Soil and seed both influence bacterial diversity in the microbiome of the Cannabis sativa seedling endosphere.

Introduction: Phytobiomes have a significant impact on plant health. The microbiome of Cannabis sativa is particularly interesting both because of renewed interest in this crop and because it is commercially propagated in two different ways (i.e. clonally and by seed). Angiosperms obtain a founding population of seed-borne endophytes from their seed-bearing parent. This study examines the influence of both seed and soil-derived bacteria on the endospheres of cannabis seedlings of both hemp- and drug-types. Methods: A multi-factorial metagenomic study was conducted with three cannabis genotypes and two soil sources, which were tested both before and after autoclave sterilization. Seedlings were grown on soil, then rinsed and surface-sterilized, and 16S rDNA amplicons from seedling endophytes were sequenced, taxonomically classified, and used to estimate alpha- and beta-diversity in Qiime2. The statistical significance of differences in seedling microbiomes across treatments was tested, and PiCRUST2 was used to infer the functional relevance of these differences. Results: Soil was found to have a profound effect on the alpha-diversity, beta-diversity, relative abundance, and functional genes of endophytic bacteria in germinating cannabis seedlings. Additionally, there was a significant effect of cannabis genotype on beta diversity, especially when genotypes were grown in sterilized soil. Gammaproteobacteria and Bacilli were the two most abundant taxa and were found in all genotypes and soil types, including sterilized soil. Discussion: The results indicated that a component of cannabis seedling endosphere microbiomes is seed-derived and conserved across the environments tested. Functional prediction of seedling endophytes using piCRUST suggested a number of important functions of seed-borne endophytes in cannabis including nutrient and amino acid cycling, hormone regulation, and as precursors to antibiotics. This study suggested both seed and soil play a critical role in shaping the microbiome of germinating cannabis seedlings.

Hybrid Genome Assembly of Berkeleyomyces rouxiae, an Emerging Cannabis Fungal Pathogen Causing Black Root Rot in an Aeroponic Facility.

The resurged interest in cultivation of Cannabis sativa has presented an array of new challenges. Among them are the difficult-to-control pests and pathogens that infect cannabis plants. The limited methods for disease control available to cannabis growers necessitates early detection of plant pathogens, something that molecular techniques such as DNA sequencing has greatly improved. This study reports for the first time the fungal plant pathogen Berkeleyomyces rouxiae causing black root rot in high THC-containing cannabis. Aeroponically grown cannabis plants at a licenced production facility in Cranbrook BC, Canada, rapidly displayed root discoloration and rot symptoms despite testing negative for all commercially available pathogen tests. Developing sequencing-based disease diagnostics requires genomic information, so this study presents the first whole genome sequence of the multihost, widespread black root rot pathogen B. rouxiae. Hybrid genome assembly using Oxford Nanopore long-reads and Illumina short-reads yielded a genome size of 28.2 Mb represented over 404 contigs with an N50 of 267 kb. Genome annotation predicted 6,960 protein-coding genes with 59,477 functional annotations. The availability of this genome will assist in sequence-based diagnostic development, comparative genomics, and taxonomic resolution of this globally important plant pathogen.

Cannabis Seedlings Inherit Seed-Borne Bioactive and Anti-Fungal Endophytic Bacilli.

Throughout the hundreds of millions of years of co-evolution, plants and microorganisms have established intricate symbiotic and pathogenic relationships. Microbial communities associated with plants are in constant flux and can ultimately determine whether a plant will successfully reproduce or be destroyed by their environment. Inheritance of beneficial microorganisms is an adaptation plants can use to protect germinating seeds against biotic and abiotic stresses as seedlings develop. The interest in Cannabis as a modern crop requires research into effective biocontrol of common fungal pathogens, an area that has seen little research. This study examines the seed-borne endophytes present across 15 accessions of Cannabis grown to seed across Western Canada. Both hemp and marijuana seedlings inherited a closely related group of bioactive endophytic Bacilli. All Cannabis accessions possessed seed-inherited Paenibacillus mobilis with the capacity to solubilize mineral phosphate. Additionally, seeds were found to carry genera of fungal isolates known to be Cannabis pathogens and post-harvest molds: Alternaria, Penicillium, Cladosporium, Chaetomium, Aspergillus, Rhizopus, and Fusarium. Thirteen seed-borne endophytes showed antibiotic activity against Alternaria, Aspergillus, Penicillium, and Fusarium. This study suggests both fungal pathogens and bacterial endophytes that antagonize them are vectored across generations in Cannabis as they compete over this shared niche.

Mammalian Melatonin Agonist Pharmaceuticals Stimulate Rhomboid Proteins in Plants.

Melatonin is a human neurotransmitter and plant signalling metabolite that perceives and directs plant metabolism. The mechanisms of melatonin action in plants remain undefined. We hypothesized that roots have a melatonin-specific receptor and/or transporter that can respond to melatonin-mediating pharmaceuticals. To test this hypothesis Arabidopsis seedlings were grown with melatonin pharmaceutical receptor agonists: ramelteon and tasimelteon, and/or antagonists: luzindole and 4-P-PDOT. Ramelteon was found both to mimic and competitively inhibit melatonin metabolism in plants. Due to the higher selectivity of ramelteon for the MT1 receptor type in humans, a sequence homology search for MT1 in Arabidopsis identified the rhomboid-like protein 7 (RBL7). In physiological studies, Arabidopsis rbl7 mutants were less responsive to ramelteon and melatonin. Quantum dot visualizations of the effects of ramelteon on melatonin binding to root cell membranes revealed a potential mechanism. We propose that RBL7 is a melatonin-interacting protein that directs root architecture and growth in a mechanism that is responsive to environmental factors.

Ancient Relatives of Modern Maize From the Center of Maize Domestication and Diversification Host Endophytic Bacteria That Confer Tolerance to Nitrogen Starvation.

Plants can adapt to their surroundings by hosting beneficial bacteria that confer a selective advantage in stressful conditions. Endophytes are a class of beneficial bacteria that exist within the internal spaces of plants and many species can improve plant nitrogen use efficiency. Nitrogen is an essential plant macronutrient, and is often a limiting factor to plant growth, especially in cereal crops such as maize. Every year farmers apply over 100 million metric tonnes of synthetic nitrogen fertilizer to meet the growing demand for stable food crops. Breeding efforts in maize over the past several decades has focused heavily on yield in response to nitrogen inputs, and so may have selected against adaptations that allow plants to survive in nitrogen stressed conditions. Data suggests that our heavy dependence on synthetic nitrogen fertilizer is not sustainable in the long term, and so there is on-going research efforts to reduce and replace this currently essential part of modern agriculture. Bacteria that improve plant tolerance to nitrogen stressed environments would allow farmers to reduce the amount of fertilizer they apply. The selection of maize under high nitrogen conditions to create modern varieties may have caused the plant to lose these beneficial bacteria that allowed wild maize ancestors to thrive in low nitrogen soil. Here in this study, we examine the root and shoot microbiomes of the wild ancestor of all maize, Parviglumis, and an ancient Mexican landrace (Mixteco) from Oaxaca, the area of early maize diversification. Both of these maize genotypes have thrived for thousands of years with little to no nitrogen inputs and so we hypothesized that they host beneficial bacteria that allow them to thrive in nitrogen stressed conditions. We identified multiple root endophyte species from each ancient maize relative that increased the growth of annual ryegrass (model maize relative) under nitrogen starvation. Furthermore, research infers these strains were vertically transmitted to new generations of plants, potentially through seed, indicating selection pressure for Parviglumis and Mixteco to maintain them in their microbiome.

Draft Genome Sequence of Enterobacter cloacae 3D9 (Phylum Proteobacteria).

Presented here is the draft genome sequence of Enterobacter cloacae 3D9. This candidate seed endophyte was isolated from Zea nicaraguensis. The genome contains 4,653,375 bp in 28 contigs.

Draft Genome Sequence of Enterobacter cloacae 3F11 (Phylum Proteobacteria).

Presented here is the draft genome sequence of Enterobacter cloacae 3F11. This seed endophyte solubilizes rock phosphate and was isolated from Zea nicaraguensis. The genome contains 4,579,108 bp in 264 contigs.