Cocofibre can be the source of fungal contamination in Cannabis

Largest Cannabis microbiome study to date

Zamir Punja gave a startling presentation at CannMed 2025 where he performed over 100 Cannabis microbiome sequencing projects to track down the source of various Cannabis pathogens. To our surprise, he demonstrated his Cocofibre was contaminated with Aspergillus, Fusarium, Penicillium and Rhizophagus.

And these contaminations persisted all the way to flowering. You can see this in the below figure where Mother plants, Cuttings, vegetative plants, flowering plants and seeds are all sampled in Roots, Stems and Leaves and whole genome sequencing is performed. If you search Kannapedia.net for Zamir, you’ll see 111 whole genomes with microbiome analysis performed. A few samples he highlighted in Yellow and Green he expands on in the video above.

A few noteworthy observations:

Aspergillus was detected in Flowering plants, Mother plants, cuttings, vegetative plants in Roots, Stems, Leaves and into the seeds. It was also found in the Cocofibre.

Rhizophagus and Penicillium chryosogenum were found in every plant sample and may in fact be beneficial microbes.

Zamir goes on to dissect the plants and use SEM to identify where in the plant these microbes are hiding. Whole genome sequencing grinds up tissues and sequences all DNA but does not spatially pinpoint where in the plant each of these genomes come from. SEM provides another angle on this data.

The large tubes you see above are known as Vessel elements and transport nutrients up and down the stems. A lateral cross section of these is seen below. You can see 5-10um holes these known as Pits. That diameter is going to be important in regards to which microbes can cross this barrier.

Below you can see microbial spores in the Vessel elements with the Pits in the background.

Some spores are too large to cross the pits and are free to flow up and down the stem.

Other spores can cross the pits and can move but vertically and horizontally in the plant.

As you get closer to the flowers these Vessel elements are chock full of spores. Biopsy taken at the blue line with arrow.

Fungal mycelium is also found growing inside these vessel elements.

Meristem tissue culture can eliminate these ….But….

This leaves an empty niche which is quickly colonized by pathogenic Fusarium… unless you have the beneficial Trichoderma around.

If you expose Meristem cleaned tissue culture to Fusrium, over 90% of the microbiome reads are Fusarium. These plant ultimately died unless they were treated with Trichoderma.

When you add in Trichoderma, it dominates the microbiome (over 50%).

Zamir has shown strong evidence that the route of transmission of many of these endophytes in the flowers is through the substrate you grow in. This is an important observation as many grows who are lobbying to get rid of Aspergillus testing are making the assumption that the Aspergillus spores are ubiquitous in the air and their ubiquity implies futility in testing. We have challenged this assumption as the studies used to monitor Aspergillus spores in the air use cubic meters of air sampling and fail to speciate the 400+ Aspergillus species, most of which will not colonize cannabis.

As a comparison, cannabis flowers are testing 1 gram or 1ml sample. There are 1 million grams in a cubic meter so these studies are quantifying 1 million X larger volume and including all 400 Species when cannabis is only screened for 4 pathogenic species. What is most apparent from Zamir’s data is that these pathogens are coming from the CocoFibre the plant is sprouted from.

We decided to follow this up and went to our local grow store in Mass and explained the problem. They were kind enough to give us a few samples of their most popular Cocofibre and Rockwool brands. This was very helpful, otherwise I would have been walking out with three 25 pound bags when we only test a 1g sample:)

We first tested 3 different Coco products for Total Yeast and Mold using the Medicinal Genomics qPCR platform. This is such a broad ITS screen that we expect to find something but it may not be viable. Dead DNA could persist on Coco-fibre so we follow up the experiments with plating on DRBC and before and after enrichment qPCR.

These qPCR results are Pre-Enrichment as that is how TYM is run in the cannabis industry since the regulations are set so high (10K to 100K CFU/g). By comparing these results to TYM qPCR after growth in a Fungal broth with antibiotics, we can assess if any of these fungal spores are viable. The qPCR signal should decrease in CT if the spores grow.

After enrichment, everything is positive. This is not surprising but it also shows us some of these TYMs are alive as the CT drops after growth.

Since Aspergillus is a Presence/Absence test (<1 CFU/g), qPCR is only performed after enrichment. They are performed at 2 different dilutions with 1gram being wetted in 9ml and 19ml of broth. This test used the Medicinal Genomics Aspergillus 5 color kits.

The 19ml Coir Coco samples likely overloaded the test as all the other 19ml broth samples had very low CTs after 48hr enrichment and the 9ml samples were positive at CT of 19-20.

These samples were plated before enrichment

We will be sequencing some of these colonies to ID them.

The Coco samples were also qPCR tested for Fusarium (10 species) and Pythium (5 species) and were clean of these organisms.

This underscores the importance of testing your substrate for pathogens that trigger compliance testing failures. Zamir’s work demonstrated some of these organisms persist into flowering and even into seeds of the next generation.

Some beneficial microbes like Trichoderma did not survive curing. Penicillium did survive curing however.

We grew these colonies out a few more days to get more material for colony isolation and sequencing.

Our next experiments will look at if we can sterilize this Coco with various chemical treatments or infusion of Trichoderma.

In conclusion, testing your Substrate for the compliance pathogens may be the easiest methods you can introduce to track and eliminate contamination sources in your facility. The Coco Coir samples in this study were the cleanest and will likely result in higher pass rates for TYM or Aspergillus testing.

Never has it been easier to run these types of qPCR tests on site, in your grow.