Enhanced production of select phytocannabinoids in medical Cannabis cultivars using microbial consortia

Abstract

The root microbiome of medical cannabis plants has been largely unexplored due to past legal restrictions in many countries. Microbes that live on and within the tissue of Cannabis sativa L. similar to other plants, provide advantages such as stimulating plant growth, helping it absorb minerals, providing protection against pathogen attacks, and influencing the production of secondary metabolites. To gain insight into the microbial communities of C. sativa cultivars with different tetrahydrocannabinol (THC) and cannabidiol (CBD) profiles, a greenhouse trial was carried out with and without inoculants added to the growth substrate. Illumina MiSeq metabarcoding was used to analyze the root and rhizosphere microbiomes of the five cultivars. Plant biomass production showed higher levels in three of five cultivars inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis and microbial suspension. The blossom dry weight of the cultivar THE was greater when inoculated with R. irregularis and microbial suspension than with no inoculation. Increasing plant biomass and blossom dry weight are two important parameters for producing cannabis for medical applications. In mature Cannabis, 12 phytocannabinoid compounds varied among cultivars and were affected by inoculants. Significant differences (p ≤ 0.01) in concentrations of cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabigerol (CBG), cannabidiol (CBD), and cannabigerolic acid (CBGA) were observed in all Cannabis cultivars when amended with F, K1, and K2 inoculants. We found microbes that were shared among cultivars. For example, Terrimicrobium sp., Actinoplanes sp., and Trichoderma reesei were shared by the cultivars ECC-EUS-THE, CCL-ECC, and EUS-THE, respectively. Actinoplanes sp. is a known species that produces phosphatase enzymes, while Trichoderma reesei is a fungal train that produces cellulase and contributes to organic matter mineralization. However, the role of Terrimicrobium sp. as an anaerobic bacterium remains unknown. This study demonstrated that the use of inoculants had an impact on the production of phytocannabinoids in five Cannabis cultivars. These inoculants could have useful applications for optimizing cannabis cultivation practices and increasing the production of phytocannabinoids.

Keywords: Cannabis; arbuscular mycorrhizal fungi; bioinoculant; microbiome; phytocannabinoids; rhizosphere.

Figure 1

Quantitative determination of phytocannabinoids. Cumulative percent recovery of a subset of cannabinoids under different treatments in five Cannabis cultivars: (A) cannabidivarin (CBDV), (B) cannabidivarinic acid (CBDVA), (C) cannabidiolic acid (CBDA), (D) cannabigerol (CBG), (E) cannabidiol (CBD), and (F) cannabigerolic acid (CBGA). The bar represents the concentration in milligrams per kilogram of raw material. Values with the same letters are not significantly different by a Tukey’s range test.

Figure 2

Quantitative determination of phytocannabinoids. Cumulative percent recovery of a subset of cannabinoids under different treatments in five Cannabis cultivars: (A) cannabinol (CBN), (B) cannabinolic acid (CBNA), (C) cannabichromene (CBC), (D) delta-9-tetrahydrocannabinol-A (△9-THCA-A), (E) cannabicyclolic acid (CBLA), and (F) cannabichromenic acid A (CBCA). The bar represents the concentration in milligrams per kilogram of raw material. Values with the same letters are not significantly different by a Tukey’s range test.

Figure 3

Microbial diversity and structure of the bacterial community. The analysis of alpha diversity measured by Shannon, Simpson, Chao, and Pielou of the bacterial community in roots (A) and rhizosphere soil (B). The analysis included four Cannabis cultivars: CBD Therapy (THE), Euforia (EEA), Critical (CCL), CBD Sweet and Sour Widow (ECC), and CBD US (EUS) under different treatments with Ferticann (F), microbial suspension (K1), of R. irregularis mixed with microbial suspension (K2), and control (K0).

Figure 4

Microbial diversity and structure of the fungal community. The analysis of alpha diversity measured by Shannon, Simpson, Chao, and Pielou of the fungal community in roots (A) and rhizosphere soil (B). The analysis included four Cannabis cultivars: CBD Therapy (THE), Euforia (EEA), Critical (CCL), CBD Sweet and Sour Widow (ECC), and CBD US (EUS) under different treatments with Ferticann (F), microbial suspension (K1), of R. irregularis mixed with microbial suspension (K2), and control (K0).

Figure 5

Taxonomic composition at the order level for bacterial communities. (A) Taxonomic representation at order level in the roots. (B) Relative abundance of top 10 bacterial orders in the root. (C) Taxonomic representation at order level in rhizosphere soil. (D) Relative abundance of top 10 bacterial orders in rhizosphere soil.

Figure 6

Taxonomic composition at the order level for fungal communities. (A) Taxonomic representation at order level in the roots; (B) relative abundance of top 10 fungal orders in the roots. (C) Taxonomic representation at order level in rhizosphere soil. (D) Relative abundance of top 10 fungal orders in rhizosphere soil.

Figure 7

Core microbiota pattern among Cannabis cultivars. The Venn diagram shows eco-microbiota shared among Cannabis cultivars under different treatments for bacterial community (A) and fungal community (B). Bacterial core microbiota in the root biotope (C) and soil biotope (D). Fungal core microbiota in the root biotope (E) and in the rhizosphere soil biotope (F).