Endophytic Fungi In Cannabis

Endophytic fungi are fungi that grow throughout the plant. Practically, they can most likely be encouraged by encouraging other fungal growth - adding some calory source (oat flakes, bananas) covered by soft carbon (hemp bedding) encourages fungal growth in the medium.

https://link.springer.com/article/10.1007/s13225-012-0216-3

- A total of 30 different fungal endophytes were isolated from all the plant tissues

- The Menhinick’s index revealed that the buds were immensely rich in fungal species, and Camargo’s index showed the highest tissue-specific fungal dominance for the twigs.

- The most dominant species was Penicillium copticola that could be isolated from the twigs, leaves, and apical and lateral buds.

- The fungal endophytes were challenged by two host phytopathogens, Botrytis cinerea and Trichothecium roseum, devising a dual culture antagonistic assay on five different media.

- We observed 11 distinct types of pathogen inhibition encompassing a variable degree of antagonism (%) on changing the media.

Wow! Do you have that in layman's terms?

Fungal Diversity
May 2013, Volume 60, Issue 1, pp 137–151 | Cite as

Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens

• Authors
• Authors and affiliations

• Parijat Kusari
• Souvik Kusari
• Michael Spiteller
• Oliver Kayser

• Parijat Kusari
  • 1
• Souvik Kusari
  • 2
• Michael Spiteller
  • 2
• Oliver Kayser
  • 1
  Email author

1. 1.Department of Biochemical and Chemical EngineeringChair of Technical Biochemistry, TU DortmundDortmundGermany
2. 2.Institute of Environmental Research (INFU) of the Faculty of ChemistryChair of Environmental Chemistry and Analytical Chemistry, TU DortmundDortmundGermany

ArticleFirst Online: 19 November 2012

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Abstract

The objective of the present work was isolation, phylogenetic characterization, and assessment of biocontrol potential of endophytic fungi harbored in various tissues (leaves, twigs, and apical and lateral buds) of the medicinal plant, Cannabis sativa L. A total of 30 different fungal endophytes were isolated from all the plant tissues which were authenticated by molecular identification based on rDNA ITS sequence analysis (ITS1, 5.8S and ITS2 regions). The Menhinick’s index revealed that the buds were immensely rich in fungal species, and Camargo’s index showed the highest tissue-specific fungal dominance for the twigs. The most dominant species was Penicillium copticola that could be isolated from the twigs, leaves, and apical and lateral buds. A detailed calculation of Fisher’s log series index, Shannon diversity index, Simpson’s index, Simpson’s diversity index, and Margalef’s richness revealed moderate overall biodiversity of C. sativa endophytes distributed among its tissues. The fungal endophytes were challenged by two host phytopathogens, Botrytis cinerea and Trichothecium roseum, devising a dual culture antagonistic assay on five different media. We observed 11 distinct types of pathogen inhibition encompassing a variable degree of antagonism (%) on changing the media. This revealed the potential chemodiversity of the isolated fungal endophytes not only as promising resources of biocontrol agents against the known and emerging phytopathogens of Cannabis plants, but also as sustainable resources of biologically active and defensive secondary metabolites.

Good to see endophyte reseach in Cannabis, though that paper has some flaws.

Firstly they only isolated a paltry 30 isolates, all from an indoor grow in the Nietherlands. For horizontally aquired endophytes, the environment in which they are grown will largely determine the endophyte community.

They did a lot of secondary analysis (diversity indexes) that have no relevance based on their sampling methods (culture based from an indoor grow). If they wanted those measurements and analysis to have any meaning they should have used next-gen sequencing and dilution-to-extinction culturing to determine a more acurate endophyte population in natural stands (or at least outdoor farms) and at different times.

It also seemed to put a lot of work into culturing each antagonism trial on 5 different media types, yet they only used a single race of each pathogen from a laboratory accession. They should use multiple races of each pathogen known to be virulent on Cannabis; specific pathogen-endophyte interactions are going to vary with pathogen race as well as media type. I would think consistent inhibition of a virulent pathogens is more important that consistent inhibition of a single pathogen across media. But their point that media affects inhibition is taken (I just don't see how to apply it, unless we know which media or combination of media best approximates activity in plants. And of course many endophyte activities only happen in planta).

For the antagonism test I am also confused that there are single numbers for each mediaXpathogen test with no statistics. So is there a single replicate per pathogen? If so I am surprised it was published. This is more a methods paper then (which it is 'old' at 2012). The diversity section is largely meaningless since it was from an indoor grow with unspecified parameters (Bedrocan BV Medicinal Cannabis). And single replicate antagonism tests with no stats are also hard to interpret as meaningful.

GrowingHigher said:

The diversity section is largely meaningless since it was from an indoor grow with unspecified parameters (Bedrocan BV Medicinal Cannabis). And single replicate antagonism tests with no stats are also hard to interpret as meaningful.

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This is western pharma science for you. Incapable of quantifying the minute variables of nature (or rather unable to attribute positive characteristics to natural products which they could not recreate in pill form), so they just wiggle there head make a funny noise, and claim they don't have time for that. Variables aren't scientific anymore. I blame the gender neutral agenda.

Penicillium is easy to smell, I just ran across some, picking up some glossy black beans from an old timer who grows in a region that stayed wet all fall. His bud wasn't penicillium based last year.

Cannabis research fell victim to pharmafuckation long ago, I nor does anyone have use for these discussions who fail to understand strains, plant evolution and the environmental factors that guide change. Every strain has different endophytes. And every strain is different because it evolved, via endophytes.. Which of those were passed down via seed? Which were passed down via guano? Etc etc. The question no one will ever answer, what endophytes are actually native to each strain, from seed.

Plants and animals literally evolved from microbes and western pharma science continues to downplay their importance, skew their message and pervert their functions, fuck em.

Endophytes of industrial hemp (Cannabis sativa L.) cultivars:
identification of culturable bacteria and fungi in leaves,
petioles, and seeds

Pathogens and Molds Affecting Production and Quality
of Cannabis sativa L. -This one also has an endophyte section, even though the title doesn't specify it.

I'mback said:

Wow! Do you have that in layman's terms?

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haven't read the paper yet, but some quick things that may make it easier to understand:

endophyte=micro organism living within a plant that is not harmfull(if it's harmfull, it's classified as pathogen). sometimes they just live there without either positive or negative effect on the plant, but they may have positive effects too. some pathogens can also exist as endophyte(for example botrytis has been shown to be able to be present sometimes within plant tissue without causing any desease symptoms), and sometimes they can then transition to a desease-causing state if the plant is stressed for example.

anytime you read phyto-something, that's something related to plants. so phytopathogen is a plant pathogen for example.

dual culture antagonistic assay: never heard of this exact term, but deducing the meaning, I guess this means they put 2 micro-organisms(1 isolated endophyte, 1 plant pathogen) on opposite sides of a petri dish then looked at if they inhibited eachother's growth as they grew towards eachother. so basically a kind of cockfighting with fungi.

- We observed 11 distinct types of pathogen inhibition encompassing a variable degree of antagonism (%) on changing the media.

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my take away from this would be that there is not just 1 mechanism, and the medium(the stuff they put in the petri dish to let the fungi grow on) matters for how big the effect is.
that means since the media they tested in petri dishes are obviously not exactly the same as the inside of the plant, that the exact numbers etc they found probably won't aply directly to the situation within the plant. but the general result that there is an effect at all matters. and that the total effect is a combination of different mechanisms each contributing some effect.

other thing, tissue-specific means for example that a specific endophyte only grows in leaves but not in the stem, or the other way around. tissue-specifity can be relevant, for example if a specific endophyte inhibits botrytis, but that endophyte only lives in the stem or roots, it's not going to help against budrot. or if a specific endophte can't live in seeds, than it must be acquired by a plant after germination(so it must be present in the growing environment), while if it can survive in a seed than it can be passed from mother to daughters.

also a thing to keep in mind reading this abstract: in this case they identified these endophytes by isolating them first and growing them in petri dishes on specific media(I'm guessing ether PDA or MEA, those are the 2 most common media to grow fungi on). then from the fungi on the plates they took some tissue to do a dna-test on to identify them(that's the ITS thing, ITS is a specific region of fungus dna that's often used for fungus identification. if I remember right it's part of the ribsomal dna, so conserved enough among species that you don't need to design a new primer for each species, bat variable enough to tell them apart).

but obviously, the natural situation within a leaf is not the same as a petri dish.

so only those fungi that are able to survive outside the leaf and grow on the media they choose to use will be isolated, so there may have been many more endophytes that they missed. simply because they never grew out of the piece of leaf onto the medium in the petri dish.

edit: reading the materials&methods now, seems like they didn't use MEA or PDA for isolation, but WA, 'water agar', which I assume is just water+agar without any nutrients. only after growing out of the leaf on that medium were they transferred to a medium with more nutrients. I guess they did that to select only for fungi that could survive from the nutrients present within the leaf-pieces(explains why it took them 4-6 weeks to grow anything out though, on standard strength MEA fungi grow way faster)

edit2: read the paper(or well, skimmed some parts and read mostly what interested me, especially the discussion and some parts materials). growinghigher makes some valid points. although the authors note this too in the discussion, this research is a start for mor research(like any research). specifically about diplicates of the pathogen x endophyte interactions, the materials&methods says this:

All control and test plates were run in duplicates.

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on the point of using only indoorgrown plants, I think this part of the discussion is relevant:

It is imperative that any plant-fungal interaction is always preceded by a physical encounter between a plant and a fungus, followed by several physical and chemical barriers that must be overcome to efficaciously establish a plant-endophyte association (Kusari et al. 2012b). It is mostly by chance encounters that particular fungi establish as endophytes for a particular ecological niche, or plant population, or plant tissue, either in a localized and/or systemic manner (Hyde and Soytong 2008). Thus, even a fungus that is pathogenic in one ecological niche can be endophytic to plant hosts in another ecosystem. It has been established for a plethora of fungi that pathogenic-endophytic lifestyles are interchangeable and are due to a number of environmental, chemical and/or molecular triggers (Eaton et al. 2011; Hyde and Soytong 2008; Schulz et al. 1999). Furthermore, groups of fungi containing large numbers of plant pathogenic species also contain large numbers of endophytic taxa. A vast majority of endophytes discovered so far are filamentous Ascomycota; this phylum comprises more than 3000 genera of mostly plant pathogens (Berbee 2001; Heckman et al. 2001; Mueller and Schmit 2007). Therefore, it is compelling that the diverse fungal isolates obtained from the tested C. sativa plants in the present work are selected towards coexistence with the hosts as endophytes. Interestingly for example, we found a number of Penicillium species exhibiting endophytic lifestyle in the associated C. sativa host plants (Table 1). Admittedly, only the ‘cultivable’ endophytic fungi could be isolated in this study and do not represent the non-culturable endophytic microorganisms of the sampled C. sativa plants. It should also be mentioned here that 5.8S-ITS analysis can sometimes underestimate the endophytic fungal ‘species diversity’ (Gazis et al. 2011), and additional parameters should be coupled to ITS rDNA sequence data before fungal isolates can be referred at the ‘species’ level. Further, it is highly desirable to compare the obtained ITS sequences with those from type species, when available, in order to authenticate the tentative species identification (Ko et al. 2011). Thus, the ITS-based species identification concept may not be in full agreement with the current classical concepts of Trichocomaceae. Nevertheless, this work can serve as the handle for further studies (both ITS-based and different other methods) on endophytes of Cannabis bioprospected from different other populations, different collection centers, and wild populations (when accessible) for a landscape or global scale diversity analysis.

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(bold parts I highlighted)

Endophytes get sucked up into the roots, get scrubbed of nutrients by a superoxide in the root hair. Then the plant ejaculates the endophytes back out to gather more food. With the Endophytes being re-released by exudates. The Endophytes are given a shopping list of the type of nutrients the plant needs and in what ratios.
Fungal Repositories provide all these super colonies for your plants to work with. See first post page 1 in forum aims for much more in depth details.