16. Conant RT, et al(2017) Effects of a Microbial Biostimulant, Mammoth PTM, on Cannabis sativa Bud Yield. J Hortic 4: 191.
  17. Punja, ZK (2018) Flower and foliage-infecting pathogens of marijuana (Cannabis sativa L.) plants. Can J Plant Path.  40(4).
  18. Miller, M (2019) Pathogens Causing Wilting in Field-grown Cannabis Identified. Analytical Cannabis.
  19. Kusari, P, et al (2017) Cannabis Endophytes and Their Application in Breeding and Physiological Fitness. Cannibis sativa L.- Botany and Biotechnology.
  20. Qadri, M,et al  (2013)  Identification and bioactive potential of endophytic fungi isolated from selected plants of the Western Himalayas. Springer Plus. 2:8.
  21. Kusari, P, et al (2014) Quorum quenching is an antivirulence strategy employed by endophytic bacteria. Appl Microbiol Biotechnol. DOI 10.1007/s00253-014-5807-3.
  22. McKernan, K, et al (2016) Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers. F1000 Research. 4:1422.
  23. Wielgusz, K & L Irzykowska (2017) Occurrence of pathogenic and endophytic fungi and their influence on quality of medicinal plants applied in management of neurological diseases and mental disorders. Herba Pol. 63(4):57-69.
  24. Bouakline, A, et al (2016)  Fungal Contamination of Food in Hematology Units. J Clin Microbiol. doi: 10.12688/f1000research.7507.2

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