In April of 2018, DigiPath Labs and Medicinal Genomics began a research program to better understand testing discordance with 3M TAC and TYM Petri-Films and PathoSEEK qPCR. Discordant samples were collected. Individual Colonies were isolated, subjected to qPCR and then DNA Sequenced to obtain 18S and 16S ITS sequence information.
Surprisingly the isolated colonies gave robust amplification with both 16S and 18S PathoSEEK primers. This implies these organisms are not being missed or under reported due to a failure of PCR primer design. DNA sequencing revealed the presence of Ralstonia which is an endofungal bacteria.
This is a plant and human pathogen that lives inside of various fungal cells.
Spraker et al. demonstrate that Ralstonia can infect many different fungi that have been documented to thrive on cannabis.
Ralstonia is known to induce Chlamydospore formation in Fungi. This is a multi-culular asexual spore clump. This clumping creates sampling bias when pipetting TSB or other broths out of a homogenized cannabis sample. This makes plate counts difficult to ascertain and can be a significant source of false negatives with qPCR. If the sample you pipetted didn’t contain a clump, you will get nothing detected. If it contains a clump you will detect large sums of DNA in the sample.
Ralstonia isn’t the only Endofungal toxigenic bacteria found on cannabis. Rhizopus has also been detected on cannabis and it can host Burkholderia bacteria that produce Rhizoxin. An excellent dissection of the impact of endofungal bacteria on food testing is published here.
Some states use TAC and TYM testing and consider Petri dishes to be the gold standard. There are three significant problems with this.
1)Filamentous Fungal cells clump making 10,000 CFU/g difficult to measure with certainty as single CFUs are difficult to discern from colonies seeded with 100 infective spores.
2)If fungal cells can harbor bacterial pathogens, then neither TAC or TYM plates will pick these up.
3)A single fungal cell can have 100s-1000s of pathogenic bacteria so a TYM sample with 9,900 CFU/g could easily have 1M pathogenic bacteria as endofungal symbiotes that fail to be detected on TAC plates.