Editor’s Note: This article contains some very technical descriptions of microbial communities and genetics. Be ye forewarned!

For many growers in the international Cannabis industry, the only memorable interaction they with the Cannabis microbiome occurs when they fail a Total Yeast and Mold test. The strict limit of 10,000 colony-forming units per gram (CFU/g) in Colorado and other US states has forced many growers to undertake stringent sterilization measures for their product to pass. This action limit approaches a no-tolerance policy on yeasts and molds for a plant grown in soil and in close quarters. Such a strict limit has also undoubtedly reduced the public’s exposure to potentially harmful fungi, forcing an otherwise unwilling industry to adapt to modern food safety regulations.

Unfortunately, counting fungi on finished products has distracted both growers and testing laboratories from studying the interactions of the microbial community on Cannabis throughout the plant’s life cycle and many processing steps. Many producers have begun to shift their focus from simply clearing regulatory hurdles to investigating how microbial communities can simultaneously contaminate products while also promoting desired plant traits.

Metagenomics is the study of DNA (or RNA) sequences from samples directly taken from the environment. These sequencing techniques rely on technologies developed during the race to sequence the human genome. During the project, all 3 billion base pairs of the human DNA code were not sequenced all at once, but split into millions of smaller DNA fragments which were iteratively sequenced. Then, computer software pieced them back together into a final human genome (Editor’s Note: This method is known as shotgun sequencing). Sequencing technology and computing power have advanced by orders of magnitude since then, and we can now apply this strategy to the microbiome; we can now repeatedly sequence DNA from environmental samples containing hundreds or thousands of microorganisms and then use software to construct the microbial metagenome.

This is how we make genetics exciting.

The small fragment of DNA that researchers look for during this type of analysis is a section of the 16S ribosomal RNA gene, which encodes an RNA molecule responsible for synthesizing proteins in all living cells. This gene is found across all life on the planet, making it the standard target for differentiating organisms and assigning taxonomies. In a next-generation sequencing run, sections of this gene and neighboring non-coding regions are targeted by sequencing primers which bind to highly conserved base pairs. These universal primers capture the sequences necessary to distinguish species.

Metagenomic analysis has been applied to countless different environments in order to tease out the varied roles that microbes play in plant development. Immune function, yield, nutrient capture, and chemotype, are a few of the many plant functions affected by microbial assemblages.

Modern Cannabis grows, however, have not had this analysis. No one in the Cannabis industry would argue that their plants can be grown in sterile soil, and there are many products on the market claiming to inoculate soil with only ‘beneficial’ microbes, but the literature is lacking. Ultimately, getting the right mix of microbes to maximize plant fitness and soil productivity can really only be established by first defining what a ‘healthy’ microbial community looks like, and then looking for deviations from that baseline.

In a system with potentially hundreds of different players, defining the optimal microbiome can be a real challenge. Consider how a microbial community in a Cannabis crop changes from seed through harvest, curing, and packaging. Then consider collecting samples for metagenomic analysis at every single stage, and you realize how significant of an undertaking this would be. Extending such a study to multiple crop cycles and between different grows while collecting data covering plant disease incidence, biomass, and chemotypes, as well as abiotic soil data would be even more daunting.

Despite the challenge involved, studies like this have been employed to address complex ecological questions - for example, how microbial community structure varies and impacts plant communities on a global scale. Fortunately, Cannabis monoculture is a much simpler system than the natural world, and the environment here can be controlled. The environment of the grow can be almost completely determined by the inputs like soil, air, water, and growth medias, and the outputs: the roots, stems, leaves and buds. Each of these sample types will have distinctly different microbial communities.

Many things can go wrong with a Cannabis crop, and the symptoms of different disorders can manifest similarly. When the cause of a particular problem is completely unknown, a metagenomic approach can be an invaluable first step in determining what the issue is, or whether it is even caused by a microbe.

At PhytaTech, we have found success applying metagenomics tools as a diagnostic, primarily for fungi. For instance, we have found high abundances of Fusarium species, one cause of root rot, infecting the roots and stems of phenotypically ‘sick’ plants. But, interestingly enough, phenotypically ‘healthy’ plants of the same strain also had a similar distribution of the same species of Fusarium, but the number of detections was much lower, suggesting a vertical transmission from the mother plant. Additionally, we have detected rare Ustilaginales species, or smut fungi, as the likely culprit of abnormal gall-like deformations on Cannabis leaves.

From our small projects, we are beginning to piece together what the industrial Cannabis fungal microbiome looks like. The first pattern that stands out is that the microbial community is drastically depending on what tissue is sampled. The rhizosphere is by far the most diverse region due to its contact with the soil. Here, endo- and ecto-mychorrhizal fungi are detected in the greatest abundances, assisting plants with nitrogen acquisition. Here we also find harmful Aspergillus sp. in the greatest abundance.

The intersection of the rhizome and stem shows some overlap. We begin to see increased abundances of plant endophytic fungi and intracellular yeasts. Here a complex interplay of poorly understood mutualistic mycoparasites attack phytoparasities, seeking to exploit the plant’s nutrient transport system.

All I can picture in my head

The ubiquitous powdery mildew (PM) genus, Golovinomyces, also begins to show up in the stem tissue, and composited leaf/bud tissue samples. PM can make up >99% of detected genomic sequences. Even in plant tissue from leaves with no visible PM, abundances are still 90-99% of detected sequences. The relationship between Cannabis and PM is quite intimate – the fungal hyphae completely vascularize the leaf tissue before the ‘powdery’ fungal candida even begin to form. So far, our research seems to indicate that if you see powdery mildew on a leaf, it is on every leaf in the greenhouse. Studying this interaction in more depth would be interesting, as some Cannabis strains appear resistant, but may still harbor the organism. Despite the dominance of Golovinomyces, we also find a lot of the fungi like Penicillium and Candida bear some responsibility for failed Total Yeast and Mold tests on the leaf/bud tissue. Finally, there is often evidence of human contact with the plants, often in the form of Trichsporon cutaneum, a fungus associated with human skin and responsible for dandruff.

Detailing the mutualistic and antagonistic interactions of the microorganisms is a major goal of our research, and one with potential applied benefits. Recently, Dyett et. Al. showed that occurrence of Pierce’s Disease in vineyards caused by the bacteria Xylella fastidiosa is negatively correlated with two other bacterial species, Pseudomonas fluorescens and Achromobacter xylosoxidans. When the abundance of these two other bacteria was high in the grapevine stem tissue, there was a significant increase in observations of the disease-free phenotype. While some biological control agents have harmful downstream effects, altering the microbiome to prevent plant disease presents a less toxic and more effective solution.

In our research so far, we have noted at least one potential mutualism between fungal Fusarium sp. and the bacteria Xanthomonas campestris, the cause of a plant disease called black rot, which is notable for its similarities to Fusarium wilt. These two species have both shown increased abundances in diseased tissues across different grows, and it would be interesting to examine how they interact to control the root rot phenotype. There is already at least one fungal species, Trichoderma viride, that is sold as a biocontrol agent for Fusarium wilt. During our survey of the diseased rhizosphere we detected this organism, but the abundance was very low, suggesting that it was insufficiently applied or had failed to thrive. Ongoing metagenomic monitoring could also be applied in such a case to gauge the efficacy of such treatments.

The type of long-term longitudinal study previously described would be a major investment, but the outcome could have major effects on the industry. By learning how to better alter the Cannabis microbiome we could develop better disease and pest management, increase yields and impact chemotypes, and understand the succession of microbial communities through harvesting and processing to pinpoint where ‘contaminating’ microbes enter into the system. Better understanding the interplay of powdery mildew and cannabis might change our perspective of it from a pest which needs to be eradicated to a biocontrol agent with a potential downside that can be managed. And finally, we want to understand how growers can harness these benefits while still being able to pass a yeast and mold test.

1. Waldrop, M P, Holloway, J M, Smith, D B, Goldhaber, M B, Drenovsky, R E, Scow, K M, Dick, R, Howard, D, Wylie, B, Grace JB. 2017. The interacting roles of climate, soils, and plant production on soil microbial communities at a continental scale. Ecology. 2017 Jul; 98(7):1957-1967. doi: 10.1002/ecy.1883.
2. Deyett, E., Roper, C.M., Ruegger, P., Yang, J., Borneman, J., Rolshausen, P.E. Microbial Landscape of the Grapevine Endosphere in the Context of Pierce’s Disease. Phytobiomes. 2017 Nov; (1) 3: 2471-2906. doi: 10.1094/PBIOMES-08-17-0033-R

Resources:

1. Want to learn more about subjects similar to those touched upon in this article? Check out our articles on subjects such as:
   1. Growers Network’s Pest Profile: Spider Mites
   2. Canna Cribs Episode 5: Honeydew Farms -- Honeydew, California
   3. Bacteria and Fungi – Friends of Cannabis
   4. Which is Better for Sleep - CBD or THC?
   5. From Flask to Field: How Microbes are Revolutionizing Big Agriculture
2. Want to get in touch with Evio Labs? They can be reached via the following methods:
   1. Website: http://eviolabs.com/
   2. Phone: 888-544-EVIO
   3. Email: info@eviolabs.com