Psilocybe cubensis microbiomes

Abstract

Psilocybe cubensis has been decriminalized in Oregon, Colorado and many other jurisdictions. Inoculation of P.cubensis spores into sterilized grains often takes 2-4 weeks for mycelium growth to mature before flushing and fruiting can occur. This long growth window is vulnerable to the growth of background contaminating microorganisms that destroy yield and potentially expose consumers to mycotoxins synthesized by contaminating fungi.

Results

We used Oxford Nanopore sequencing to identify the genus and species of a contaminating fungi that was believed to be a Trichoderma contamination of a P.cubensis mycelium growth. PCR amplification of a 1.5kb fragment of the ITS region identified Penicillium oxalicum as the organism responsible for the green contamination in a spawn jar. This fungi is known colonize corn and known to synthesize the mycotoxin secalonic acid D or SAD .

After discovering sequenced reads with homology to Penicillium oxalicum ITS sequence, we searched through our Illumina derived whole genome shotgun datasets in Psilocydia.net and only found Penicillium oxalicum contaminants in the Penicillium dataset. This classification was found using Kraken. BLAST analysis of the longer reads also identifies Penicillium solitum with equal or higher scores. Penicillium solitum is not known to produce mycotoxins so further speciation of these sequences is required using more accurate 10.4.1 flow cells or using assembly and error correction. The samples in Psilocydia.net are mostly derived from spore samples suggesting Penicillium contamination is possible at both the substrate and spore introduction steps.

Methods

DNA was purified from a pipette tip scraped across the green mycelium using a 30 minute enzymatic digestion (12.5ul of Thaumatin-Like Protein) at 37C in 250ul of ddH20. 12.5ul of MGC lysis buffer was added to the sample and vortexed and spun. 250ul Supernatant was purified using 419ul of SenSATIVAx. After 2x 200ul 70% EtOH washes, the magnetic beads were briefly dried and eluted in 50ul of MGC elution buffer. PCR used 10ul of DNA, 10ul of ITS primers, 5ul ddH20, 25ul 2 X LongAmp master mix.

The PCR amplicon was barcoded using the ONT Rapid Barcoding kit (SQK-RBK110-96) and sequenced on a vR9.4.1 flongle.

Discussion

The several week incubation time required for P.cubensis cultivation is vulnerable to background microorganism contamination and qPCR tools may be helpful for identifying contaminated spore lots prior to inoculation. Screening spores or grains prior to use may reduce the unwanted expansion of contaminating microorganisms and lead to more productive growth of target organisms.