Science, Irving K. Barber Faculty of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Dumigan, Christopher Robert
Doctor of Philosophy - PhD
University of British Columbia
Cannabis plants are hosts to a variety of fungal pathogens that have limited tools for control. Studying the microbiome of other crops shows that microorganisms can play an integral role in plant health by antagonizing pathogens, promoting plant vigor, and creating a functioning community or a “healthy” microbiome. Furthermore, plant-associated microorganisms can be passed down plant generations to germinating seedlings by their mother plant and vectored by seed. These can be both pathogenic and beneficial to their host plant. This thesis investigates the microbiome of Cannabis plants and the activities that both beneficial and pathogenic microorganisms have on this crop. Investigating 15 genotypes of Cannabis grown to seed across western Canada showed both hemp and marijuana inherit a closely related group of bioactive and anti-fungal endophytic bacilli. 13 of these isolates were able to antagonize known fungal pathogens of Cannabis that were also seed-borne, suggesting co-evolution and potential symbiosis between Cannabis and this class of bacteria. Mature Cannabis plants are infected by a growing number of fungal diseases. Documented here is the first report of Berkeleyomyces rouxiae infecting drug-type Cannabis causing black root-rot. This fungal pathogen was isolated from an aeroponics facility in Cranbrook BC and was not identified due to a lack of genomic resources on this fungal species. Here the first whole genome sequence is provided using hybrid genome sequencing and assembly. Finally, this thesis investigates the relative contributions of both seed and soil bacteria in shaping the endosphere communities in germinating Cannabis seedlings. A multifactorial metagenomic experiment was performed using three genotypes of Cannabis with two soil types, both of which were treated and untreated with autoclave sterilization. Cannabis genotype, soil type, and soil sterilization all play a role in structuring the endophyte communities of germinating Cannabis seedlings. This suggests inoculum from both seed and soil shape the microbiome of germinating Cannabis seedlings. There was however a shared element of the microbiome conserved across genotypes, soil types, and soil sterilization, indicating seed-borne endophytes are maintained across these conditions. Soil, however, had a dramatic and significant role in shaping the diversity and abundance of bacteria in this niche.
Exploring the Cannabis Microbiome to Develop Safe and Sustainable Bio-Fungicides by Christopher Robert Dumigan MSc, University of Guelph 2018 BSc, Western University 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) March 2024 © Christopher Dumigan, 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Exploring the Cannabis Microbiome to Develop Safe and Sustainable Bio-Fungicides submitted by Christopher Robert Dumigan in partial fulfillment of the requirements of the degree of Doctor of Philosophy. Dr. Michael K Deyholos, Biology, Irving K. Barber Faculty of Science Supervisor Dr. Miranda Hart, Biology, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Deanna Gibson, Biology, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Susan Murch, Chemistry, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Kirsten Wolthers, Chemistry, Irving K. Barber Faculty of Science University Examiner Dr. Zamir Punja, Department of Biological Sciences, Simon Fraser University External Examiner iii Abstract Cannabis plants are hosts to a variety of fungal pathogens that have limited tools for control. Studying the microbiome of other crops shows that microorganisms can play an integral role in plant health by antagonizing pathogens, promoting plant vigor, and creating a functioning community or a “healthy” microbiome. Furthermore, plant-associated microorganisms can be passed down plant generations to germinating seedlings by their mother plant and vectored by seed. These can be both pathogenic and beneficial to their host plant. This thesis investigates the microbiome of Cannabis plants and the activities that both beneficial and pathogenic microorganisms have on this crop. Investigating 15 genotypes of Cannabis grown to seed across western Canada showed both hemp and marijuana inherit a closely related group of bioactive and anti-fungal endophytic bacilli. 13 of these isolates were able to antagonize known fungal pathogens of Cannabis that were also seed-borne, suggesting co-evolution and potential symbiosis between Cannabis and this class of bacteria. Mature Cannabis plants are infected by a growing number of fungal diseases. Documented here is the first report of Berkeleyomyces rouxiae infecting drug-type Cannabis causing black root-rot. This fungal pathogen was isolated from an aeroponics facility in Cranbrook BC and was not identified due to a lack of genomic resources on this fungal species. Here the first whole genome sequence is provided using hybrid genome sequencing and assembly. Finally, this thesis investigates the relative contributions of both seed and soil bacteria in shaping the endosphere communities in germinating Cannabis seedlings. A multifactorial metagenomic experiment was performed using three genotypes of Cannabis with two soil types, both of which were treated and untreated with autoclave sterilization. Cannabis genotype, soil type, and soil sterilization all play a role in structuring the endophyte communities of germinating Cannabis seedlings. This suggests inoculum from both seed and soil shape the microbiome of germinating Cannabis seedlings. There was however a shared element of the microbiome conserved across genotypes, soil iv types, and soil sterilization, indicating seed-borne endophytes are maintained across these conditions. Soil, however, had a dramatic and significant role in shaping the diversity and abundance of bacteria in this niche. v Lay Summary Cannabis is a poorly researched crop due to the history of prohibition. The production of this crop of renewed interest is made more difficult by the limited tools to control disease in this plant, and the limited research on Cannabis microbiology and pathology. Plants host vast and complex communities of microorganisms that can be both beneficial and detrimental to themselves. Plants can also transfer microorganisms across plant generations by seed-to-seedling transmission. These microorganisms can be pathogenic, looking to take advantage of young and delicate plants. They, however, also can provide protection to germinating seedlings against harsh environments and plant disease during this delicate stage of a plant’s lifecycle. This thesis investigates for the first time the seed-inherited endophyte communities in Cannabis sativa. This thesis documents that Cannabis plants vector bioactive and antifungal bacteria to the next generation of Cannabis plants that live inside them. Cannabis seeds are known to transfer fungal pathogens that can opportunistically infect seedlings during this fragile stage when plants are establishing themselves. These seed-borne bacteria secrete compounds to antagonize these fungal pathogens, and so suggest that Cannabis plants may have co-evolved a relationship with these bacteria to help protect their progeny. Much about the Cannabis plant is still unknown. This thesis also documents a new pathogen of drug-type Cannabis, which was found causing black root rot in an aeroponic facility in Cranbrook BC. This fungal pathogen was isolated and tested for its ability to infect other clonally propagated Cannabis plants, which confirmed it was the causal agent of the observed black root rot. Additionally, this thesis provides the first whole genome sequence of this fungus, giving a resource for early detection and further study of this new and economically destructive pathogen. vi This thesis also sought to explore how the microbial communities living inside Cannabis seedlings are shaped and structured by microbes inherited from their mother plant, but also by microbes obtained from soil. This thesis shows that Cannabis seedlings obtain bacteria from both their mother plant and from their soil environment, and that certain types of bacteria are present regardless of what soil they are grown. This is true even if the soil has been sterilized to kill all of the microorganisms. Soil plays a large role in shaping the population numbers of certain bacteria, even across different strains of Cannabis. Interestingly, different strains of Cannabis have distinct “fingerprints” of what microbes they host, however, certain microbes are shared between them all. This thesis builds upon the existing body of research about the complex and poorly understood area of plant microbial ecology and introduces new findings into this area in an understudied crop. The seed microbes found and studied in this thesis have activities that are relevant to Cannabis production and show potential as beneficial inoculants to the Cannabis industry that does not have a well-developed tool kit to deal with plant disease. vii Preface Chapter 2 has been published as: Dumigan CR, Deyholos MK (2022) Cannabis Seedlings Inherit Seed-Borne Bioactive and Anti- Fungal Endophytic Bacilli. Plants 2022, 11(16), 2127 https://doi.org/10.3390/plants11162127 Chapter 3 has been published as: Dumigan CR, Maddock S, Bray-Stone D, Deyholos MK (2023) Hybrid Genome Assembly of Berkeleyomyces rouxiae, an Emerging Cannabis Fungal Pathogen Causing Black Root Rot in an Aeroponic Facility. Plant Disease 2023, 107(9), https://doi.org/10.1094/PDIS-11-22-2690-RE Savannah Maddock and Delaney Brae-stone co-authored the Plant Disease paper by helping with the in planta experiments and sending the infected plant material respectively. Chris Dumigan designed and performed experiments, analyzed data, and wrote all manuscripts in this thesis. Michael Deyholos edited all manuscripts and supervised the research. viii Table of Contents Abstract .................................................................................................................................... iii Lay Summary ............................................................................................................................ v Preface ..................................................................................................................................... vii Table of Contents ................................................................................................................... viii List of Tables ........................................................................................................................... xii List of Figures ........................................................................................................................ xiii Acknowledgements .............................................................................................................. xvii Chapter 1: Introduction ............................................................................................................ 1 1.1 Beneficial Plant Bacteria ............................................................................................. 1 1.2 Microbial Inheritance................................................................................................... 6 1.3 Cannabis as a Modern Crop ....................................................................................... 9 1.4 Cannabis Pathogens .................................................................................................11 1.5 Cannabis Microbiome ................................................................................................14 1.6 Current Cannabis Cultivation Practices......................................................................16 1.7 Cannabis Genomics ..................................................................................................18 1.8 Berkeleyomyces rouxiae ............................................................................................19 1.9 Conclusions ...............................................................................................................20 1.9 Objectives and Hypothesis ........................................................................................21 Chapter 2 ......................................................................................................................21 Chapter 3 ......................................................................................................................21 Chapter 4 ......................................................................................................................21 Chapter 2: Cannabis Seedlings Inherit Seed-Borne Bioactive and Anti-Fungal Endophytic Bacilli ....................................................................................................................................... 23 2.1. Abstract ...................................................................................................................23 2.2 Introduction ..............................................................................................................24 2.3 Methods ...................................................................................................................28 2.3.1 Source of Seed ..............................................................................................28 ix 2.3.2 Seed-borne Cannabis Seedling Endophyte Isolation ............................................29 2.3.3 Taxonomic Identification by 16S Gene Sequencing ..............................................29 2.3.4 Phosphate Solubilization Assay ............................................................................30 2.3.5 Pectinase Activity Pectinase Activity .....................................................................30 2.3.6 IAA Secretion........................................................................................................30 2.3.7 Seed-Borne Fungi from Cannabis Culturing .........................................................31 2.3.8 Anti-Fungal Activity of Cannabis Endophytes .......................................................31 2.4 Results ....................................................................................................................32 2.4.1 Cannabis Seedlings Inherit Seed-Borne Endophytic Bacilli ............................32 2.4.2 Endophyte Phosphate Solubilization ..............................................................37 2.4.3 Endophyte Indole-3-Acetic Acid Activity .........................................................38 2.4.4 Endophyte Pectinase Activity .........................................................................39 2.4.5 Seed-Borne Fungi ..........................................................................................39 2.4.6 Endophyte Anti-fungal Activity ........................................................................42 2.5 Discussion ...............................................................................................................44 Chapter 3: Hybrid Genome Assembly of Berkeleyomyces rouxiae, an Emerging Cannabis Fungal Pathogen Causing Black Root Rot in an Aeroponic Facility ................................... 51 3.1 Abstract ...................................................................................................................51 3.2 Introduction ..............................................................................................................52 3.3 Methods ...................................................................................................................53 3.3.1 Source of Seed ..............................................................................................53 3.3.2 Culturing microorganisms from root tissue .....................................................53 3.3.3 Taxonomic classification through colony PCR ................................................54 3.3.4 Long read sequencing ....................................................................................54 3.3.5 Short-read sequencing ...................................................................................55 3.3.6 Hybrid genome assembly ...............................................................................55 x 3.3.7 Genome annotation ........................................................................................56 3.3.8 Phylogenetic analysis .....................................................................................56 3.3.9 Confirmation of pathogenicity .........................................................................56 3.4 Results ....................................................................................................................58 3.4.1 Culturing microorganisms from root tissue ............................................................58 3.4.2 Hybrid genome sequencing, assembly, and annotation ........................................59 3.4.3 Carbohydrate metabolism gene analysis ..............................................................64 3.4.4 Fungal taxonomy ..................................................................................................65 3.4.5 Cannabis pathogenicity ........................................................................................66 3.5 Discussion ...............................................................................................................68 Chapter 4. Effect of Seed and Soil bacteria on Cannabis Seedling Microbiomes. ............. 73 4.1 Introduction ..............................................................................................................73 4.2 Methods ...................................................................................................................75 4.2.1 Source of Seeds ...................................................................................................75 4.2.2 Source of Soil and Characteristics ........................................................................76 4.2.3 Experiment Set-Up ...............................................................................................76 4.2.4 Surface Sterilization ..............................................................................................76 4.2.5 DNA Isolation........................................................................................................77 4.2.6 16S rRNA Amplicon Sequencing ..........................................................................77 4.2.7 Bioinformatics .......................................................................................................78 4.3 Results ...................................................................................................................79 4.3.1 Effect of Seed and Soil on Cannabis Seedling Microbiomes. ................................79 4.3.2 Effect of Seed and Soil on Cannabis Seedling Endosphere on Alpha Diversity ....82 4.3.3 Effect of Seed and Soil on Cannabis Seedling Endosphere on Beta Diversity ......84 4.3.4 Differential Abundance Testing with ANCOM........................................................86 xi 4.3.5 Metagenome Functional Prediction with PICRUSt2 ..............................................87 4.4 Discussion ...............................................................................................................90 4.4.1 Seed and Soil Bacteria are both Sources of Inoculum for Germinating Cannabis Seedling Endospheres ..................................................................................................90 4.4.2 Shifts in Bacterial Relative Abundance from Soil Sterilization ...............................93 4.4.3 Differential Enrichment of Functional Gene Pathways from Seed and Soil Inoculum ......................................................................................................................................95 4.5 Conclusion and Future Directions ............................................................................98 Chapter 5 Conclusion ........................................................................................................... 100 Bibliography .......................................................................................................................... 106 Appendices ........................................................................................................................... 125 Appendix A Chapter 2.1 Supplementary Materials .......................................................... 125 Appendix B: Chapter 2.2 Supplementary Materials ......................................................... 127 Appendix C: Chapter 2.3 Supplementary Materials ......................................................... 130 xii List of Tables Table 1.1. Definitions of Different Regions in the Plant Microbiome ........................................... 4 Table 2. 1. Culturable Seed-borne Endophytes from Seedling Endospheres. Green shaded cells indicate isolation from pool of 5 seedlings following germination in sterile environment and surface sterilization. ..................................................................................................................35 Table 2. 2. Summary of Anti-fungal Activity in Seed-borne Cannabis Endophytes. ...................43 Table 3. 1. Summary of annotation sources. .............................................................................62 Table 3. 2. Distribution of genes in COG functional annotation categories ................................64 Table 3. 3. Predicted virulence factors in the B. rouxiae PM34 genome……………………….65 Table 4. 1. PICRUST ANCOM Differential Abundance MetaCyc Pathways for Soil Sterilization. All Cannabis samples were pooled and compared between autoclave sterilized and natural untreated soil. Pathways shown were determined to be significantly different between treatments as determined by the W statistic. .............................................................................88 Table 4. 2. Selected Significantly Differentially Abundant MetaCyc Pathways for Genotypes in Soil Treatments. Each genotype was analyzed separately. Pathways shown were determined to be significantly different between treatments as determined by the W statistic. Selected pathways had highest percent abundance values of at least 400. The full table can be seen in Supplemental File 2. .................................................................................................................90 Supplemental Table S1. Genome assembly methods tested in this paper. ........................... 127 Supplemental Table S2. PERMANOVA Bray-Curtis Beta-diversity distance matrix significant results (bray_curtis_distance_matrix-significance.qzv). (A) Innotech Soil PERMANOVA, (B) Okanagan Soil PERMANOVA, (C) Genotype PERMANOVA……………………………………130 xiii List of Figures Figure 1.1. Visual Representation of Regions in the Plant Microbiome. Definitions for the annotated regions can be seen in Table 1. ................................................................................. 5 Figure 2. 1. (a) Geographic origin of parental Cannabis sativa accessions used for seed in this study. All experiments were performed at UBCO (Kelowna, BC, Canada). (b) Schematic for the location and isolation of seedling endophytes in this study. Both seed surface and seed endosphere contribute to seed-borne microbiome. Figure b was created with BioRender.com. 34 Figure 2. 2. Nearest-Neighbor Phylogenetic Tree of 16S Amplicons from Culturable Seed-Borne Bacterial Endophytes. Amplicons were aligned using the MUSCLE algorithm prior to nearest-neighbor phylogenetic tree construction using the Tamura–Nei model on MEGA software (Version 11.0.11). .......................................................................................................36 Figure 2. 3. Summary of in vitro Phenotyping Assays. Cannabis endophytes were assayed for the potential to solubilize mineral phosphate, secrete IAA, and hydrolyze pectin. Blue squares indicate phosphate solubilization. Red squares indicate IAA secretion. Yellow squares indicate pectinase activity. ......................................................................................................................38 Figure 2. 4. Seed-Borne Fungi from Cannabis. (a) Fungal isolates from seeds of 15 Cannabis accessions germinated on PDA for 5 days. (b) Germination rates and percent of seeds with visible mycelia growth (disease pressure) for each Cannabis accession. ..................................41 Figure 2. 5. Biocontrol Activity of 78 Seed-Borne Cannabis Endophytes Against Seed-Borne Fungi. Four fungal isolates belonging to plant pathogenic genera were selected (a) Alternaria, (b) Aspergillus, (c) Penicillium, (d) Fusarium. Diameters of zones of inhibition were measured at 7 days post-inoculation with endophytes. Color-coded letters correspond to means within the same group as calculated by ANOVA and Tukey HSD test………………………………………42 xiv Figure 3. 1. Black rot of aeroponic Cannabis root systems. A and B, High THC-containing ‘Nuken’ strain Cannabis plants rapidly displayed symptoms of root rot; images were taken 1 week apart. C, Light microscope image of root wash at 200× magnification. D, Detail of ‘Nuken’ strain infected root. E, Root pressed on carrot agar showing black fungal growth. ....................58 Figure 3. 2. Morphology of fungal isolates from aeroponically grown Cannabis. A and B, Fusarium spp. isolate PM30. C and D, Fusarium spp. isolate PM31. E and F, Trichoderma spp. isolate PM32. G and H, Fusarium spp. isolate PM33. I and J, Berkeleyomyces spp. isolate PM34. A, C, E, and G, Colonies on potato dextrose agar. I, Carrot agar. B, D, F, H, and J, Whole mount light microscopy (200× magnification) of fungi .....................................................59 Figure 3. 3. DNA plotter chart of Berkeleyomyces rouxiae PM34 annotation. The genome annotation file from the Funannotate pipeline was visualized using Artemis DNA plotter to display the distribution of coding sequences, tRNAs, assembly gaps, and GC% skew (Carver et al. 2012). ...................................................................................................................................63 Figure 3. 4. Maximum likelihood phylogenetic tree of MCM7 gene. MCM7 genes were aligned using the MUSCLE algorithm prior to construction of a maximum likelihood phylogenetic tree of MCM7 genes and the General Time Reversible model (GTR) on Mega software (version 11.0.11).....................................................................................................................................67 Figure 3. 5. Confirmation of pathogenicity for Berkeleyomyces rouxiae PM34. A and B, Crowns and roots of B. rouxiae PM34-infected Cannabis plants began to show visible dark mycelia growth matching colony morphology seen on carrot agar and C, root discoloration 5 days post inoculation. D, Light microscope of infected root wash at 200× magnification ...........................67 Figure 3. 6. Effect of PM34 inoculation on root growth. A, B, and C, Cannabis plants (control) and D, E, and F, 14 days post infection with Berkeleyomyces rouxiae PM34. ...........................68 xv Figure 4. 1. Experimental Design. (A) Three Cannabis genotypes where each grown in four soil treatments (n=4) for two independent trials. (B) Plants were grown for 7 days prior to (C) harvesting, surface sterilization, DNA isolation, and amplicon metagenomic sequencing. .........80 Figure 4. 2. Taxonomic Bar Plot for Cannabis Seedling Endospheres grown in Different Soil Conditions. Phylum level ASVs (amplicon sequence variants) from 7-day old Cannabis seedling endospheres germinated in different soil conditions. .................................................................81 Figure 4. 3. Heatmap of Taxonomies for Cannabis Seedling Endospheres. Phylum level ASVs (amplicon sequence variants) from 7-day old Cannabis seedling endospheres germinated in different soil conditions. .............................................................................................................82 Figure 4. 4. Alpha Diversity of Cannabis Genotypes Grown in Different Soil Conditions. Shannon diversity Kruskal-Wallis (pairwise). .............................................................................83 Figure 4. 5. Principle Coordinate Analysis of Bray-Curtis Distances in Experiment 2. A) All treatments individually coloured on PCOA. B) Soil Treatment coloured to show clustering of samples withing soil groups. .....................................................................................................85 Supplemental Figure S1. 16S Phylogenetic Tree of Culturable Seed-borne Cannabis Seedling Endophytes Across Hemp X59 Generations. Strains of Bacillus megaterium and Paenibacillus mobilis were cultured from two successive generations of Hemp X59 grown to seed. Generation-1 was grown outdoors in soil in Vegreville, AB, Generation-2 was grown indoors in Sunshine Mix in Kelowna, BC. P. mobilis isolates are outlined in teal. B. megaterium isolates are outlined in orange. Amplicons were aligned using MUSCLE algorithm prior to nearest-neighbor phylogenetic tree was construction using Tamura-Nei model on MEGA software (Version 11.0.11) .................................................................................................................... 125 Supplemental Figure S2. Altair Seed-Associated Fungi. Alternaria and Penicillium growth on germinating Altair hemp seeds 4 days after planting on PDA .................................................. 126 Supplemental Figure S3. Genome assembly method scorings. .............................................. 128 xvi Supplemental Figure S4. Genome assembly k-mer spectrum plot from Merqury with short reads. ...................................................................................................................................... 128 Supplemental Figure S5. Trial 2 of Cannabis plant inoculated with Berkeleyomyces rouxiae PM34……………………………………………………………………………………………………129 xvii Acknowledgements First, I would like to thank my Supervisor Mike Deyholos for bringing me to his lab, especially during his tenure as Department Head when his time is limited. I would like to thank the course coordinators Brendan D’Souza and Soheil Mahmoud who put full trust in me to run the lab sections of their courses which I enjoyed greatly. My lab colleague Savannah Maddock who taught me many things about the Cannabis industry. Dan Durall for use of his sequencer and facility, and graduate students in the BRAES lab for their support, encouragement, soil expertise, and for use of their facility. Finally, I would like to thank Jan Slaski from Innotech Alberta for the hemp seeds used in this dissertation. 1 Chapter 1: Introduction 1.1 Beneficial Plant Bacteria Discovering the importance of the human microbiome in regards to health, disease, and nutrition was a major milestone for medicine in the 21st century (The NIH HMP Working Group, 2009; Ursell et al., 2012). The combination of low-cost sequencing technologies and the development of computer science are what made these paradigm shifting discoveries possible. The field of agriculture has mirrored medical research, using the same technologies to discover the importance of the plant microbiome to crop health. The field of plant microbiome research has exploded within the past-decade as governments, growers, and society as a whole search for sustainable ways to fertilize our crops, fight plant disease, and minimize environmental degradation (Waltz, 2017). Plants can adapt to their environment by hosting beneficial bacteria and fungi that confer selective advantages to their hosts in stressed conditions (Johnston-Monje and Raizada, 2011b). These microorganisms can contribute to overall plant health by assisting in nutrient acquisition, secreting plant growth promoting phytohormones, assisting in host immune responses, or antagonizing pathogens to prevent disease (Hu et al., 2003; Johnston-Monje and Raizada, 2011b; Mousa et al., 2016; Shehata et al., 2017). Microorganisms that exist within plants are known as “endophytes”, and current research has elucidated how important these microorganisms are with respect to plant health (Berg et al., 2017; Khalaf and Raizada, 2018). The internal spaces of plants are unsurprisingly ideal habitats for many microorganisms. With a rich supply of carbon and other nutrients, as well as protection from harsh environments, plants host vast communities of microorganisms that can have a beneficial or pathogenic effect on their host. An example of such a relationship is described in detail in Shehata et al., (2017). Zea 2 nicraguensis is a variety of maize found thriving in a volcanic swap of Nicaragua. The soil of this swamp is rich in insoluble rock phosphate that is not bioavailable to plants in this form. Interestingly, the seeds of this maize variety were found to host an endophytic microbe that localizes to root hairs and secretes organic acids to solubilize rock phosphate and make it bioavailable to its plant host, providing a selective advantage in this environment that lacks readily available phosphate. Endophytes can assist in disease suppression in their host by inducing plant resistance genes, competing for the same niche, or by secreting antimicrobial compounds (Haas and Défago, 2005; Mousa and Raizada, 2015; Waller et al., 2005). There are examples of pathogen-tolerant plants that have been shown to possess beneficial endophytes in their microbiome that contribute to this phenotype. Finger millet is a grain crop that is cultivated in Africa and South Asia (Goron and Raizada, 2015) and has been reported by subsistence farmers to have resistance to many fungal pathogens including fusarium (Sundaramari, 2015). Finger millet is known to produce high concentrations of an anti-fungal polyphenol (Chandrashekar and Satyanarayana, 2006; Siwela et al., 2010), however, this resistance is also attributed in part to beneficial endophytes in the finger millet microbiome (Mousa et al., 2016). An endophytic Enterobacter species (M6) isolated from finger millet can create a physiochemical barrier to protect roots from invading fusarium and secrete anti-fungal compounds to antagonize this pathogen, conferring tolerance to its plant host (Mousa et al., 2016). Pathogenic microorganisms are responsible for around $220 billion in crop loss every year (Chakraborty and Newton, 2011), and in general, growers are often heavily reliant on the use of chemicals to control plant pathogens. The widespread use of pesticides is often costly to growers, the environment, and human health. The use of beneficial microorganisms to fight plant pathogens has proven to be an effective, safe, and more-sustainable approach to combat 3 this problem (Berg et al., 2017; Khalaf and Raizada, 2018; Mousa et al., 2016; Shehata and Raizada, 2017). A variety of commercial products exist that use beneficial microorganisms to combat plant pathogens. Serenade® was developed by AgriQuest.Inc (now owned by Bayer) and contains a species of Bacillus subtills that produces a class of anti-fungal compounds known as lipopeptides. Serenade® is effective at controlling a variety of pathogens including: fungi such as botrytis (Percival et al., 2017), Fusarium (Jimenez-Quiros et al., 2022) powdery mildew (Matzen et al., 2019), yellow rust (Reiss and Jørgensen, 2017) and early blight (Abbasi and Weselowski, 2014); and protists such as clubroot (Lahlali et al., 2011), and the oomycetes Pythium and Rhizoctonia (Jo et al., 2014). In addition to bacterial symbionts, there is a diversity of fungi that assist in plant health. Trichoderma is a genus of fungus in the Ascomycota phylum that has been widely applied in agriculture as a beneficial plant microorganism that antagonizes pathogenic fungi and other pests (reviewed in Verma et al., 2007). Species of Trichoderma have been commercialized for their mycoparasitic activity, secretion of antibiotic metabolites, and ability to outcompete pathogenic fungi in their plant host (Verma et al., 2007). Trichoderma is a soil-borne fungus that symbiotically interacts with plant rhizospheres and in addition to biocontrol activity, also acts as a plant-growth promoter, suggesting additional beneficial phenotypes and modes of action (Natsiopoulos et al., 2022; Ousley et al., 1994; Zhang et al., 2016) In plant microbiome research, the phytosphere is used to describe the whole plant as an ecosystem. Researchers compartmentalize different tissues and associated areas of plants as they show distinct microbial communities from one another. A summary of the various regions of the plant microbiome can be seen in Table 1.1 and Figure 1.1. 4 Table 1.1. Definitions of Different Regions in the Plant Microbiome Region of Plant Microbiome Definition Phytosphere All regions of a plant. Phyllosphere Above ground plant tissue surfaces. Anthosphere Flowers and reproductive tissues Spermosphere Region of soil surrounding germinating seed. Root Microbiome Below ground plant tissue. Rhizsosphere Narrow region of soil on the surface of roots. Endosphere The internal tissues of plants. 5 Figure 1.1. Visual Representation of Regions in the Plant Microbiome. Definitions for the annotated regions can be seen in Table 1. 6 1.2 Microbial Inheritance In addition to the microorganisms obtained from the environment, plants inherit a founding microbiome of microorganisms via seed to seedling vertical transmission (Ferreira et al., 2008). Developments in our understanding of the seed microbiome and its role in plant ecology have been slow in comparison to that of the interaction of soil, the rhizosphere, and the plant microbiome as a whole (Nelson, 2018). Seed-vectored microorganisms are of particular interest as they act as the starting point for the assemblage of new microbial communities for germinated seedlings during this critical stage in the plant life-cycle (Shahzad et al., 2018). Research suggests that the microbial communities associated with seeds planted in soil actually originate primarily from the mother plant rather than from their soil environment (van Overbeek et al., 2011). This seed microbiome is an important source of inoculum for developing seedlings, and plants appear to place strong selective forces on seed-borne microorganisms during germination by shaping what species proliferate and colonize (Barret et al., 2015). Microorganisms that have effectively colonized plant tissues have the potential to exert a “barrier effect” against other microorganisms, preventing their colonization (Bacilio-Jiménez et al., 2001; Massalha et al., 2017). This potentially explains why seed-vectored microorganisms, especially endophytes, are not simply overtaken by the ocean of soil-borne microorganisms and can persist across plant generations (Hardoim et al., 2012). Soil is a well-established driver of the composition of plant associated microbial communities. However, understanding of a role for seed-vectored microorganisms in the plant life cycle has grown substantially throughout the past decade (Nelson, 2018). While research has suggested that the bacterial communities of rhizospheres are primarily influenced by the surrounding soil (Normander and Prosser, 2000), there is also evidence that microbial inheritance plays an important role in forming early rhizosphere communities. One such study found that while soil type was the main driver of bacterial group structure in the rhizosphere, the 7 most common bacterial cells (Proteobacteria and Bacteroidetes) in juvenile maize rhizospheres across three soil types appeared to be seed derived. (Johnston-Monje et al., 2016). This phenomenon of microbial inheritance appears to play a major role in determining microbial communities in plant endospheres. Bacterial endophytes of maize have been suggested to be primarily seed inherited rather than originating from their soil environment (Johnston-Monje et al., 2014). Given that this phenomenon of microbial inheritance appears to play a major role determining microbial communities in plant endospheres, it is interesting to speculate about alternative ways that selective advantages are inherited from parental plants. There is likely selection pressure for microorganisms to be maintained and transmitted to offspring plants to protect them against biotic and abiotic stresses. Researchers have suggested that certain microorganisms can protect developing seedlings from pathogens in their new environment by altering seedling redox status (Pitzschke, 2016). Furthermore, a diversity of seed endophytes isolated from cucurbit species showed antibiotic activity against common greenhouse pathogens such as powdery mildew and Pythium (Khalaf and Raizada, 2016, 2018). In addition to protection from pathogens, research has suggested that endophytes can be transferred across plant generations to confer tolerance to abiotic stresses such as environmental metal contamination (Sánchez-López et al., 2018; Truyens et al., 2016). Moreover, a core microbiome of seed-vectored endophytes that provide stress tolerance can emerge across several plant generations (Sánchez-López et al., 2018). Researchers have demonstrated that seed-vectored microbial communities are partitioned to different tissues in germinated seedlings (Abdelfattah et al., 2021). Acorn seeds were surface sterilized and germinated in gnotobiotic conditions removing all environmental microbes. Despite this, the researchers found a diversity of plant associated bacteria, including endophytes, in the newly growing seedlings. This same study also showed that the phyllosphere 8 and rhizosphere of oak inherited different microorganisms, and that the relative abundance of genera varied in different compartments of the acorn. Because every plant species examined to date shows the presence of endophytes, it is not unreasonable to assume that microbial inheritance of seed-vectored endophytes is a widespread process and an important adaptation for seedling vigor (Shahzad et al., 2018; Smith et al., 2008). An interesting example of how seed-vectored endophytes can alter the environment of their host plant describes a species of invasive grass that transmits seed-vectored endophytic pseudomonads to new seedlings (White et al., 2018). These bacterial endophytes were found to promote seedling vigor and disease resistance and were released into the rhizosphere to create an environment favorable for their host grass while killing off competing plant species. Microbial inheritance via seed to seedling vertical transmission occurs through microbial colonization of external and internal seed tissue in developing flowers (Dutta et al., 2014; Mitter et al., 2017). In fact, microscopic analysis shows that the inner surface of flower petals are densely populated by a diversity of bacteria, including species that show biocontrol activity (Fürnkranz et al., 2012). These species are in close contact and on the surface of the seed coat and have the potential to be passed down to developing seedlings as they germinate in their new environment (Nelson, 2018). In addition to colonizing the surface of seeds via the seed coat, microorganisms can colonize the seed or fruit endosphere via xylem transport to plant reproductive organs prior to seed-transmission (Compant et al., 2011)(reviewed in Aleklett and Hart, 2013). In addition to the vertical transmission of plant microbiomes, research has also demonstrated a process known as “microbiota transfer”, whereby microbial communities are taken from one plant genotype and given to another. This is analogous to horizontal transmission. Rhizosphere transfer from a resistant variety of tomato has been shown to alter community structure of susceptible varieties and confer tolerance to bacterial wilt (Choi et al., 9 2020; Kwak et al., 2018). Altering microbial community structure could be a powerful method of increasing pest tolerance in agriculturally relevant crops while not sacrificing other beneficial genetic traits such as yield or flavour profiles in bred resistant varieties. This process would be of particular interest to growers of Cannabis where specific strains are desired for terpene and cannabinoid profiles but may not have genetic resistance to pathogens. 1.3 Cannabis as a Modern Crop Cannabis sativa is an annual dioecious flowering plant that originates from the Himalayan foothills of central Asia (Wills, 1998) and has been cultivated by ancient peoples for thousands of years (Andre et al., 2016). Historically, Cannabis was primarily cultivated for its seed and its fibers, which were used as textiles throughout Europe and Asia (Andre et al., 2016; M. Polne-Fuller, 2020). Phytochemical and genetic investigation into a 2700 year old well-preserved cache of Cannabis demonstrated that this cultivar also contained a variety of cannabinoids including tetrahydrocannabinol (THC), as well as enzymes required for its synthesis, suggesting ancient people may have cultivated Cannabis as a medicinal or psychoactive agent (Russo et al., 2008). There is an arbitrary distinction between “hemp” and “marijuana”, both of which are Cannabis sativa, where a THC content below 0.3% classifies the cultivar as “hemp” and above 0.3% a cultivar is designated as “marijuana (Dolgin, 2019; Schluttenhofer and Yuan, 2017; Small and Cronquist, 1976). In Canada, Cannabis was added to the restricted list under the Narcotics Drug Act Amendment Bill in 1923 (House of Commons of Canada, 1923), and became legal as recently as 2018. The cultivation of industrial hemp in Canada was prohibited from 1938 to 1998. In the United States, the 2018 Farm Bill legalized production of industrial hemp nationwide (US Food and Drug Administration, 2019), and to date, eleven States have legalized the cultivation and use of recreational and/or medicinal Cannabis. The legal landscape 10 of this crop has changed rapidly in recent years and will almost certainly continue to change, and the development of both “hemp” and “marijuana” as modern crops poses many challenges and opportunities. In this proposal, the name “Cannabis” will be used to refer to any plant from this genus, regardless of its intended use. Cannabis has generated renewed research interest because of the large variety of secondary metabolites it produces. Over 150 unique terpene molecules and around 100 cannabinoids have been found in the resin of various Cannabis cultivars (Booth and Bohlmann, 2019; Hanuš et al., 2016). Recently, the connection between Cannabis genotype and chemotype has been called into question (Lange and Zager, 2022). Research into pharmacological properties of various cannabinoids has been ongoing since the 1940s (Pertwee, 2006) and the importance of endogenous cannabinoid receptors in regard to disease has highlighted the potential therapeutic value of these compounds. Cannabinoids such as cannabidiol (CBD), cannabigerol (CBG), Cannabigerivarin (CBGV), and D9-tetrahydrocannabinol (THC) have been shown to have analgesic, anti-inflammatory, and anti-cancer effects by interacting with the mammalian endocannabinoid system (De Petrocellis et al., 2011; Di Marzo and Piscitelli, 2015). The production of new industrial hemp varieties in North America is rapidly accelerating. Hemp oilseed along with the medicinal non-intoxicating cannabinoid, CBD, are among the most promising focuses for production as a North American field crop (Cherney and Small, 2016). Improvements in hemp cultivars have been made, such as the introduction of traits for shattering resistance which greatly increase seed yield. The non-shattering variety “Finola” is currently the most popular cultivar in Canada (reviewed in Schluttenhofer and Yuan, 2017). The tendency for mature hemp seeds to shatter off their bracts means growers harvest around 70% seed maturity and this is an area where large improvements in breeding can be made, as Finola seeds are roughly half the size as other shattering varieties (Schluttenhofer and Yuan, 2017). 11 The success of hemp as a modern crop is dependent on reasonable regulation, realistic expectations, and the development of productive cultivars and production technology (Cherney and Small, 2016). 1.4 Cannabis Pathogens The emerging global Cannabis market is predicted to reach 30 billion USD by 2025 (New Fronteir Data, 2019) and the demands of growing a new crop on this scale will present an array of problems that require creative and sustainable solutions. Given the nature of the market, little research has been done on Cannabis cultivation relative to other crops, and so there are many opportunities to answer important questions that will benefit Canadian growers. Speaking to commercial growers in both California and Canada, both areas where recreational and medicinal Cannabis has been recently legalized, the control of fungal pathogens such as powdery mildew, botrytis, fusarium, and Pythium are among the biggest issues their crop is faced with. Furthermore, synthetic fungicides are forbidden for both recreational and medicinal Cannabis in Canada and California (https://bit.ly/2BMfIYt, https://bit.ly/2OkvT4T). Consumer demand for safe, organically grown Cannabis products means that growers need effective bio-control agents for fungal pathogens, an area that has seen little to no research in Cannabis. A study by Punja et al., (2019) describes the different types of fungal pathogens and molds affecting marijuana plants grown hydroponically, in greenhouses, and in field conditions. The researchers isolated multiple species of Fusarium from root tissue, as well as the oomycete pathogens Pythium from roots and Rhizopus from coco fiber substrates where plants were rooted. Pathogens of these genera have been previously associated with root and crown rot in field grown Cannabis causing wilting at a farm in Northern California (Punja et al., 2018). Recently, Recently, multiple Pythium species have been found to cause root and crown rot in Cannabis grown in a commercial greenhouse (Punja et al., 2022). Additionally, Fusarium 12 proliferatum, and F. oxysporum have been shown to cause crown/stem rot and pith necrosis in eight Cannabis licensed production facilities across Canada and one in California (Punja, 2020a). These pathogens are spread by air and water-borne spores and can be devastating to plant growth, causing wilting, stunted growth, yellowing of leaves, and even plant death. Because these typically root-associated fungal pathogens can spread through hydroponic solution and even infect stems tissue, re-circulation of nutrient solution without treatment can cause infections of other plants, including propagated plant cuttings that are being rooted (Punja et al., 2019). Pathogens causing root rot such as Fusarium can be devastating to licensed production facilities with limited means to control these pathogens. The major fungal pathogen associated with foliar Cannabis tissue is Golovinomyces cichoracearum causing powdery mildew (Punja et al., 2019). Although this pathogen can also affect stems and flowers, it most commonly presents itself as white powdery mycelia growth on leaves (Punja, 2018). This pathogen typically spreads via air-borne spores that affect growth of vegetative tissues and can spread via clonal propagation and can also infect flowers causing a form of “bud-rot”, making the end product unusable (Punja, 2018). Golovinomyces species are obligate biotroph pathogens and affect a wide variety of fruits and vegetables such as cucumbers, melons, and tomatoes (Braun et al., 2019; Lebeda et al., 2016). In addition to airborne disease dispersal, Cannabis plants are known to be affected by seed-borne fungal disease such as Alternaria and Stemphylium which can cause stem-canker and leaf spot disease in mature plants (Roberts and Punja, 2022). Botrytis cinerea is another major fungal pathogen that can infect flowering tissue and cause “bud-rot”, often appearing as a brown rust-like growth (Punja, 2018). The pathogen-host relationship of Botrytis was recently reviewed, and in greenhouse cannabis, this pathogen can quickly destroy inflorescence when conditions are optimal (>70% relative humidity and moderate temperatures 17ºC-24ºC) (Mahmoud et al., 2023). In addition to affecting actively 13 growing flowers, Botrytis can manifest as a post-harvest pathogen on dried flowers, decaying the tissue and making the product unusable (Punja, 2020b). Moreover, many fungal plant pathogens have the potential to produce harmful mycotoxins that make consumption unsafe (Perrone and Susca, 2017). Testing of Cannabis flowers for post-harvest yeast and molds has become standard practice. Interestingly, levels of yeast and molds on both fresh and dried Cannabis flowers are influenced by plant genotype, their environment, as well as post-harvest protocols (Punja et al., 2023). As previously mentioned, conventional fungicides are prohibited for use on Cannabis intended for the recreational and medicinal markets. This means that growers must detect pathogens quickly and accurately or face total crop destruction. Additionally, limited research on Cannabis, Cannabis cultivation, and Cannabis pathogens means growers of this crop have limited knowledge, tools, and systems to create effective operations and businesses. Several Cannabis cultivation businesses in Canada have had entire crops destroyed due to widespread pathogens, and no ability to control their spread. The use of microorganisms to control plant pathogens in Cannabis is already a widespread practice in the industry as part of a holistic approach to pest management, however, there has been limited research on the efficacy of various products on Cannabis and the ability of these microorganisms to colonize Cannabis plants. A previous study evaluated various methods to control powdery mildew in Cannabis (Scott and Punja, 2020). The researchers found that a variety of control measures that could be approved within current Canadian Cannabis regulations were effective at controlling pathogen spread, including beneficial Bacillus species (Rhapsody and Stargus). These however were outperformed by other methods such as hydrogen peroxide (ZeroTol), and so improvements could likely be made as these products we not developed specifically for Cannabis. Additionally, species of Trichoderma and Bacillus have been shown to have biocontrol activity against Fusarium and Pythium (Scott and Punja, 2023). 14 To date there has been limited bioprospecting from both hemp and drug-type Cannabis plants to find organisms well adapted to this host crop and its pathogens. The regulation surrounding pathogen control, the demand from the consumer for safe product, and the limited available tools for controlling Cannabis pathogens outlines a clear opportunity for research into effective management using biological control and beneficial microorganisms to create a more resilient crop. 1.5 Cannabis Microbiome Across the field of agriculture, restrictions on the genetic modification of crops has pushed companies to find new ways to improve traits, and an increasing concern about the sustainability of modern agricultural practices has caused governments to tighten regulation of chemical use. The use of beneficial microorganisms to stimulate plant growth, assist in nutrient acquisition, and fight plant disease has been a growing trend in the agriculture-biotech space and continues to take an increasing portion of the 200 billion dollar global fertilizer/agriculture chemicals market (Waltz, 2017). The microbiome of both industrial and drug type Cannabis has seen comparatively little research for crops of similar market size. A few recent articles have begun to investigate the microbiome of Cannabis, but several important questions remain unanswered. A recent study found a diversity of epiphytic and endophytic fungi associated with Cannabis stem and flower tissue (Punja and Scott, 2023). Additionally, an evaluation of the microbiome of field grown hemp found a variety of microorganisms associated with pathogen resistance, and significant variation in the bacterial and fungal communities across plant tissues. (Barnett et al., 2020). Comeau et al., (2020) looked at how the microbiome of drug-type Cannabis cultivars change in various tissues throughout the growth cycle in a commercial production setting. Unsurprisingly, there was a large portion of Proteobacteria and Ascomycota, as is often the case with plant 15 associated microorganisms. The researchers also claim a cultivar dependent effect on the below-ground populations of microorganisms. These results align with an earlier study. Winston et al., (2014) investigated the effects of both soil and cultivar on the microbiome of Cannabis. This research again demonstrated that cultivar type had a significant effect on the bacterial endophyte communities present in Cannabis, whereas bulk soil and samples from the rhizosphere were more similar to each other than samples from the endosphere of roots. The data suggests that in Cannabis, the soil environment is the primary determinant of the rhizosphere, whereas cultivar type is the primary determinant of the root endosphere. Several recent papers have demonstrated that plant genotype has a significant effect on associated microbial community structure, especially in regards to endophyte populations (Ding et al., 2013; Johnston-Monje et al., 2014, 2016; Lundberg et al., 2012; Peiffer et al., 2013; Walitang et al., 2018). This “genotype effect” causes plants grown in different soil conditions to have a conserved element of their microbiome despite the different environments. Microbial inheritance via seed transmission has been suggested to play a major role in the observed plant genotype effect on endophyte communities, giving rise to conserved endosphere species even when plants are grown in different soils (Johnston-Monje et al., 2014). Alternatively, the genotype effect could be entirely dependent on host traits, leading plants of a given genotype to acquire certain species of microbes from the surrounding reservoir of soil These two explanations of an observed genotype effect were investigated in corn with a soil swapping experiment. Johnston-Monje et al., (2014) showed that the root and shoot endophyte communities were more similar when the same host plant genotype was grown on different soil types than host plants grown within the same soil type. Additionally, a large portion of endophytic bacteria from isolated corn roots and shoots was shared with both seeds and plants that were grown in sterile sand, suggesting these endophytes were vertically transmitted rather being acquired from the soil environment. 16 Currently there is no definitive evidence demonstrating Cannabis has vertically transmitted endophytes, nor whether the observed genotype affect is a result of a core element of the Cannabis endosphere being inherited from mother plants. Research into this phenomenon and the potential role it plays in Cannabis plant health would assist in developing this crop of renewed interest. Seed endophytes have been isolated from three varieties of industrial hemp, many of which showed biocontrol activity (Scott et al., 2018). There is, to date, no published evidence that THC drug-type marijuana plants grown for the recreational or medicinal markets have vertically transmitted endophytes, although it is reasonable to assume that such endophytes exist, given the similarity of drug-type Cannabis to industrial hemp. The discovery of seed-vectored endophytes in Cannabis and the role they play in plant health could have implications for the current clone-based cultivation methods of this crop. Because there is partitioning of inherited endophytes to different plant tissues (Abdelfattah et al., 2021), clonally propagated plants from nodal or meristem segments could possibly lack some beneficial seed-vectored endophytes that, for example, are normally localized to roots and protect against root pathogens or other stresses. 1.6 Current Cannabis Cultivation Practices Cannabis plants intended for the recreational and medicinal markets (i.e. marijuana) are not typically cultivated from seed and are instead vegetatively propagated from cuttings (Small, 2018). Clonal plant tissue is then typically grown under supplemental light to stimulate rooting and vegetative growth. Once plants reach an appropriate size, they are then transferred to a reduced photoperiod to induce flowering. Cannabis cultivars show a high degree of heterozygosity and genetic diversity (Henry et al., 2020) and seeds from the same plant can give offspring with wildly different phenotypes. The consumer demand for a high-quality 17 consistent product necessitates more costly propagation methods. Clonally propagating Cannabis plants ensures genetic homogeneity for commercial production; however, the operating costs are greater when compared to seed-grown production at field scale. Pathogens from mother plants can be transferred to genetically identical and therefore equally susceptible clones if proper measures are not taken to minimize microbial transmission. An alternative method to circumvent this problem uses tissue-culture micropropagation in aseptic environments to mass produce pathogen and virus-free plants for production (M. Polne-Fuller, 2020). The microbiome of clones or micropropagated plants from tissue culture is likely highly divergent from Cannabis plants grown from seed as the former likely lacks seed-vectored microorganisms that act as the founding community in new plants. However, as there are limited tools available to control pathogens in Cannabis, these methods make sense to ensure growers are starting with plants free of pathogens, even if they are potentially more susceptible to new pathogens. The re-introduction of Cannabis-specific microorganisms that are associated with pathogen suppression could prove to be a powerful tool for Cannabis growers in indoor production facilities. Rather than being grown in greenhouses, hemp is typically grown from seed in a field using large equipment in a similar fashion to grain crops. The large amount of genetic and phenotypic variability among seed derived plants (Henry et al., 2020; Lata et al., 2010) is largely not an issue for seed, oil, fiber, and extract based products, and the lower production cost push growers to outdoor field growth (Crini and Lichtfouse, 2020). However, a challenge faced in the field is less control over pathogens such as fungi and insects. Additionally, there are currently no conventional fungicides labelled as safe for hemp (Mark et al., 2020). Row crops such as corn, wheat, and soy are often planted with seed coatings containing beneficial microorganisms that assist in overall plant health. Seed coating microorganisms are typically robust endospore 18 forming species of firmicutes that can survive and lay dormant on seeds for multiple years and can be co-applied with seed chemistries. 1.7 Cannabis Genomics Cannabis plants display considerable amounts of phenotypic plasticity, whereby plants from the same parental line can show large amounts of phenotypic variation. Cannabis sativa has a genome size of around 8.18 Mbp with the first draft genome was published in 2011 (van Bakel et al., 2011). The phylogenetic classification of the wide variety of Cannabis cultivars grown across the world is poorly understood. Cannabis genomes are extremely heterozygous. A recent study suggested the Cannabis genome has a single nucleotide polymorphism (SNP) once every 50 to 100 base pairs (McKernan et al., 2020). Furthermore, Cannabis genomes display large amounts of copy number variations in the genes responsible for cannabinoid production, and the genes that confer fungal pathogen resistance (McKernan et al., 2020). Government restrictions have resulted in limited research, irregular breeding efforts, and a poor understanding of Cannabis genomics (Henry et al., 2020). Historically, the taxonomy of Cannabis was divided among three species: Cannabis sativa, Cannabis indica, and Cannabis ruderalis. However, genetic evidence suggests there is little support for these taxonomic classifications for cultivars sold in commercial dispensaries (Schwabe and McGlaughlin, 2019). Instead this classification reflects the geographical origin of these three groups (Henry et al., 2020; Schwabe and McGlaughlin, 2019). More recently, genetic analysis of polymorphisms in the Cannabis genome has been used to cluster different Cannabis cultivars into five groups based on genetic markers associated with terpene, cannabinoid, fibre, and resin production (Henry et al., 2020). Improved understanding of the genetic diversity and phylogenetic relationships present in currently produced Cannabis cultivars is an essential step in the development of this crop. 19 Firstly, there are over 2000 “strains” of drug type Cannabis currently available to consumers, however the relationships of these strains are poorly understood and there is large amounts of genetic inconsistency even within strains (Schwabe and McGlaughlin, 2019). This can lead to phenotypic differences and unexpected effects for the consumer of both recreational and medicinal Cannabis. Improved understanding and consistency of the genetics of commercially available “strains” would be especially beneficial to patients reliant on this medication. Additionally, identification of polymorphisms could be used for genome wide association studies (GWAS) and marker-assisted selection (MAS), ultimately assisting the breeding of desirable and more stable traits. Finally, developments in the understanding of the phylogenetic relationships would assist in cultivar fingerprinting and seed-to sale tracking, protecting the intellectual property and efforts of breeders. Low Coverage Whole Genome Sequencing (LCWGS) is a methodology that could be applied to further the understating of genome-wide polymorphisms in Cannabis, particularly for the assessment of polygenic traits (Homburger et al., 2019). The technology has shown an improvement over SNP genotyping arrays in identifying low frequency variants and circumvents the high cost associated with high-depth sequencing GWAS studies (Gilly et al., 2019). Furthermore, modeling has shown that sequencing larger samples at the cost of sequencing depth results in improved power for genome association studies (Alex Buerkle and Gompert, 2013). By sequencing many of Cannabis genomes at a low sequencing depth, insights into the genetic variants present and phylogenetic relationships among industrial, drug type, and landrace Cannabis could be better understood. 1.8 Berkeleyomyces rouxiae Limited knowledge of Cannabis fungal pathogens, including their genetic resources, severely limits the ability of growers to effective control outbreaks. DNA based techniques allow 20 rapid and accurate identification of potentially devastating disease, however, there are fewer fungal genomes available compared to other kingdoms on the tree of life. Therefore, there is incentive for the sequence of new fungal genomes, especially economically relevant ones such as pathogens. Berkeleyomyces rouxiae is a widespread multi-host hemibiotrophic fungal pathogen known to cause black root rot in many crop plant species. Black root rot was first described in 1850 (Berkeley and Broome, 1850) and since it’s discovery has had a tumultuous taxonomic history driven by conventional morphological methods for fungal species classification (Nel et al., 2019). Until recently, this fungal pathogen was thought to belong to the genus Theilavopsis, and has now been reclassified using DNA-based methods (Nel et al., 2018). 1.9 Conclusions There is a clear demand in the Cannabis industry for effective, safe, and sustainable control of fungal pathogens. Both field-grown hemp and drug type marijuana have limited options for the control of fungal pathogens. Additionally, there has been limited research on the microbiomes of hemp and marijuana cultivars and the seed-vectored endophytes that have the potential to confer tolerance to pathogens in the next generation of plants. The current clonal production methods of indoor-grown drug type Cannabis may inadvertently alter microbiome structure, especially root endophytes, and result in plants that are more susceptible to disease. These clonal plants may lack seed-vectored endophytes that protect the seedlings from pathogens in their new environment and leave plant-niches to be filled with potentially harmful microorganisms. This thesis consists of three chapters that intend to answer important questions regarding the genotyping of commercially available Cannabis cultivars, the seed-transmission and inheritance of endophytes in Cannabis and investigate potential methods to control pathogens affecting Canadian growers, and those around the world. 21 1.9 Objectives and Hypothesis Chapter 2 The initial hypothesis in this chapter is that there are bacterial and fungal endophytes in Cannabis plants that are seed derived and that can be vertically transmitted to new plants across generations. These microorganisms can exist within the internal tissues of growing Cannabis plants without causing disease symptoms and are not derived from the environment and are instead passed down from parental plants. These types of endophytes are particularly interesting because they act as the founding microbiome of new plants and there is therefore likely selection pressure for certain beneficial endophytes to be maintained if they confer a selective advantage in certain environments. Additionally, this chapter aims to identify bioactivities of these endophytes that may contribute to overall plant health. Note that there is no expectation that the Cannabis endophytes found in this study will necessarily be present in all Cannabis plants; the objective is only to identify seed-derived endophytes that are present in the samples studied and demonstrate that these endophytes can be vertically transmitted. Chapter 3 The hypothesis of this chapter is that the black root rot found in the aeroponic Cannabis production facility in Cranbrook BC is a previously uncharacterized fungal pathogen in Cannabis. The objective is to identify and characterize the pathogenicity of this microorganism, and to sequence the first genome of this species. Chapter 4 The hypothesis of this chapter is that both seed-borne and environmental bacteria contribute to Cannabis seedling endosphere microbiomes. Additionally, we hypothesis a 22 component of this microbiome will be seed-derived and independent of soil conditions. The objective is to better understand how seed and soil bacteria influence the community structure in germinating Cannabis seedlings using 16S-based amplicon metagenomics. 23 Chapter 2: Cannabis Seedlings Inherit Seed-Borne Bioactive and Anti-Fungal Endophytic Bacilli 2.1. Abstract 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. 24 2.2 Introduction Plants can adapt to their environment by hosting beneficial bacteria and fungi that confer selective advantages to their host in stressed conditions (Johnston-Monje and Raizada, 2011b). These microorganisms can contribute to overall plant health by assisting in nutrient acquisition, secreting plant-growth-promoting phytohormones, eliciting host immune responses, or antagonizing pathogens to prevent disease (Hu et al., 2003; Johnston-Monje and Raizada, 2011b; Mousa et al., 2016; Shehata et al., 2017). Microorganisms that exist within plants are known as “endophytes”, and current research has elucidated how important these microorganisms are with respect to plant health (Berg et al., 2017; Khalaf and Raizada, 2018). The internal spaces of plants are unsurprisingly ideal habitats for many microorganisms: with a rich supply of carbon and other nutrients, as well as protection from harsh environments, plants host vast communities of microorganisms that can have a beneficial or pathogenic effect on their host (Barret et al., 2015; Hu et al., 2003; Johnston-Monje and Raizada, 2011b; Mousa et al., 2016; Shehata et al., 2017). In addition to the microorganisms obtained from the environment, plants inherit a founding microbiome via seed-to-seedling vertical transmission (Ferreira et al., 2008). Developments in our understanding of the seed microbiome and its role in plant ecology have been slow in comparison to that of the interaction with soil, the rhizosphere, and the plant microbiome as a whole (Nelson, 2018). Seed-borne microorganisms are of particular interest as they act as the starting point for the assemblage of new microbial communities for germinated seedlings during this critical stage in the plant life cycle (Shahzad et al., 2018). Research suggests that the microbial communities associated with seeds planted in soil originate primarily from the mother plant rather than from their soil environment (Johnston-Monje et al., 2021; van Overbeek et al., 2011). This seed microbiome is an important source of inoculum for developing seedlings, and plants appear to place strong selective forces on seed- 25 borne microorganisms over soil-borne microorganisms during germination by shaping what species proliferate and colonize emerging seedlings (Barret et al., 2015). Microorganisms that have effectively colonized plant tissues have the potential to exert a “barrier effect” against other microorganisms, preventing their colonization (Bacilio-Jiménez et al., 2001; Massalha et al., 2017). This potentially explains why seed-borne microorganisms, especially endophytes, are not simply overtaken by the ocean of soil-borne microorganisms and can persist across plant generations (Hardoim et al., 2012). Soil is a well-established driver of the composition of plant-associated microbial communities. However, understanding of the role for seed-borne microorganisms in the plant life cycle has grown substantially throughout the past decade (Nelson, 2018). While research has suggested that the bacterial communities of rhizospheres are primarily influenced by the surrounding soil (Normander and Prosser, 2000), there is also evidence that microbial inheritance plays an important role in forming early rhizosphere communities. One such study found that while soil type was the main driver of bacterial group structure in the rhizosphere, the most common bacterial cells (Proteobacteria and Bacteroidetes) in juvenile maize rhizospheres across three soil types appeared to be seed-derived. (Johnston-Monje et al., 2016). These results are further supported by a recent study showing that soil adds microbial diversity to early plant rhizospheres, while seeds contribute the most abundant bacteria found during the early stages of a plant’s life cycle (Johnston-Monje et al., 2021). This phenomenon of microbial inheritance appears to play a major role in determining microbial communities in plant endospheres (Johnston-Monje et al., 2014). Bacterial endophytes of maize have been suggested to be primarily seed-borne rather than originating from their soil environment (Johnston-Monje et al., 2014). Given that this phenomenon of microbial inheritance appears to play a major role in determining microbial communities in plant endospheres, it is interesting to speculate about alternative systems outside of genetics by 26 which selective advantages are inherited from parental plants. There is potentially selection pressure for microorganisms to be maintained and transmitted to offspring plants to protect them against biotic and abiotic stresses. Researchers have suggested that certain microorganisms can protect developing seedlings from pathogens in their new environment by altering seedling redox status (Pitzschke, 2016). Furthermore, a diversity of seed endophytes isolated from cucurbit species showed antibiotic activity against common greenhouse pathogens such as powdery mildew and Pythium (Khalaf and Raizada, 2016, 2018). It is clear that plants and microorganisms have co-evolved over hundreds of millions of years and that plants have selected for symbionts that are vertically transmitted across generations to provide a selective advantage to germinating seedlings (Johnston-Monje et al., 2021; Nelson, 2018). The emerging global Cannabis market is predicted to reach 30 billion USD by 2025 (New Fronteir Data, 2019) and the demands of growing a new crop on this scale will present an array of problems that require creative and sustainable solutions. Given the nature of the market, little research has been done on Cannabis cultivation relative to other crops, and so there are many opportunities to answer important questions that will benefit growers in Canada and around the world. Cannabis growers are challenged by an array of fungal pathogens with the potential to destroy entire harvests. Furthermore, synthetic fungicides are forbidden for both recreational and medicinal Cannabis in Canada and California (https://bit.ly/2BMfIYt (accessed on March 1 2021), https://bit.ly/2OkvT4T (accessed on March 1 2021). Consumer demand for safe, organically grown Cannabis products means that growers need effective biocontrol agents for fungal pathogens, an area that has seen little research in Cannabis. Recent research on pathogens of Cannabis has documented what growers have struggled with long before the recent legalization of Cannabis in Canada: roots and stems of Cannabis plants are commonly infected by species of Fusarium and Pythium (Punja, 2020a; Punja et al., 2018, 2019), both of which cause wilting and stem rot—symptoms colloquially 27 known as “damping off”. The dominant foliar pathogen associated with Cannabis tissue is Golovinomyces cichoracearum, which causes powdery mildew (Punja et al., 2019). Although this pathogen can also infect stems and flowers, it most commonly presents itself as white powdery mycelial growth on leaves (Punja, 2018). The revived interest in Cannabis is primarily driven by the secondary metabolites produced in flowering tissue. Cannabis female inflorescences (commonly known as “buds”) are infected by several pre- and post-harvest pathogens with the potential to make the final product unusable. Botrytis, Penicillium, and Fusarium are causes of “bud rot” in both pre- and post-harvest tissues (Punja, 2018, 2020b). Species of well-studied plant pathogens, Cladosporium, Alternaria, and Aspergillus, have also been isolated from flower tissue grown indoors and in greenhouses (Punja, 2018; Punja et al., 2019). These genera of flower-infecting fungi are known to have toxigenic species that produce mycotoxins that cause illness, or, at the very least, lead to quality control failures (Imolehin, 1983; McKernan et al., 2015). In general, microbial inheritance via seed-to-seedling vertical transmission occurs through microbial colonization of external and internal seed tissue in developing flowers (Dutta et al., 2014; Mitter et al., 2017). In fact, microscopic analysis shows that the inner surface of flower petals is densely populated by a diversity of bacteria, including species that show biocontrol activity (Fürnkranz et al., 2012). These species are in close contact on the surface of the seed coat and have the potential to be passed down to developing seedlings as they germinate (Nelson, 2018). Given that a diversity of fungal pathogens readily colonize Cannabis flowers both pre-and post-harvest, we hypothesize that Cannabis seeds vector opportunistic fungal pathogens as well as bacterial symbionts that protect them against biotic and abiotic stresses as they germinate into seedlings. Throughout our report, the mature Cannabis fruits (achenes) are referred to as “seed” for simplicity and because they are commonly referred to as such in the hemp and marijuana industry. This whole Cannabis fruit is what is typically sown in 28 fields or naturally shed from Cannabis plants when ripe (Maity et al., 2021; Schluttenhofer and Yuan, 2017) and so is analogous to seeds of other crops in the context of seed-borne microorganisms (Johnston-Monje et al., 2021). The objective of this study is to survey seed-borne endophytes found inside juvenile Cannabis seedlings. A selection of 15 accessions of hemp and marijuana types were grown to maturity in different locations across Western Canada before seeds were germinated in the absence of environmental microorganisms. A total of 136 bacterial isolates were cultured, identified, and phenotyped for their capacity to solubilize mineral phosphate, secrete indole-3-acetic acid (IAA), and hydrolyze pectin. A selection of unique isolates from each accession (78 endophytes) were then tested for their ability to antagonize seed-borne fungi that had been isolated from germinating Cannabis seeds. We demonstrated that the Cannabis accessions in this study inherited a conserved group of Bacilli with bioactivites that included suppression of fungal growth. This study suggests co-evolution between Cannabis and vertically transmitted, endophytic Bacilli. 2.3 Methods 2.3.1 Source of Seed Bethune-0 was obtained from Kernen Farm in Saskatoon. Altair, Alyssa, CAN3797, FIN34, Grandi, Katani, LKCSD, VIR577, and X59 generation-1 were from InnoTech Alberta. Canda was from Triple S Seeds LTD in Grandview, Manitoba. Afghani, BC Big Bud, God’s Bud, God’s Pink, and White Widow are from a commercial supplier in Qualicum Beach, British Columbia. X59 generation-2 was grown to produce seed at UBCO Kelowna, British Columbia. 29 2.3.2 Seed-borne Cannabis Seedling Endophyte Isolation An overview of the isolation design can be seen in Figure 2.1. Seedling surface sterilization was based on established methods for seeds with modification (Johnston-Monje and Raizada, 2011a). Five seeds of each Cannabis accession were germinated in sterile environments of Petri plates on autoclaved wet paper towel and placed in the dark for 5 days at 20 °C. Once germinated, seedlings were cleaned by washing with filter-sterilized 0.1% triton X for 10 min. Samples were then surface-sterilized by washing with 3% sodium hypochlorite for 20 min. The samples were then drained and rinsed with autoclaved, distilled water, then washed in 70% ethanol for 10 min. The ethanol was removed, and samples were rinsed 5 times with autoclaved, distilled water. To check for surface sterility, 150 μL of the final wash was spread on R2A and PDA, and one piece of tissue per treatment was transiently placed on sterile agar plates, which were all incubated for 10 days at 25 °C. Surface-sterilized seedlings were ground in 2 mL sterile phosphate buffer using a sterile mortar and pestle. An amount of 150 μL of this solution was spread on plates: R2A + 20 μg/mL nystatin, PDA + 20 μg/mL tetracycline, and TSA+ 20 μg/mL nystatin and incubated at 25 °C. Morphologically unique colonies from each Cannabis accession were isolated and subcultured from each media type. Isolates were grown in R2B for 3 days at 25 °C and stored in 20% glycerol at −20 °C. All research was conducted in a Health Canada licensed facility (LIC-0IW2D2L5JY-2019). 2.3.3 Taxonomic Identification by 16S Gene Sequencing Colony PCR of bacterial isolates was performed on 1 μL of R2B culture using primers 341F and 785R. PCR was conducted with the following settings: initial denaturation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min; a final extension phase was performed at 72 °C for 10 min. Amplicons were run on a 1% TAE gel to confirm successful amplification prior to sending to BioBasic (Markham) for Sangar Sequencing. Forward and reverse sequences were aligned and 30 consensus sequences were identified using BLAST against the 16S ribosomal RNA sequences database. Highest scoring alignments were assigned as isolate taxonomy. Sequences were submitted to Genbank under accession numbers OP087663-OP087800. 2.3.4 Phosphate Solubilization Assay Solubilization of mineral phosphate activity was tested by growing isolates on tricalcium phosphate media (10 g/L glucose, 2 g/L NH4CL, 0.41 g/L MgSO4, 0.295 g/L NaCl, 0.003 FeCl3, 0.7 g/L Ca3HPO4, 12 g/L Agar) and identifying halos around colonies after 3 days of growth at 25 °C (Johnston-Monje and Raizada, 2011a; Rodríguez et al., 2000). Glycerol stocks were used to inoculate R2B medium which was grown for 2 days shaking at 20 °C. Cultures were inoculated on tricalcium phosphate media using a 96-pin replicator; this assay was done in triplicate. 2.3.5 Pectinase Activity Pectinase Activity Pectinase activity was tested by growing isolates on R2A media supplemented with 0.2% citrus pectin (#J61021, Alfa Aesar) and 0.1% triton X-100. Glycerol stocks were used to inoculate R2B medium, which was grown for 2 days shaking at 20 °C prior to inoculating pectin media in triplicate using a 96-pin replicator. Pectin plates were placed in the dark at 25 °C for 3 days followed by flooding with Gram’s Iodine, where isolates displaying pectinase activity were surrounded by clear halos (Johnston-Monje and Raizada, 2011a). 2.3.6 IAA Secretion Indole-3-acetic acid secretion was tested by growing isolates on R2A media supplemented with 5 mM L-tryptophan. Glycerol stocks were used to inoculate R2B media, which was grown for 2 days shaking at 20 °C prior to inoculating tryptophan-supplemented R2A 31 in triplicate using a 96-pin replicator, which was then grown for 2 days at 25 °C. Plates were then covered with a nitrocellulose membrane and placed at 4 °C overnight, allowing for metabolite transfer. Nitrocellulose membranes were then removed from plates and soaked in Salkoski reagent (0.01 M ferric chloride in 35% perchloric acid) for 30 min. Reddish pink halos were visualized on an illuminated background and indicate bacterial IAA production (Bric et al., 1991). 2.3.7 Seed-Borne Fungi from Cannabis Culturing Five seeds of each Cannabis accession were planted on PDA + 20 μg/mL tetracycline (inhibits bacterial growth), in a biosafety cabinet and incubated for 5 days at 25 °C to allow for germination and/or growth of fungal colonies. Morphologically unique colonies of fungi associated with seeds and seedlings were subcultured and isolated on PDA. Seed germination and number of seeds/seedlings engulfed in fungal colonies were recorded. Fungal isolates were then subjected to colony PCR with primers ITS86F and ITS4R using the following settings: initial denaturation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 1 min; a final extension phase was performed at 72 °C for 10 min. Amplicons were run on a 1% TAE gel to confirm successful amplification prior to sending to BioBasic (Markham) for Sangar Sequencing. Taxonomy was determined by BLAST against the ITS ribosomal RNA sequences database; highest identity alignments were assigned as isolate taxonomy. Amplicon sequences were submitted to Genbank and assigned accession numbers OP18318-OP183206 2.3.8 Anti-Fungal Activity of Cannabis Endophytes Unique bacterial endophytes from each Cannabis accession were selected to assay for anti-fungal activity against seed-borne Cannabis pathogens: Alternaria PM35 from Altair, Aspergillus PM40 from CAN3797, Penicillium PM52 from God’s Bud, and Fusarium PM60 from 32 VIR577 (Supplemental File S1). Modifying an established assay (Shehata et al., 2016), bacterial endophytes were streaked on R2A plates from glycerol stocks and grown for 2 days at 25 °C. Single colonies where then used to inoculate TSB liquid medium which was grown at 20 °C for 2 days shaking at 150 RPM. Fungal cultures were grown on PDA for 5 days prior to harvesting spores by pipetting 1.3 mL sterile 0.1% Tween-20 on plates and harvested by gentle scraping with an L-spreader. An amount of 1 mL of this solution was used to inoculate 2 L of molten PDA cooled to 50 °C, which was poured into 150 mm × 15 mm plates to solidify. A sterilized Pasteur pipette was used to bore 14 holes in each agar plate. An amount of 100 μL of bacteria liquid culture was pipetted into holes into the agar plate. All 78 isolates were tested in triplicate across different agar plates. Uninoculated TSB was used as a control in this experiment. Plates were incubated for 7 days at 25 °C to allow fungi to grow, after which zones of inhibition surrounding bacterial cultures were measured and recorded. Zones with uneven diameters were measured using the shortest distance that bisected the agar hole. Statistical analysis was done using ANOVA and Tukey’s HSD test on RStudio (2021.09.2 + 382). Means not statistically different from each other are denoted by the same letter groups. 2.4 Results 2.4.1 Cannabis Seedlings Inherit Seed-Borne Endophytic Bacilli We germinated seeds from 15 different accessions of Cannabis including both hemp and marijuana types (Figure 2.1a). These seeds were germinated in sterile Petri plates, and the culturable endophytes within the germinated seedlings were identified (Figure 2.1b). Because the culture environment had been sterilized, the endophytes present inside seedling tissue must have been acquired from the seed surface and seed endosphere (Johnston-Monje et al., 2021). Prior to detection of endophytes, all seedlings were tested for surface sterility by plating a final 33 wash onto nutrient agar; no growth was observed on any plate, which confirms that seedling surfaces were sterile. A total of 136 bacterial isolates were obtained from the seedling endosphere and were assigned to taxa using 16S sequencing. Although the seeds came from four different locations and growing conditions in Canada, we observed unexpected conservation of some of the culturable, seed-borne seedling endophytes (Table 1). Moreover, seeding endospheres of these 15 Cannabis accessions appeared to be dominated by endospore-forming bacteria in the class Bacilli. Notably, Paenibacillus mobilis isolates were cultured from endospheres of 100% (15/15) of accessions, despite the different conditions in which their mother plants were grown (Figure 1a). The well-studied and agriculturally important soil bacterium, Bacillus subtills, was found in the majority (8/15) of Cannabis accessions and was present in both hemp and marijuana. 34 (a) (b) Figure 2. 1. (a) Geographic origin of parental Cannabis sativa accessions used for seed in this study. All experiments were performed at UBCO (Kelowna, BC, Canada). (b) Schematic for the location and isolation of seedling endophytes in this study. Both seed surface and seed endosphere contribute to seed-borne microbiome. Figure b was created with BioRender.com. 35 Table 2. 1. Culturable Seed-borne Endophytes from Seedling Endospheres. Green shaded cells indicate isolation from pool of 5 seedlings following germination in sterile environment and surface sterilization. Seed from field-grown flax, Linum usitatissimum var.Bethune, was included in this experiment as an out-group for comparison and as a control for contamination. Only one culturable seed-borne endophyte, Bacillus megaterium, was isolated from flax seedlings (Table 2.1). This bacterium was found in 47% of the Cannabis genotypes (7/15). Seed-borne Paenibacillus mobilis was not isolated from any flax endospheres in our experiments. Phylogenetic analysis of the 16S gene from the 136 seed-borne Cannabis bacterial endophytes further demonstrated that many of the culturable bacterial taxa that colonized Seed-Borne Seedling Endophytes Hemp Cultivars Marijuana Cultivars Flax Field Field Field Field Field Field Field Field Field Indoor Indoor Indoor Indoor Indoor Indoor Field Bacterial Species Altair Alyssa CAN 3797 Canda FIN34 Grandi Katani LKCSD X-59 X-59 Gen 2 Afghani BC Big Bud God's Bud God's Pink White Widow Bethune Bacillus aerius Bacillus circulans Bacillus ginsengihumi Bacillus inaquosorum Bacillus megaterium Bacillus stratosphericus Bacillus subtilis Bacillus velezensis Bacillus wiedmannii Bacillus zhangzhouensis Bacillus simplex Brevibacillus choshinensis Paenibacillus azotifigens Paenibacillus humicus Paenibacillus illinoisensis Paenibacillus mobilis Paenibacillus pabuli Paenibacillus polymyxa Paenibacillus senegalensis Paenibacillus sinopodophylli Paenibacillus taohuashanense Paenibacillus terreus Paenibacillus vulneris Paenibacillus yunnanensis Pantoea agglomerans Psychrobacter pulmonis 36 Cannabis seedling endospheres were well-conserved across seed accessions (Figure 2.2). Endophytes belonging to the genera Bacillus (28.26%) and Paenibacillus (67.39%) made up the majority of isolates. Bacillus endophytes were isolated from 80% of Cannabis genotypes, while endophytes belonging to the genus Paenibacillus were isolated from all 15 (100%) Cannabis accessions. In the absence of environmental microorganisms, the Cannabis genotypes in this study appeared to inherit a group of closely related endophytes from parental seed. Details on all isolates obtained from the seed-borne endospheres can be found in Supplemental File S1. Figure 2. 2. Nearest-Neighbor Phylogenetic Tree of 16S Amplicons from Culturable Seed-Borne Bacterial Endophytes. Amplicons were aligned using the MUSCLE algorithm prior to nearest-neighbor phylogenetic tree construction using the Tamura–Nei model on MEGA software (Version 11.0.11). 37 Hemp variety X59 was represented in our analysis by two samples from two different generations, grown under different conditions. The first generation (X59 Gen-1) was harvested from field-grown plants in Vegreville, Alberta. Some of these seeds were subsequently sown in soilless commercial potting mix and grown to maturity indoors in Kelowna, British Columbia, and the seeds produced by these plants were labeled X59 Gen-2. Analysis of endophyte 16S amplicons showed that the strains of Bacillus megaterium and Paenibacillus mobilis isolated from seedling endospheres across these generations were identical despite growing in vastly different environments (Supplemental Figure S1). 2.4.2 Endophyte Phosphate Solubilization Plants have been known to harbor root endophytes that secrete organic acids to solubilize rock phosphate in the surrounding environment. Seed-borne Cannabis endophyte isolates were assessed for their ability to solubilize mineral phosphate by growing on tricalcium phosphate media and creating clear halos around the culture—interestingly, phosphate-solubilizing bacteria were isolated from the endosphere of all 15 Cannabis accessions (100%). In total, 10 different bacterial species were found to solubilize phosphate. Ubiquitous across all Cannabis seedings in this study was phosphate-solubilizing Paenibacillus mobilis (Figure 2.3). 38 Figure 2. 3. Summary of in vitro Phenotyping Assays. Cannabis endophytes were assayed for the potential to solubilize mineral phosphate, secrete IAA, and hydrolyze pectin. Blue squares indicate phosphate solubilization. Red squares indicate IAA secretion. Yellow squares indicate pectinase activity. 2.4.3 Endophyte Indole-3-Acetic Acid Activity Plant-associated bacteria have been known to secrete phytohormones that interact with their host plant with the potential to modulate plant growth. Microbial production of the auxin, indole-3-acetic acid (IAA), can promote root growth and colonization, and circumvent plant immune responses (Spaepen et al., 2007; Spaepen and Vanderleyden, 2011). Cannabis endophytes were assayed for in vitro IAA production and secretion using a colorimetric assay 39 (Bric et al., 1991). IAA production was detected in six different bacterial species isolated from 53.3% (8/15) of Cannabis accessions in this study (Figure 2.3). 2.4.4 Endophyte Pectinase Activity Pectin is a structural heteropolysaccharide that is a major component of plant cell walls. Pectinase activity of seed-borne Cannabis endophytes was assessed by growing on agar supplemented with citrus pectin and visualizing halos by staining with Gram’s Iodine. Pectinase activity was found in four different bacterial species across five Cannabis accessions (33%) (Figure 2.3). Pectinase activity was limited to endophytes of hemp, as no marijuana accessions hosted species with this phenotype. 2.4.5 Seed-Borne Fungi Seedling endospheres are expected to harbor fungi (Shahzad et al., 2018). However, following seedling surface sterilization, only a single accession of either Cannabis or flax produced culturable fungus on PDA with tetracycline. This isolate was obtained from the God’s Bud accession and was identified as PM17- Penicillium (Supplemental File S1). In contrast, bacterial endophytes were cultured from seedling endospheres of every accession, totaling 136 bacterial isolates. Seeds are a well-known vector of both fungal symbionts and pathogens in many plant species (Imolehin, 1983; Verma et al., 2018; Wang et al., 2022). The absence of fungi from surface-sterilized seedlings does not imply that they are absent from seeds and seedling surfaces. Therefore, Cannabis accessions were examined without seedling surface sterilization where seeds were instead directly germinated on PDA with tetracycline to assess germination and presence of seed-associated fungi. In this experiment, “VIR577” was substituted for “God’s Pink” due to availability of seeds. 40 Fungal isolates were cultured from seeds/seedlings of 73.3% (11/15) of Cannabis accessions. From each accession, morphologically unique colonies were subcultured and identified by ITS region sequencing. A total of 24 seed-associated fungal isolates were found from this experiment (Figure 2.4a). Of interest, 83.3% (20/24) of these isolates were identified as genera of known Cannabis pathogens and post-harvest molds: Alternaria, Penicillium, Cladosporium, Chaetomium, Aspergillus, Rhizopus, and Fusarium. The germination rates and number of seeds engulfed by mycelia (disease pressure) in this experiment can be seen in Figure 2.4b. Notably, there were large fluctuations in the germination rates of the various Cannabis accessions as well as three cultivars with 100% germination and 0% of seeds with any fungal disease pressure: Afghani, Alyssa, and White Widow. 41 Figure 2. 4. Seed-Borne Fungi from Cannabis. (a) Fungal isolates from seeds of 15 Cannabis accessions germinated on PDA for 5 days. (b) Germination rates and percent of seeds with visible mycelia growth (disease pressure) for each Cannabis accession. 42 2.4.6 Endophyte Anti-fungal Activity A total of 78 seed-borne bacterial endophytes were selected based on unique taxonomies from each accession of Cannabis in this study. These isolates were tested for their ability to antagonize growth of four different seed-borne fungi belonging to genera of known Cannabis pathogens: Alternaria PM35, Aspergillus PM40, Penicillium PM52, and Fusarium PM60 (Figure 2.5). Thirteen bacterial isolates covering four different species were found to have anti-fungal properties and were capable of creating zones of inhibition when co-cultured on PDA with these fungi. Anti-fungal activity was found in bacterial isolates from 60% (9/15) of the Cannabis accessions examined in this study. A summary of these data can be seen in Table 2.2 Figure 2. 5. Biocontrol Activity of 78 Seed-Borne Cannabis Endophytes Against Seed-Borne Fungi. Four fungal isolates belonging to plant pathogenic genera were selected (a) Alternaria, (b) Aspergillus, (c) Penicillium, (d) Fusarium. Diameters of zones of inhibition were measured at 7 days post-inoculation with endophytes. Color-coded letters correspond to means within the same group as calculated by ANOVA and Tukey HSD test. 43 Table 2. 2. Summary of Anti-fungal Activity in Seed-borne Cannabis Endophytes. Strain ID Taxonomy Accession Host Antagonizes OKM96 Bacillus inaquosorum BC Big Bud Marijuana Alternaria OKM115 Bacillus subtilis BC Big Bud Marijuana Alternaria, Fusarium OKM121 Bacillus subtilis God’s Bud Marijuana Alternaria, Fusarium OKM128 Bacillus subtilis CAN 3797 Hemp Alternaria, Fusarium, Penicillium OKM141 Bacillus velezensis CAN 3797 Hemp Alternaria, Aspergillus, Fusarium, Penicillium OKM147 Paenibacillus polymyxa Altair Hemp Alternaria, Aspergillus, Fusarium, Penicillium OKM155 Paenibacillus polymyxa Katani Hemp Alternaria, Aspergillus, Fusarium, Penicillium OKM162 Bacillus subtilis Grandi Hemp Alternaria, Fusarium OKM163 Bacillus subtilis Altair Hemp Alternaria, Fusarium OKM169 Bacillus subtilis LKCSD Hemp Alternaria, Fusarium OKM170 Paenibacillus polymyxa FIN34 Hemp Alternaria, Aspergillus, Fusarium, Penicillium OKM173 Bacillus subtilis FIN34 Hemp Alternaria, Fusarium OKM186 Bacillus subtilis Alyssa Hemp Alternaria, Fusarium Alternaria PM35 (Figure 2.5a) was the fungal isolate that was antagonized by the largest number of bacterial isolates tested, with 13 bacterial isolates inhibiting Alternaria PM35. Bacillus subtills OKM128 was the strongest antagonist and created an average zone of inhibition equal to 38.6 ± 11.02 mm in diameter after 7 days of co-culturing. In contrast, Aspergillus isolate PM40 (Figure 5b) was inhibited by only four of the bacterial isolates tested, making it the fungus that showed the highest level of resistance in this study (Table 2.2). Anti-Aspergillus isolates were limited to the species Paenibacillus polymyxa and Bacillus velezensis; the largest average zone of inhibition was 11.0 ± 1.0 mm from Bacillus velezensis OKM141. Five isolates comprising three different species were able to antagonize Penicillium PM52: Bacillus subtilis, Paenibacillus polymyxa, and Bacillus velezensis. Bacillus subtills OKM128 produced, again, the largest zone of inhibition with an average diameter of 18.33 ± 2.5 mm. Zones of inhibition against Fusarium PM60 were produced by 12 isolates (three different species), the greatest of which was an average of 17.67 ± 2.3 mm by Paenibacillus polymyxa 44 OKM170. All negative controls (uninoculated TSB liquid medium) showed no zones of inhibition, with fungal hyphae growing over holes in agar. ANOVA and Tukey’s HSD test for significance were conducted across bacterial isolates (including negative control) for antagonism of each fungus. Letter values (Figure 2.5) were assigned to means to distinguish groups significantly different from one another (α = 0.05). 2.5 Discussion Plant microbiome research has been heavily focused on the role of soil on microbial community structure. Until recently, the dominance of seed-borne microorganisms in early community establishment was poorly understood. New research demonstrates that seed-borne microorganisms are the primary colonizers of juvenile angiosperms, and soil microorganisms only colonize young plants efficiently when the seed microbiome is disrupted by surface sterilization (Johnston-Monje et al., 2021; Moroenyane et al., 2021). Importantly, most previous research on the contributions that plant genotype, seed microorganisms, and soil communities have on the plant microbiome has used surface-sterilized seeds, disrupting spermosphere/seed-coat microorganisms that play an important role in vertical transmission (Lundberg et al., 2012). As has been done in more current studies, we deliberately did not surface sterilize Cannabis seeds, prior to seedling surface sterilization, to avoid artificially disrupting seed-surface microorganisms that may be heritable (Johnston-Monje et al., 2014, 2021; Moroenyane et al., 2021; Nelson, 2018). Despite the diverse geographic origins of seed production, Cannabis seedlings in this study, when germinated in the absence of environmental microbes, inherited a closely related group of endophytic Bacilli with a variety of interesting phenotypes that may benefit seeds as they germinate in a new environment (Table 2.1, Figure 2.3). 45 The phenomenon of microbial inheritance is well-studied in many plant species (Shahzad et al., 2018). Our report documents this process in Cannabis and identifies some of the relevant functions of seed-borne seedling endophytes. To further support the previous observation, a second generation of the hemp cultivar X59 was grown to maturity to produce seed indoors in Sunshine soil mix (Sun Gro Horticulture, Agawam, MA, USA) at the University of British Columbia, Okanagan. This environment differs greatly from the outdoor fields, where X59 generation-1 was grown to maturity to produce seed at InnoTech Alberta (Figure 2.1). The clear preservation of two identical species of seed-borne endophytes across this generation suggests that X59 (and Cannabis in general) selects for certain microorganisms to be maintained in its microbiome and passed down subsequent plant generations (Supplemental Figure S1). Other plants such as rice and corn have conserved and vertically transmitted core microbial groups that appear to be maintained across generations from ancestral relatives to modern cultivars (Johnston-Monje and Raizada, 2011a; Walitang et al., 2018). Future studies should investigate what core microbial species are found in Cannabis seeds throughout its evolution, domestication from the Himalayan foothills of central Asia, and thousands of years of cultivation (Andre et al., 2016; Wills, 1998). Such a study would rely on improved phylogenetic classification of ancestral, landrace, and modern Cannabis accessions grown today, an area that is currently being researched (Henry et al., 2020). All 15 accessions of Cannabis, including hemp and marijuana, inherited seed-borne Paenibacillus mobilis with the capacity to solubilize mineral rock phosphate. The out-group flax cultivar, Bethune-0, did not harbor this species of phosphate-solubilizing Paenibacillus mobilis (Figure 2.3). Phosphate is a vital macronutrient and plants have been documented to host endophytic bacteria in root zones that secrete organic acids that solubilize mineral phosphate, increasing bioavailability, and promoting root growth (Mohabeer et al., 1997; Richardson et al., 2009; Shehata et al., 2017) . Of note, an endophyte was isolated from seeds of a wild maize 46 relative growing in a volcanic swamp with high levels of insoluble mineral phosphate, and was likely selected to be vertically transmitted across plant generations to assist seedlings in this harsh environment (Shehata et al., 2017). The observation that all accessions of Cannabis hosted seed-vectored Paenibacillus mobilis with the potential to convert tricalcium phosphate to a more plant-bioavailable form leads to speculation about what selection pressure nature has exerted on Cannabis throughout its evolution and domestication. Indoor and field-grown hemp and marijuana hosted this endophyte. Phosphorous and nitrogen have been documented to be the most important macronutrients contributing to Cannabis flower yield (Bevan et al., 2021), and phosphorous nutrition can have an effect on cannabinoid profile (Shiponi and Bernstein, 2021); however, research in this area is still developing. Seed germination and seedling establishment is sensitive to oxidative imbalances. Reactive oxygen species (ROS) act both as an elicitor to release seed dormancy, a defense mechanism against disease, and toxic metabolites when accumulated in plant cells (Bailly et al., 2008; Lamb and Dixon, 1997). These conflicting effects create an “oxidative window for germination” that is tightly controlled by auxin hormone signaling pathways such as IAA to promote healthy germination (Chen et al., 2014; Ivanchenko et al., 2013; KrishnaMurthy and Rathinasabapathi, 2013). IAA-secreting bacterial endophytes were found in 53.3% of Cannabis accessions examined in this study (Figure 2.3). Seed-borne endophytic bacteria that produce IAA have previously been documented to alter seedling development in rice (Verma et al., 2017). This same study showed that when these seed-associated bacteria were removed, there were lower levels of ROS as well as a detrimental effect on root hair development and seedling size compared to rice with an intact seed-borne microbiome. It is speculated that the increased ROS production from seed-borne endophytes may also serve to protect seedlings against invading pathogens (Verma et al., 2017). IAA-producing endophytes have been previously isolated from industrial hemp cultivars (Scott et al., 2018). It is unsurprising that the Cannabis 47 accessions examined here, similar to other plants, inherit bacteria that secrete IAA and likely contribute to seedling germination and establishment. Further testing of these endophytes and their effect on seed germination and establishment should be examined. Hydrolysis of plant cell walls by enzymes such as pectinase, xylanase, and cellulase is thought to be a mechanism by which some endophytes gain entry into plant tissues (Kandel et al., 2017). A small number of bacteria in this study were able to grow on and hydrolyze citrus pectin (Figure 4). Further testing of this library of endophytes should examine other cellulolytic enzyme activity, as well as other sources of pectin. It should be noted that citrus pectin has been documented to have powerful antibiotic activity against both gram-negative and gram-positive bacteria (Presentato et al., 2020). There appeared to be growth inhibition of many endophytes in the pectinase assay which would lead them to score negative; these endophyte species may well be able to hydrolyze other forms of pectin. The role of seeds in dispersal of fungal pathogens in Cannabis has not seen extensive research (Punja, 2021); however, seed-borne fungal diseases are well-documented in established crops (Imolehin, 1983; Kumar and Gupta, 2020; Verma et al., 2018; Wang et al., 2022). This study found that 73.3% (11/15) of Cannabis accessions hosted at least one seed-borne fungal isolate (Figure 2.4). The observation that 83.3% (20/24) of these isolates were identified as genera of known Cannabis pathogens and post-harvest molds: Alternaria, Penicillium, Cladosporium, Chaetomium, Aspergillus, Rhizopus, and Fusarium, is unlikely a coincidence. Rather, Cannabis seeds likely act as a vector for fungal pathogens to spread and proliferate. Of particular interest is the observation that Penicillium, Fusarium, Cladosporium, Alternaria, and Aspergillus have been previously identified infecting Cannabis flowers (Punja, 2018, 2020b; Punja et al., 2019). Isolates belonging to these pathogenic genera account for 70.8% (17/24) of total isolates. Seed-bone Alternaria has also recently been documented as a pathogen of mature Cannabis (Roberts and Punja, 2022). Taken together, this suggests that 48 Cannabis flowers act as an infection point for opportunistic fungi to be vectored across plant generations via seed transmission. Observationally, the fungi isolated here from germinating seeds appeared to colonize and destroy seed and seedling tissue (Supplemental Figure S2); however, further testing must be done to investigate their potentially pathogenic behavior to mature Cannabis plants. Given the evidence that Cannabis seeds vector both a conserved group of endophytic Bacilli as well as fungi belonging to potentially pathogenic genera, there is likely competition and co-evolution for the shared niche of germinating Cannabis seeds, as well as selection pressure on Cannabis to maintain beneficial microorganisms with biocontrol in their microbiome. Members of class Bacilli produce a variety of antimicrobial secondary metabolites (Cochrane and Verderas, 2014; Ongena and Jacques, 2008). The genera Bacillus and Paenibacillus are currently the best characterized for producing lipopeptides and polyketides that have immense commercial value in agriculture for their capacity to antagonize pathogenic fungi and suppress crop disease. A total 60% (9/15) of Cannabis accessions in this study hosted seed-borne endophytic Bacilli that inhibited growth of seed-associated fungi belonging to plant pathogenic genera (Figure 2.5, Table 2.2). Four fungal isolates were selected for this assay based on their taxonomy belonging to the genera Alternaria, Aspergillus, Penicillium, and Fusarium. All of these are known plant pathogens and have been documented to infect Cannabis flowers and cause disease in Cannabis plants (Punja, 2018, 2020b; Punja et al., 2019; Roberts and Punja, 2022). Of the 78 selected endophytes isolated from the 15 Cannabis accessions, 13 isolates showed antagonism against seed-associated Cannabis fungi (Figure 2.5, Table 2.2). Four endophytes showed antagonism against all fungi assayed: Bacillus velezensis OKM141, Paenibacillus polyxma OKM147, OKM155, and OKM170. These endophytes will be further studied for their ability to control fungal pathogens in mature Cannabis plants, as well as on seed. Given the observation of highly variable rates of germination, it is interesting to speculate 49 about the contribution of these various fungi to the diminished germination seen in seeds of certain Cannabis accessions (Figure 2.4b). Seed-borne Fusarium, Penicillium, Rhizopus, and Aspergillus are associated with decreased seed germination in rice (Imolehin, 1983). Future testing of seed-coating anti-fungal bacterial isolates on low-germination seed will be done to investigate antagonism of seed-associated fungal pathogens, and the resulting effect on germination. Additionally, these anti-fungal bacteria will be tested for their capacity to protect seeds by flower and foliar applications and establishment in the subsequent plant generation as seedling endophytes. A variety of commercial products exist that use beneficial microorganisms to combat plant pathogens. Serenade® was developed by AgriQuest.Inc (now owned by Bayer) and contains a species of Bacillus subtills that produces a variety of antimicrobial lipopeptides. Serenade® is effective at controlling a variety of pathogens including: fungi such as botrytis (Percival et al., 2017), powdery mildew (Matzen et al., 2019), yellow rust (Reiss and Jørgensen, 2017) and early blight (Abbasi and Weselowski, 2014); and protists such as clubroot (Lahlali et al., 2011) and the oomycetes Pythium and Rhizoctonia (Jo et al., 2014). Researchers have begun to investigate the potential of Bacillus species to control fungal pathogens and promote plant growth in Cannabis with promising results (Balthazar et al., 2022; Comeau et al., 2021; Punja and Ni, 2021). What makes Bacillus and Paenibacillus particularly attractive to commercial farming is their capacity to form desiccation-resistant endospores, allowing formulation and survival as seed coats (Ongena and Jacques, 2008). The seed-borne endophyte library generated in this study is dominated by endospore-forming bacteria and so may possess strains with the potential to be useful as seed-coating biologicals in field-grown hemp. Furthermore, drug-type Cannabis (marijuana) is typically clonally propagated to mitigate variation in phenotypes observed when plants are grown from seed (Campbell et al., 2019; Caplan et al., 2017, 2018; Monthony et al., 2021). The results presented here lead to 50 speculations about the different microbiomes of clonally propagated and seed-grown Cannabis plants. Whether these seed-borne anti-fungal seedling endophytes are absent in clonally propagated Cannabis plants would be of interest to growers of this horticultural crop with limited tools for control of fungal pathogens. In conclusion, this study demonstrates that Cannabis seeds inherit bioactive and anti-fungal Bacilli that colonize seedling endospheres. Additionally, Cannabis seeds appear to be associated with fungi belonging to genera of known floral pathogens, suggesting that fungi may opportunistically use flowers as a transmission route to infect germinating Cannabis seedlings. The production of antimicrobial secondary metabolites that antagonize seed-associated fungi by these bacterial endophytes suggests competition for the shared niche of germinating Cannabis seeds. Taken together, this paper suggests co-evolution and potential symbiosis between Cannabis and seed-borne Bacilli endophytes. 51 Chapter 3: Hybrid Genome Assembly of Berkeleyomyces rouxiae, an Emerging Cannabis Fungal Pathogen Causing Black Root Rot in an Aeroponic Facility 3.1 Abstract The resurgent 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 from a licenced production facility in Cranbrook BC, Canada, rapidly displayed root discolouration and rot symptoms despite testing negative for all commercially available pathogen tests. Developing sequencing-based disease diagnostics requires genomic information, therefore this study presents the first whole genome sequence of the multi-host, 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 6960 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. 52 3.2 Introduction Several fungal pathogens infect leaves, flowers, stems, and roots of Cannabis sativa, including both low-THC types (i.e. hemp) and high-THC types. Cannabis roots are infected by species of Fusarium such as F. oxysporum, F. brachygibbosum, and F. solani, and oomycetes including Pythium species P. dissotocum, P. myriotylum, and P. aphanidermatum (Punja et al., 2018, 2019). These root pathogens cause root, crown, and pith rot and discolouration, progressing to stunting, wilting, and potentially host death (Punja et al., 2018, 2019). Because of prohibitions on Cannabis during most of the 20th century, formal studies of its cultivation, pathogens, and biocontrol have until recently been very limited. Following legalization, most jurisdictions have regulated the pesticides that may be used on commercial Cannabis. For example, in both Canada and California, synthetic fungicides are prohibited for use on Cannabis for medicinal and recreational markets (https://bit.ly/2BMfIYt https://bit.ly/2OkvT4T). This leaves growers with limited tools to control fungal outbreaks, which may rapidly spread and result in complete crop destruction. Therefore, monitoring and detection of disease is paramount in commercial Cannabis production and is a prerequisite for effective integrated pest management (IPM) programs. Diagnosis of disease may be possible based on visual criteria alone, but molecular technologies have the potential to be more sensitive and accurate. Incomplete knowledge about the DNA sequences of fungal pathogens of Cannabis limits the development of diagnostic tests. This study was motivated by the observation of an unknown root pathogen in an aeroponic facility growing high THC-type Cannabis in Cranbrook BC, Canada. Infected plants were subjected to all available Cannabis pathogen tests, but no positive identification was made. Preliminary morphological characterization indicated that a microorganism abundant on the surface of infected plants was likely a member of the Berkeleyomyces genus, which is known to cause black rot in many agricultural plants (Nel et al., 2019). Berkeleyomyces is a 53 recently described genus of ascomycetes in the Ceratocystidaceae family with only a single available genome for B. basicola (GenbBank accession: GCA_003671435.1) Berkeleyomyces was not previously reported to infect high THC-containing Cannabis plants, however, during the writing of this manuscript it was described as a pathogen of greenhouse-grown hemp in Kentucky, USA (Rahnama et al., 2022), suggesting it may already be a widespread Cannabis pathogen. We were therefore motivated to further characterize this pathogen, including its complete genomic sequence and its ability to infect high THC-type Cannabis in greenhouse environments. 3.3 Methods 3.3.1 Source of Seed Clonally propagated Cannabis plants ‘Nuken’, ‘Blueberry x Northern Lights’, ‘Coastal Sage’ and ‘Strawberry Guava’ were grown from cuttings aeroponically using Aetrium-4 Aeroponic Growing System (Aessence, Santa Clara) at a commercial facility located in British Columbia, Canada. Plants were grown in four rows of nine tubes at 28°C, 60-75% relative humidity, under a light intensity of 1200 µmol/m2/s. Plants were grown using General Hydroponics 6-part Flora Series fertilizer solution (General Hydroponics, Santa Rosa) at pH 5.8 using manufacturer-recommended concentrations throughout the production cycle. Plants followed a weekly pre-determined nutrient dosing cycle. 3.3.2 Culturing microorganisms from root tissue Plants were grown aeroponically at a licensed Cannabis production facility (Cranbrook, BC, Canada). Plants initially were screened for pathogens by sending to A&L Canada Laboratories (London, Canada). Diseased roots of the “Nuken” strain were shipped to the University of British Columbia, Okanagan campus (Kelowna, BC, Canada) for pathogen testing. Root tissue was aseptically cut into small segments prior to plating on R2A + 20 μg/mL nystatin, 54 PDA + 20 μg/mL tetracycline, and carrot agar + 20 μg/mL tetracycline (Medina and Platt, 1999). Carrot agar was made by simmering 200 g of chopped fresh carrots in water for 5 minutes followed by blending. This paste was then squeezed through muslin, supplemented with 12 g agar, and diluted to a final volume of 1 liter prior to autoclaving. These plates were incubated at 25°C for 24 to 48 hours for microbial growth. Morphologically unique microorganisms were subcultured on corresponding media to isolate pure cultures. 3.3.3 Taxonomic classification through colony PCR Isolated fungal cultures were subjected to colony PCR by heating mycelia-water suspension at 60°C for 10 minutes. 1 µL of this suspension was used as a template for PCR to amplify the ITS2 region with primers ITS86F (GTGAATCATCGAATCTTTGAA) and ITS4R (TCCTCCGCTTATTGATATGC) (Vancov and Keen, 2009) using the following settings: initial denaturation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 1 min; a final extension phase was performed at 72 °C for 10 min. Amplicons were run on a 1% TAE gel to confirm amplification prior to Sanger sequencing using SeqStudio Genetic Analyzer (ThermoFisher) with BigDye™ Terminator v3.1 Cycle Sequencing Kit (ThermoFisher). Identification of fungal isolates was determined by using a BLASTn query against an ITS ribosomal sequences database (rRNA_typestrains/ITS_RefSeq_Fungi) and assigning the highest identity alignments to the isolates. All isolates were >98% similarity to top match. 3.3.4 Long read sequencing Carrot agar cultures of B. rouxiae PM34 were grown for 1 week at 25°C prior to harvesting by pipetting 1.3 mL of sterile 0.1% Tween20 and scraping tissue with an L-spreader. 500 mg of tissue was flash frozen in liquid nitrogen and ground in a sterile mortar and pestle. High-molecular weight (HMW) DNA was extracted using CTAB method according to an online 55 protocol (https://dx.doi.org/10.17504/protocols.io.bwr8pd9w). DNA quality was assessed using a Nanodrop and Qubit Fluorometer. 1 μg of fungal DNA was used to prepare a Nanopore Sequencing Library using the Genomic DNA by Ligation (SQK-LSK110) protocol. Sequencing was carried out with two FLO-FLG001 R9.4.1 flow cells on a MinION for 72 hours with no basecalling. MinION generated fast5 files were basecalled after sequencing using Guppy 6.0.1 Superior Basecalling Algorithm (dna_r9.4.1_450bps_sup.cfg) conducted on an NVIDIA 3060ti. Reads with a q-score below 9 were discarded. 3.3.5 Short-read sequencing 1 μg of fungal DNA was sent to CD genomics (Shirley, NY) for paired end library preparation for PE150 sequencing on Illumina HiSeq X Ten System. 160,979,852 clean reads were assessed for quality using FastQC (Andrews, 2010). 3.3.6 Hybrid genome assembly A total of 9 genome assembly methods were tested for contiguity and genome completeness using BUSCO scores with the sordariomycetes_odb10 database (Supplemental Fig. 1, Supplemental Table S1). The highest quality genome assembly was produced with Canu (Koren et al., 2017) and ONT long-reads with the following settings: (genomeSize=30m correctedErrorRate=0.105 -nanopore) prior to error correction using POLCA genome polishing software (https://github.com/alekseyzimin/masurca) (Zimin and Salzberg, 2020) and Illumina short-reads. Merqury was used to create k-mer spectrum plots to assess the quality of this assembly using short reads (Rhie et al., 2020). 56 3.3.7 Genome annotation Genome annotation was conducted using the Funannotate pipeline (v1.8.12) run on CentOS Linux 7, 8 cores, ~ 124 GB RAM. Python: 3.8.12 (https://github.com/nextgenusfs/funannotate)(Palmer, 2016). First, the assembly was cleaned of repetitive contigs using minimap2, followed by softmasking repeats using TANTAN (Frith, 2011). Ab initio gene predictions were done using Augustus (V3.3.3)(Stanke et al., 2006). Functional annotation of predicted genes was subsequently carried out by parsing protein-coding models and comparing to the Pfam domains(El-Gebali et al., 2019), Uniprot (2022_02) using DIAMOND (2.0.15)(Buchfink et al., 2014), CAZymes (Cantarel et al., 2009), secreted proteins, proteases (MEROPS)(Rawlings et al., 2009), BUSCO(Waterhouse et al., 2018), InterProScan5(Mitchell et al., 2019), Eggnog (emapper v2.1.9)(Cantalapiedra et al., 2021; Ferrés and Iraola, 2018; Huerta-Cepas et al., 2019), and antiSMASH (v6)(Medema et al., 2011) databases. Annotation files were concatenated with separation by 1kb of in-frame stop codons, then visualized and analyzed with Artemis software and DNAPlotter (Carver et al., 2012). 3.3.8 Phylogenetic analysis The phylogeny of B. rouxiae PM34 was determined by constructing a maximum likelihood phylogenetic tree of MCM7 genes and the General Time Reversible model (GTR) on Mega software (version 11.0.11). The MCM7 gene data set used to place B. rouxiae PM34 on Ceratocystidaceae phylogenetic tree were obtained from a previously published dataset (Nel et al., 2018; Wingfield et al., 2018). 3.3.9 Confirmation of pathogenicity To satisfy Koch’s postulates, B. rouxiae PM34 was tested for pathogenicity on Cannabis plants. Nodal segments of the genotype “Sour Diesel” were propagated in tissue culture prior to ex-vitro acclimation and rooting in a custom substrate made of 4 parts peat moss, 2 parts sand, 1 part vermiculite and ½ part Turfex. For consistency with cultural acclimation protocols in the 57 commercial setting, this substrate was not autoclaved. Once established, the plant roots were cleaned by washing in water and then cut to equal lengths immediately before treatment. The treatment plants were soaked in 500 mL of 1.17E+07 spore solution of B. rouxiae PM34 for 20 minutes. Briefly, spore solutions were created by pipetting 1.3 mL 0.1% sterile Tween20 on a fungal agar plate and gently scraping spores before pipetting concentrated solution to an Eppendorf tube. This solution was diluted to make spore solution. Control plants were soaked in sterile RO water. Treated and control plants were re-potted in autoclaved vermiculite and grown under white fluorescent light with a photoperiod of 18 hours of light and 6 hours of dark. Plants were watered every 3-4 days and fertilized weekly with Plant-Prod MJ Grow supplemented with the Diablo Monster-cal. The nutrient solutions were adjusted to a pH range of 5.8-6.2. Pathogenicity was assessed after 5 and 14 days after inoculation. Infection was confirmed through visual assessment, light microscopy, and culturing. This trial was repeated twice with three biological replicates for each treatment. All research was conducted in a Health Canada licensed facility (LIC-0IW2D2L5JY-2019). 58 3.4 Results Figure 3. 1. Black rot of aeroponic Cannabis root systems. A and B, High THC-containing ‘Nuken’ strain Cannabis plants rapidly displayed symptoms of root rot; images were taken 1 week apart. C, Light microscope image of root wash at 200× magnification. D, Detail of ‘Nuken’ strain infected root. E, Root pressed on carrot agar showing black fungal growth. 3.4.1 Culturing microorganisms from root tissue Cannabis plants displayed extensive black growth of a suspected pathogen on the surface of aeroponically-grown roots (Figure 3.1). Further inspection showed an abundance of mycelia with dark, septate conidia (chlamydospores). Following culture of root segments on PDA, fungi with four distinct morphologies were labeled PM30-33 and were subcultured and identified according to species using light microscopy and ITS sequencing (Figure 3.2). None of these four isolates displayed the distinctive segmented pigmented aleuriospores (chlamydospores) observed on infected root tissue. Therefore, an additional medium made from freshly ground carrots was tested and black 59 mycelia were observed growing from the pressed Cannabis root tissue. This black fungus was subcultured, isolated, and found to produce dark, septate conidia (chlamydospores) (Figure 3.2). Sequencing of the ITS region of this isolate (PM34) identified it as a member of the Berkeleyomyces genus. Figure 3. 2. Morphology of fungal isolates from aeroponically grown Cannabis. A and B, Fusarium spp. isolate PM30. C and D, Fusarium spp. isolate PM31. E and F, Trichoderma spp. isolate PM32. G and H, Fusarium spp. isolate PM33. I and J, Berkeleyomyces spp. isolate PM34. A, C, E, and G, Colonies on potato dextrose agar. I, Carrot agar. B, D, F, H, and J, Whole mount light microscopy (200× magnification) of fungi 3.4.2 Hybrid genome sequencing, assembly, and annotation All of the isolates except for Berkeleyomyces PM34 represented common pathogens of Cannabis, for which abundant genome sequence information is available. Because of the lack of equivalent resources for Berkeleyomyces spp., and to aid in further characterization and classification of isolated B. rouxiae PM34, we conducted whole-genome sequencing using Illumina and Oxford Nanopore Technology (ONT) platforms. Approximately1.08 gigabases (Gb) of raw sequence were pooled from two runs on the ONT Flongle. A total of 781.1 megabases 60 (Mb) were successfully base-called. The same genomic DNA extract produced 160,979,852 clean Illumina reads (paired end PE150), yielding 24.15 Gb of sequence. Sequence reads are available at NCBI under Bioproject accession PRJNA898645. We applied nine different assembly methods to the ONT and Illumina reads and compared the results based on contiguity (N50) and BUSCO scores (Supplemental Figure S3, Supplemental Table S1). Based on these metrics, the highest-quality final assembly was obtained using Canu (Koren et al., 2017) and POLCA (Zimin and Salzberg, 2020) error correction with short reads. This assembly contained 28,194,266 bp in 404 contigs, with an N50 of 267 kb, and a Consensus QV score of 30.20 (99.904%). BUSCO scores using sordariomycetes_odb10 database for this assembly were: 90.0% complete and single copy, 0.9% complete and duplicated, 1.8% fragmented, and 7.3% missing. Genome sequence depth with ONT long reads was 22.51x, and POLCA error correction was performed with 856.56x short reads. K-mer spectrum plots for this assembly were also created using Mercury (Supplemental Figure S4). The plots of the k-mers in both the raw reads and in the assembly shows a large number of unincorporated reads at low multiplicity, followed by a very large, distinct peak centered around 250 k-mer multiplicity, corresponding to 1-copy k-mers, with no other peaks at higher multiplicities. This is as would be expected for a high read-depth assembly based on the largely hyphal, haploid tissue used for the DNA extraction. There were also very few assembly-only k-mers (<1%), which is consistent with a low assembly error rate. This genome was submitted to Genbank under the accession number JAOUZH000000000. Genome annotation of the POLCA polished Canu assembly was done using the Funannotate annotation pipeline. After scaffolds were cleaned and sorted, the 401 remaining contigs were subjected to soft masking using TANTAN (Frith, 2011), which resulted in 1,407,716 bp being masked, or 5% of the assembly. 61 Ab initio gene prediction found a total of 7848 gene models; one gene model that was shorter than the minimum threshold, and 887 transposable elements were filtered leaving a total of 6960 predicted protein-coding genes. 488 tRNA genes were identified with tRNAscan-SE (v.2.0.9), yielding a final count of 7448 gene models. Functional annotation through the Funannotate pipeline added 59,477 valid annotations to the Genbank file, details can be seen in Table 3.1 and Figure 3.3. 62 Table 3. 1. Summary of annotation sources. Program/Database Annotations Added EggNog and COG 13,771 Pfam 8,179 Diamond and UniProt DB version 2022_02 689 valid gene/product annotations from 900 total MEROPS 261 CAZYme 204 BUSCO2 and dikarya_obd9 1270(C:96.0%[S:95.0%, D:1.0%],F:2.1%,M:1.9%,n:1312) InterProScan and InterPro 5668 antiSMASK and smCOG 12 clusters, 23 biosynthetic enzymes 63 Figure 3. 3. DNA plotter chart of Berkeleyomyces rouxiae PM34 annotation. The genome annotation file from the Funannotate pipeline was visualized using Artemis DNA plotter to display the distribution of coding sequences, tRNAs, assembly gaps, and GC% skew (Carver et al. 2012). 64 Table 3. 2. Distribution of genes in COG functional annotation categories COG category Annotated genes (count) A: RNA processing and modification 298 B: Chromatin structure and dynamics 107 C: Energy production and conversion 213 D: Cell cycle control, cell division, chromosome partitioning 165 E: Amino acid transport and metabolism 319 F: Nucleotide transport and metabolism 96 G: Carbohydrate transport and metabolism 306 H: Coenzyme transport and metabolism 162 I: Lipid transport and metabolism 206 J: Translation, ribosomal structure and biogenesis 358 K: Transcription 300 L: Replication, recombination and repair 212 M: Cell wall/membrane/envelope biogenesis 50 N: Cell motility 6 O: Posttranslational modification, protein turnover, chaperones 509 P: Inorganic ion transport and metabolism 163 Q: Secondary metabolites biosynthesis, transport and catabolism 172 S: Function unknown 1684 3.4.3 Carbohydrate metabolism gene analysis The Funannotate genome annotation pipeline predicted 204 putative Carbohydrate Active Enzyme (CAZymes) annotations (Cantarel et al., 2009)(Table 3.1). Among these CAZymes, there were 96 glycoside hydrolases (GH), 53 glycosyltransferases (GT), 17 carbohydrate esterases (CE), 5 polysaccharide lyases (PL), 30 auxiliary activity (AA), and 3 carbohydrate-binding module (CBM) family genes. Additionally, 306 coding sequencings were 65 found to have the COG function for carbohydrate transport and metabolism (G) (Table 3.2). Carbohydrate metabolism genes are important for plant pathogenic fungi-host infection (Aylward et al., 2017; Klosterman et al., 2011). 12 genes were found to be predicted virulence factors by InterProSan and all were involved in carbohydrate metabolism with the majority being involved in pectin degradation. (Table 3.3). Table 3. 3. Predicted virulence factors in the B. rouxiae PM34 genome. GeneID Contig CAZyme Enzyme Activity InterPro Activity FUN_000346 scaffold_3 GH28 polygalacturonase virulence factor FUN_002406 scaffold_17 GH55 exo-β-1,3-glucanase virulence factor FUN_002457 scaffold_18 PL3 pectate lyase virulence factor FUN_002547 scaffold_19 GH55 exo-β-1,3-glucanase virulence factor FUN_005028 scaffold_53 PL1 pectate lyase virulence factor FUN_005138 scaffold_56 CE8 pectin methylesterase virulence factor FUN_005139 scaffold_56 pectinesterase virulence factor FUN_006333 scaffold_88 PL1 pectate lyase virulence factor FUN_006496 scaffold_97 PL1 pectate lyase virulence factor FUN_007306 scaffold_283 CE8 pectin methylesterase virulence factor FUN_007308 scaffold_283 CE8 pectin methylesterase virulence factor 3.4.4 Fungal taxonomy To more precisely classify the taxonomy of Berkeleyomyces PM34, the minichromosome maintenance complex component 7 (MCM7) gene was extracted from the annotation file and a phylogenetic tree was constructed using a previously gathered dataset (Nel et al., 2018; 66 Wingfield et al., 2018). Placing Berkeleyomyces PM34 on this Ceratocystidaceae phylogenetic tree identifies PM34 species as B. rouxiae (Figure 3.4). Figure 3. 4. Maximum likelihood phylogenetic tree of MCM7 gene. MCM7 genes were aligned using the MUSCLE algorithm prior to construction of a maximum likelihood phylogenetic tree of MCM7 genes and the General Time Reversible model (GTR) on Mega software (version 11.0.11). 3.4.5 Cannabis pathogenicity To satisfy Koch’s postulates and confirm the pathogenicity of this B. rouxiae isolate, post-tissue culture Cannabis plants were inoculated with PM34 and grown for 2 weeks on 67 sterilized vermiculite. Beginning 5 days post-inoculation (dpi), plants showed visible fungal growth on their crowns (Figure 3.5). Visible fungal growth from infected crowns was scraped and examined under a compound light microscope at 200x magnification and additionally plated on carrot agar. Chlamydospores observed by light microscopy were indistinguishable from our cultured B. rouxiae PM34. After 14 dpi, plants treated with a B. rouxiae PM34 spore solution displayed stunted root growth, discolored lesions on roots, and blackened crowns (Figure 3.6). Untreated control plants displayed healthy root architecture, robust root growth and vibrant white colour. Crowns of control plants displayed no black discoloration. These results were consistent across two trials (Supplemental Figure S5) Figure 3. 5. Confirmation of pathogenicity for Berkeleyomyces rouxiae PM34. A and B, Crowns and roots of B. rouxiae PM34-infected Cannabis plants began to show visible dark mycelia growth matching colony morphology seen on carrot agar and C, root discoloration 5 days post inoculation. D, Light microscope of infected root wash at 200× magnification 68 Figure 3. 6. Effect of PM34 inoculation on root growth. A, B, and C, Cannabis plants (control) and D, E, and F, 14 days post infection with Berkeleyomyces rouxiae PM34. 3.5 Discussion Black root rot was first described in 1850 (Berkeley and Broome, 1850) and has had a convoluted taxonomic history driven by classical morphological methods (Nel et al., 2019). The advent of genetic technologies has changed the field of fungal taxonomy and shifted our understanding of fungal lineages. Although previously known as Thielaviopsis, DNA based phylogeny has demonstrated that black root rot is actually caused by two cryptic sister species 69 in the recently described Berkeleyomyces genus, which is distinct from other genera in the Ceratocystidaceae family (Nel et al., 2018). Black root rot pathogens B. rouxiae and B. basicola are commonly identified by their dark, segmented chlamydospores and have been reported to infect over 170 cultivated plant species (Long et al., 2022; Nel et al., 2019; Pereg, 2013). This pathogen is particularly devastating to cotton and has even been declared a pandemic in Australia (Pereg, 2013). These asexually reproducing fungi are hemibiotrophs that invade living growth (Mims et al., 2000). This causes plant roots to form black discoloration and impairs water and nutrient uptake causing eventual plant death (Nehl et al., 2004). The high THC-containing Cannabis plants that motivated our study showed visible root discoloration (Fig. 1) and tested negative using all available Cannabis pathogen tests. The detection of Berkeleyomyces black rot in Cannabis, which at the time of these experiments, was not a known Cannabis pathogen, highlights the need for continued research to close the knowledge gap that exists between this crop of renewed interest and other cultivated plants. Species of Fusarium and Trichoderma were also cultured from the diseased Cannabis roots (Figure 3.2), however, Fusarium species are already well known pathogens of Cannabis, and Trichoderma is a widely used biocontrol in Cannabis (Punja, 2018, 2020a, 2020b, 2021; Punja et al., 2018, 2019; Punja and Ni, 2021). Notably, B. rouxiae PM34 was not culturable on PDA and grew only on carrot agar made from fresh carrots commonly used to culture plant pathogens such as Phytophthora (Medina and Platt, 1999). Historically, fungi have been classified morphologically, but the convergent evolution of similar body plans creates challenges for accurate taxonomic identifications based on morphology alone (Berbee et al., 2017; Lücking et al., 2021). DNA barcode-based methods for fungal taxonomy, frequently based on the ribosomal internal transcribed spacer (ITS) sequence, have resulted in extensive revisions of the fungal tree of life (Lücking et al., 2021). Yet even these barcode-based classification methods have limitations (Lücking et al., 2020). Specifically, 70 different communities of researchers rely on different DNA-barcodes to classify fungi (Lücking et al., 2021). This highlights the need for increasing the number of fungal genomes available to systematists (Grigoriev et al., 2014; James et al., 2020). The novelty of black root rot infection of high-THC Cannabis and the lack of an available B. rouxiae genome prompted whole genome sequencing. Fragmented genomes can lead to fragmented open reading frames and can confound comparative genomic analysis (Klassen and Currie, 2012). Hybrid genome sequencing has become a relatively inexpensive way to improve assembly contiguity. Extraction of Berkeleyomyces DNA was challenging and several protocols were attempted with negligible DNA yields before success was found using a specific CTAB HMW DNA extraction protocol (https://dx.doi.org/10.17504/protocols.io.bwr8pd9w) (Jones, 2021). Sequencing B. rouxiae yielded a genome size of 28.2 Mbp with an N50 of 267 kbp. This genome size is similar to the estimated size of B. basicola, 25.4 Mbp, and within the expected range for species in the Ceratocystidaceae family (Wingfield et al., 2016, 2017, 2018). The highest quality genome was obtained using CANU (Koren et al., 2017) followed by error correction with POLCA (Zimin and Salzberg, 2020). Of note, using MASURCA, 1 Gbp of long-read data improved assembly contiguity 3.73-fold compared to an assembly made with short-reads alone (Zimin et al., 2013)(Supplemental Figure S3). This amount of long-read data can be obtained at very low cost (90$USD) using a single Nanopore Flongle device. Genome annotation done by the Funannotate pipeline found 7848 gene models and a total of 6960 protein-coding genes. This annotation should assist in comparative genome analysis with other Ceratocystidaceae fungi and give insight into the evolution and pathogenicity of B. rouxiae. Genes involved in host infection such as cell surface proteins and cell wall degrading enzymes are an important element in a pathogen’s life cycle, and fungi with pathogenic lifecycles are known to have expansion in carbohydrate metabolism genes (Aylward et al., 2017; Klosterman et al., 2011). Specifically, an increase in pectinolytic enzymes is associated 71 with pathogens of dicot plants (Olson et al., 2012), and pathogens that infect a wide variety of plants encode a greater diversity of carbohydrate degrading enzymes (Aylward et al., 2017; Islam et al., 2012). The genome of B. rouxiae PM34 encodes 204 putative Carbohydrate Active Enzymes (CAZymes) as predicted by the Funannotate pipeline (Table 3.1)(Cantarel et al., 2009). Among these CAZymes, there were 96 glycoside hydrolases (GH), 53 glycosyltransferases (GT), 17 carbohydrate esterases (CE), 5 polysaccharide lyases (PL1, PL3 and PL4). Of importance, this pathogen encoded polygalacturonase (family GH28) which is critical for pectin degradation in fungal plant pathogens (Zhao et al., 2014). Additionally, pathogenic fungi encode significantly more PL1 enzymes than saprophytic fungi (Zhao et al., 2014). The B. rouxiae PM34 genome encoded 3 PL1 genes, in line with the median number encoded in plant pathogenic fungi (Zhao et al., 2014). Fungal taxonomy via DNA barcoding uses a number of gene markers depending on the community of study (Lücking et al., 2020, 2021). The most common marker for fungal DNA-barcode based taxonomy is the ITS region of the rRNA genes, however, the ITS region is not informative for species in the Berkeleyomyces genus (Nel et al., 2019). Therefore, the MCM7 gene was extracted from the annotation and used to place B. rouxiae on the Ceratocystidaceae family tree using a previously prepared dataset from researchers who study black root rot (Nel et al., 2018; Wingfield et al., 2018). The MCM7 gene had 99.51% BLAST similarity to isolate CMW5472 from ground nuts (GenBank Accession: MF967080) (Nel et al., 2018). The MCM7 from B. rouxiae PM34 has been submitted to GenBank under accession number BankIt2664086 Berkeleyomyces OQ260012. Black root rot infection of high THC-type Cannabis plants has not been previously documented. Therefore, Cannabis plants from axenic tissue culture, in the vegetative stage of growth, were infected with B. rouxiae PM34. This method was based on a recent method used to confirm black root rot pathogenicity in lettuce (Nakane et al., 2019). Infected 72 plants displayed visible root discoloration and diminished root growth compared to plants soaked in sterile water as a control (Figure 3.6). Fungal growth was also visible on Cannabis crowns (Figure 3.5). Microscopic analysis confirmed the presence of black root rot identified by distinctive dark-coloured segmented chlamydospores (Figure 3.5). This study provides the first whole genome sequence of the agriculturally important multi-host black root pathogen B. rouxiae. Along with the recently released genome of its cryptic sister species Berkeleyomyces basicola (Wingfield et al., 2018), this genome should assist geneticists, mycologists and plant pathologists in understanding this globally important pathogen and give insight into the distinction between these two species. Additionally, we document for the first-time black root rot in high THC-containing Cannabis in Canada, alerting growers, governments, and plant pathologists of this potentially devastating fungal pathogen and the utility of DNA sequencing in pathogen detection. 73 Chapter 4. Effect of Seed and Soil bacteria on Cannabis Seedling Microbiomes. 4.1 Introduction The co-evolution of plants and microorganisms has resulted in complex associations that play critical roles their lifecycles. As a mechanism to adapt to terrestrial biomes, nature has selected for plants that host and maintain beneficial microorganisms that provide selective advantages against biotic and abiotic stresses. Plants themselves are ecosystems, and our understanding of the plant “holobiome” has rapidly developed within recent decades. Science is beginning to unravel the link between microbial ecology and host-plant physiology (Van and Dufresne, 2015), however, these interactions are complex and our current knowledge is limited. There is considerable economic incentive to understand how microbial ecology can affect plant health. Plant pathogenic microorganisms cause over $220 billion in annual crop loss (Chakraborty and Newton, 2011). Plant microbial communities contribute some level of resistance to plant pathogens, and some distinctions have been found between susceptible and resistant cultivars (Wolfgang et al., 2020). In fact, microbiome transfer from pathogen-resistant cultivars to susceptible cultivars can provide disease tolerance (Choi et al., 2020; Kwak et al., 2018). Beneficial plant bacteria and fungi have been widely used as inoculants in agriculture. These “biologicals” are a growing market of around $12 billion in 2022 (Fortune Business Insights, 2022). Endophytes are microorganisms that live inside plant tissue, and due to their intimate associations with their hosts, they are a major research subject in plant microbiome science (Hardoim et al., 2008, 2012, 2015; Johnston-Monje and Raizada, 2011b; Truyens et al., 2015). Endophytes can promote plant growth by providing nutrients, increasing nutrient uptake, modulating and secreting phytohormones, and defending against plant-pathogens (Hu et al., 2003; Johnston-Monje and Raizada, 2011b; Mousa et al., 2016; Shehata et al., 2017). Plants 74 appear to select endophytes, especially during seedling emergence (Barret et al., 2015). Plant endosphere (and microbiomes) can be derived from both the environment and from the mother plant, although the relative contribution of each is not always clear (Aleklett and Hart, 2013). A significant portion of endophyte species in juvenile maize plants are seed derived and inherited from their mother plant (Johnston-Monje et al., 2014, 2016; Aleklett and Hart, 2013) Some microbiota appear to be widely conserved in angiosperms, independent of soil environments, and even when grown on sterile substrates. This suggest that at least some plant-associated microbes are seed derived (Johnston-Monje et al., 2014, 2021). Moreover, related plant species appear to have a “core” microbiome of bacterial taxa that are shared between them (Sánchez-López et al., 2018). Recent research suggests that plants select specific bacteria that are vectored across generations to protect seedlings against environmental stresses (Abdelfattah et al., 2021; Pitzschke, 2016; Rochefort et al., 2021; Shahzad et al., 2018; Truyens et al., 2015). This phenomenon of microbial inheritance in Cannabis was recently demonstrated for the first time (Dumigan and Deyholos, 2022). This study showed that hemp and marijuana cultivars grown to seed at several locations in western Canada vectored bioactive and antifungal endophytes to the next generation of seedlings. Furthermore, specific bacteria from the class Bacilli were common to all Cannabis genotypes examined. However, this study was limited to culturable microorganisms, and so the unculturable seed-borne seedling endophyte communities of Cannabis were not identified. Unculturable bacteria are expected to make up a high proportion of most microbiomes (Steen et al., 2019). Therefore, the majority of the seed-borne bacteria in Cannabis seedlings are currently unknown. Additionally, the previous study of Cannabis seedling endophytes was conducted in axenic conditions, and so the effect of soil on community structure was not considered. Plants microbiomes are dependent on both environmental (soil) and inherited seed-borne inoculum to assemble communities throughout their lifecycle. The importance of seed- 75 borne microorganisms have been historically overlooked (Barret et al., 2016), but more recent research has suggested they play a critical role in plant health especially in regard to juvenile plants (Johnston-Monje et al., 2016, 2021; Johnston-Monje and Raizada, 2011a; Truyens et al., 2015). The importance of soil-borne or environmental inoculum to plants as a whole has been well studied for decades. Both seed and soil can vector pathogenic and beneficial microorganisms to plants, and the relative contributions, importance, and functions of these is very much unknown in Cannabis. Many better-studied plant systems have shown core, seed-borne endophyte communities that are maintained across environmental conditions and between genotypes (Johnston-Monje et al., 2014; Truyens et al., 2015; Walitang et al., 2018). Therefore, the hypothesis of the present study is that Cannabis seedlings will likewise host a conserved element of their endosphere microbiome that is seed-borne, conserved between genotypes, and will be consistent across soil conditions. This report describes an experiment to determine the contributions of both seed and soil inoculum to Cannabis seedling endospheres, and how altering plant genotype, soil type, and soil sterilization affects endophyte community assembly in Cannabis seedlings. 4.2 Methods 4.2.1 Source of Seeds Cannabis seeds were obtained from three locations across western Canada. Drug-type accession BC Big Bud was purchased from a commercial supplier in Qualicum Beach, British Columbia. The hemp accessions Katani and X59 were obtained from InnoTech Alberta. Canda seeds were obtained from Triple S Seeds LTD in Grandview, Manitoba. All research was conducted in a Health Canada licensed facility (LIC-0IW2D2L5JY-2019). The three cannabis 76 genotypes were selected because of their current use in Canadian production, the availability of seed, and to select both drug and industrial type Cannabis 4.2.2 Source of Soil and Characteristics “Innotech” topsoil was collected from a field at InnoTech Alberta (Vegreville, AB) (53.504377, -112.088324) in which the hemp cultivar Silesia was been grown. “Okanagan” soil was harvested from Westpoint Vineyard in (Kelowna, BC) (49.821298, -119.470140) growing Chardonnay’ (Vitis vinifera L.) wine grape Clone 99 (121) on S04 (V. vinifera) rootstock. These soils were selected to have balance between soil that has grown cannabis and soil that has not grown cannabis. To destroy soil microorganisms, a small volume of each soil type was sealed in 2 L glass mason jars and sterilized by two rounds of autoclave sterilization for 55 minutes at 121ºC. These rounds of autoclaving were 24 hours apart. 4.2.3 Experiment Set-Up BC Big Bud, Katani, and X59 seeds were planted in 5 cm of each soil type in autoclave sterilized glass test tubes sealed with cotton balls. Both soil types were sterilized in an autoclave. An unsterilized control of each soil type was also used. Seedlings were germinated and grown under white-fluorescent light for 7 days prior to removal from their soil environment, which was followed by surface sterilization. Two independent trials were conducted each with four biological replicates (seedlings) per genotype. 4.2.4 Surface Sterilization After removal from soil, seedlings were first washed with filter-sterilized 0.1% Triton-X for 10 min, and then 3% sodium hypochlorite for 20 min. Samples were drained and rinsed with autoclaved, deionized water, followed by treatment with 70% ethanol for 10 minutes. Samples 77 were rinsed 5 times to remove ethanol before being frozen for DNA isolation. An aliquot of the final rinse was plated on R2A media to test sterility. DNA from whole 7-day old seedlings was isolated using the Qiagen DNeasy Plant Pro kit according to the manufacturer’s instructions and using a Powerlyser24 homogenizer at 1500 rpm for 1 min, then 2000 rpm for 1 min. 4.2.5 DNA Isolation Frozen and surface sterilized seedling DNA was isolated using the Qiagen DNeasy Plant Pro kit according to manufacturer instructions with Powerlyser24 homogenizer at 1500r pm for 1 minute, then 2000 rpm for 1 additional minute. 4.2.6 16S rRNA Amplicon Sequencing The V4 hypervariable region of the 16S rRNA gene was amplified using primers barcoded primers with Illumina adapters: 515FB: GTGYCAGCMGCCGCGGTAA, 806RB: GGACTACNVGGGTWTCTAAT. 1 μM pPNA and 1 μM mPNA (PNA Bio Inc, Newbury Park, CA) were included in the PCR to inhibit amplification of plant chloroplast and mitochondria sequences. The PCR cycle was as follows: 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 50 s, and then extended at 72 °C for 10 min. Amplicons were visualized on a 1% TAE agarose gel. Amplicons were cleaned using Mag-Bind® TotalPure NGS magnetic beads (Omega), followed by indexing PCR with i7 and i5 primers. PCR cycle was as follows: 95°C for 3 minutes followed by 8 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, followed by 72°C for 5 minutes. Indexed amplicon libraries were bead-cleaned a second time before quantification using a Nanodrop spectrophotometer. Equimolar samples were then pooled and sent to Genome Quebec for DNA sequencing using Illumina MiSeq Reagent Kit v3 (2x300 cycles). 78 4.2.7 Bioinformatics Demultiplexed reads were analyzed using the qiime2-2022.2 pipeline running on a CentOS Linux 7 server (8 cores, 124 GB RAM, Python: 3.8.12)(Evan Bolyen et al., 2019). Paired-end reads were trimmed and truncated to maximize sequence quality, dereplicated and denoised into amplicon sequence variants (ASVs) using DADA2 (Callahan et al., 2016). The resulting feature table was taxonomically classified using the q2-feature-classifier and the machine learning based classification method scikit-learn trained against the Green Genes Database and with primers 515F and 806R (gg-13-8-99-515-806-nb-classifier.qza) (Caporaso et al., 2011; DeSantis et al., 2006). Chloroplast, mitochondria, and taxa without phylum-level classification were removed using qiime2 filter-seq. Sequences were aligned using the MAFFT plugin and phylogenetic trees were conducted using the FastTree plugin (Katoh et al.; Price et al., 2010). Alpha and beta diversity was assessed using qiime2 diversity core-metrics-phylogenetic plugin following sample rarefaction to an even sampling depth. Significance was tested using Kruskal-Wallis test. Principle coordinate analyses were conducted using qiime2 with Bray-Curtis diversity metrics. PERMANOVA and PERMDISP were used to assess significance in the differences in seed-borne cannabis seedling endosphere microbiomes across treatments (Anderson, 2017; Anderson et al., 2006). ANCOM differential abundance testing was conducted using Qiime2 rarefied to minimum frequency of 50 (--p-min-frequency 50). Significance was tested for differentially abundant bacteria using qiime2 by running a series of pairwise tests where the W statistic is a count of the number of times the null hypothesis that there is no difference was rejected. PiCRUST2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to infer functional genes in treatments based on 16S marker sequences and the corresponding genomic data for the respective amplicon sequence variants (ASVs)(Douglas et al., 2013). Qiime2 artifacts were converted to publication quality plots using Qiime2R plugin and RStudio version 4.1.2. 79 4.3 Results 4.3.1 Effect of Seed and Soil on Cannabis Seedling Microbiomes. To investigate the relative contributions of both seed and soil to the microbiota of the cannabis seedling endosphere, we profiled the microbiomes of seedlings from three different genotypes under four different soil conditions. The cannabis genotypes that were selected for testing were: BC Big Bud (indoor grown drug-type); X59 (field-grown hemp); and Katani (field-grown hemp). These genotypes were selected to represent diverse germplasm. Soil was collected from the surface of two diverse sources: an eluviated eutric brunisol from a vineyard in the Okanagan valley (BC, Canada), and black chernozem from a hemp field at a research station in Vegreville (AB, Canada). Non-sterilized and sterilized (autoclaved) soils from each location were used in a full multifactorial design (n=4, for each of the twelve combinations of seed genotype x soil source x sterilization status; replicated twice for a total of 96 seedlings)(Figure 4.1). The soil microbiome was not analyzed in this experiment as the interest was in understanding how seed and soil factors influence the endosphere microbiome of juvenile Cannabis plants. 80 Figure 4. 1. Experimental Design. (A) Three Cannabis genotypes where each grown in four soil treatments (n=4) for two independent trials. (B) Plants were grown for 7 days prior to (C) harvesting, surface sterilization, DNA isolation, and amplicon metagenomic sequencing. Amplicon sequencing yielded 15.1 million reads with an average quality score of 32.1 across 96 samples; again, chloroplast ASVs were removed from further analysis. Metagenomic analysis of this experiment showed both seed and soil inoculum have an effect on microbial composition of 7-day old Cannabis seedling endospheres (Figure 4.2, Figure 4.3). Additionally, abiotic soil characteristics have an effect in that the same Cannabis genotype grown in different sterilized soil displayed differences in microbiomes. Of note, even with large fluctuations in relative abundance, there was a core microbiome of bacterial endophytes conserved across genotypes and soil conditions. Among all reads that were classified, Gammaproteobacteria and 81 Bacilli were the two most abundant taxa and were found inall genotypes and soil treatments. Within genotypes, the abundance of Gammaproteobacteria and Bacilli appeared to be inversely correlated across soil treatments. Additionally, Betaproteobacteria were widespread, although at generally lower abundances than either Gammaproteobacteria or Bacilli (Figure 4.2). Figure 4. 2. Taxonomic Bar Plot for Cannabis Seedling Endospheres grown in Different Soil Conditions. Phylum level ASVs (amplicon sequence variants) from 7-day old Cannabis seedling endospheres germinated in different soil conditions. 82 Figure 4. 3. Heatmap of Taxonomies for Cannabis Seedling Endospheres. Phylum level ASVs (amplicon sequence variants) from 7-day old Cannabis seedling endospheres germinated in different soil conditions. 4.3.2 Effect of Seed and Soil on Cannabis Seedling Endosphere on Alpha Diversity When pooling genotypes across soil types, untreated soil had significantly higher alpha diversity as compared to autoclave-sterilized soil (p=0.03730) (Figure 4.4C), suggesting the soil inoculum increased microbial diversity in seedling endospheres. When looking at genotypes pooled across all soil treatments, BC Big Bud had significantly higher alpha diversity compared to both Katani and X59, which were similar in endosphere microbiome diversity (p= 0.00333, 0.01516 respectively) (Figure 4.4A). Alpha diversity between Innotech and Okanagan soil was not significantly different when pooling genotypes and soil treatments (p= 0.19651) (Figure 4.4B). Growing Katani seedlings in sterilized Okanagan soil resulted in a significant drop in Shannon alpha diversity (p=0.0016) when compared to untreated soil from either Okanagan or 83 Innotech, which indicated this genotype obtained a more diverse microbiome from soil as opposed to strictly seed-borne inoculum (Figure 4.4D, Supplemental Table S2). Other genotypes of Cannabis did not show significantly different changes in alpha diversity across sterilized and unsterilized soil of the same type (Figure 4.4D). Figure 4.4 Alpha Diversity of Cannabis Genotypes Grown in Different Soil Conditions. Shannon diversity Kruskal-Wallis (pairwise). (A) Katani and X59 had significantly lower alpha diversity when genotypes are pooled across soil treatments (p= 0.00333, 0.01516 respectively). (B) Innotech and Okanagan soil showed no significant difference in alpha-diversity when pooling genotypes and soil sterilization treatments (p= 0.19651). (C) Soil sterilization resulted in a significant drop in alpha-diversity when pooling genotypes and soils (p=0.03730). (D) Investigating effect of soil treatment within genotypes showed only Katani plants grown in Okanagan soil saw a reduction in alpha-diversity from soil-sterilization (p=0.001629). 84 4.3.3 Effect of Seed and Soil on Cannabis Seedling Endosphere on Beta Diversity Soil (Okanagan and Innotech), soil treatment (sterilized and untreated), and Cannabis genotype all had an effect on the bacterial beta diversity of juvenile Cannabis seeding endospheres as measured by the Bray-Curtis distance (Figure 4.5, Supplementary Table S2). An interesting observation in this experiment was Beta diversity measured using Bray-Curtis of all genotypes grown in untreated Innotech soil did not significantly differ between genotypes (p=0.189, 0.14, 0.124) (Figure 4.5, Supplementary Table S2). This indicates that soil microorganisms strongly influenced endosphere microbiome structure and masked genotype effects on microbial diversity. However, in Okanagan soil, Bray-Curtis distance was significantly different between genotypes (p=0.032, 0.002, 0.001) (Figure 4.5, Supplementary Table S2). Different genotypes grown in the sterilized soil from the same source had significantly different beta-diversity, showing a genotype effect on microbial diversity in Cannabis seedling endospheres when soil inoculum was absent (PERMANOVA p= 0.001, PERMDISP p= 0.727) (Figure 4.5, Supplementary Table S2). Soil autoclaving had a significant effect on the beta diversity of Cannabis seedling endospheres across all three genotypes, demonstrating the importance of soil bacteria in shaping juvenile Cannabis endospheres. Additionally, the same Cannabis genotype grown in different autoclaved soils were also significantly different from each other indication abiotic factors and soil characteristics are influencing endosphere microbiome assembly (p= 0.005, 0.004, 0.006) (Figure 4.5, Supplementary Table S2). 85 Figure 4. 5. Principle Coordinate Analysis of Bray-Curtis Distances in Experiment 2. A) All treatments individually coloured on PCOA. B) Soil Treatment coloured to show clustering of samples withing soil groups. 86 4.3.4 Differential Abundance Testing with ANCOM Having observed that factors derived from both seed and soil can affect the diversity of microbiota in the cannabis seedling endosphere, we sought to describe the effects of cannabis genotype, soil source, and soil sterilization on the abundance of specific bacterial taxa. When the effect of soil sterilization is was tested across all genotypes and soil sources, five taxonomic classes of bacteria showed significant differential abundance. Generally, cannabis seedlings planted in non-sterilized soil had endospheres that were enriched with Gammaproteobacteria [5.24x fold, W= 72], Flavobacteriia [47.21x fold, W=70], and Alphaproteobacteria [7.69x fold, W=66], as compared to sterilized soil. Conversely, endospheres of cannabis seedlings grown in sterilized soil were enriched with Betaproteobacteria [1.41x fold, W=65] and non-significantly with Bacilli (49x fold, W=5] (Supplemental File S2). Each cannabis genotype was also tested individually in terms of the effect of soil sterilization and soil source on the abundance of its endosphere taxa. This analysis can help to identify genotype-specific effects on the endosphere microbiome. BC Big Bud grown in non-sterilized Okanagan soil had far more Betaproteobacteria, Gammaproteobacteria, Flavobacteriia, and Sphingobacteriia than BC Big Bud grown in sterilized Okanagan soil [12.87x, W=50, 40.39x, W=52, 47.22, W=52, 8.68x, W=50 respectively] BC Big Bud grown in non-sterilized Innotech soil had far more Gammaproteobacteria as compared to sterilized Innotech soil [7.73x, W=52] (Supplemental File S2). The seedling endosphere of the Katani genotype grown in non-sterilized Okanagan soil had significantly more Actinobacteria, Gammaproteobacteria, Flavobacteriia, and Sphingobacteriia than Katani grown in sterile Okanagan soil [1.51x, W=42, 5.24x, W=41, 9.17x W=40, 100.48x, W=39, respectively]. Sterilized Okanagan soil from which viable microbes had been removed resulted in significantly more abundance of Bacilli [1.42x, W=41] Endospheres of Katani 87 seedlings grown in non-sterilized Innotech soil were significantly enriched in Actinobacteria, and Gammaproteobacteria when compared to seedlings of the same genotype grown in sterilized Innotech soil [41.23x W=42, 8.99x W=41, respectively]. Sterilizing soil to remove environmental microbes resulted in significantly greater relative abundance of Bacilli [13.10x, W=41] (Supplemental File S2). The seedling endosphere of the X59 genotype grown in non-sterilized Innotech soil had more Betaproteobacteria, Gammaproteobacteria, and Flavobacteriia than X59 grown in sterilized Innotech soil [29.23x, W=45, 10.31x, W=42, 901x W=46, respectively]. X59 seedlings grown in non-sterilized Okanagan soil were enriched in these same three taxonomies when compared to plants grown in sterilized Okanagan soil [7.78x, W=45, 11.26x, W=42, 986x, W=46]. X59 seedlings grown in sterile Okanagan soil were significantly enriched in Bacilli when compared rare taxonomies were filtered [4.39x, W=6] (Supplemental File S2). 4.3.5 Metagenome Functional Prediction with PICRUSt2 The observed changes in bacterial endosphere communities when Cannabis genotypes are grown with and without soil inoculum suggests this niche may be occupied by bacteria with different functionalities depending on their environment. Therefore, metagenome functional prediction was conducted using PICRUSt2 (phylogenetic investigation of communities by reconstruction of unobserved states) (Douglas et al., 2013). First, to get a high-level picture about how seed and soil bacteria shape the functional profile of emerging Cannabis seedling endospheres, all genotypes and soils were pooled to compare autoclave sterilized and untreated soil using ANCOM differential abundance testing (Table 4.1). Four gene pathways were found to have significantly different relative abundance between these two treatments. Cannabis seedlings germinated in untreated soil with living microorganisms were enriched in the octane oxidation (P221-PWY); a pathway commonly found and well-studied in 88 Pseudomonas species (Chakrabarty et al., 1973). Cannabis seedling grown in autoclaved soil without environmental inoculum where significantly enriched in the pathways: L-lysine biosynthesis II (PWY-2941), lactose degradation I (LACTOSECAT-PWY), and S-methyl-5-thio-α-D-ribose 1-phosphate degradation I (PWY-4361). Table 4. 1. PICRUST ANCOM Differential Abundance MetaCyc Pathways for Soil Sterilization. All Cannabis samples were pooled and compared between autoclave sterilized and natural untreated soil. Pathways shown were determined to be significantly different between treatments as determined by the W statistic. Gene Pathway MetaCyc Pathway Name Sterilized Relative Abundance Natural Relative Abundance P221-PWY octane oxidation 37295 102195 PWY-2941 L-lysine biosynthesis II 158749 2468 LACTOSECAT-PWY lactose degradation I 3648 1448 PWY-4361 S-methyl-5-thio-α-D-ribose 1-phosphate degradation I 137054 1473 Next, samples were examined within genotypes between different soil treatment to examine how the functional genes shift in emerging Cannabis seedling endospheres. BC Big Bud planted in sterile Innotech soil were significantly enriched in the methanogenesis pathway to produce methane from H2 and CO2 (METHANOGENESIS-PWY) and the reductive acetyl coenzyme A pathway I (CODH-PWY) compared to all other soil treatments. The CODH-PWY is 89 associated with autotrophic bacteria who can produce the energetic compound acetyl-COA by fixing CO2. In Katani, many gene pathways showed significant differential abundance, however a select few with higher relative abundance estimates are mentioned here. The pathway for 4-hydroxyphenylacetate degradation (3-HYDROXYPHENYLACETATE-DEGRADATION-PWY) was significantly enriched in both untreated soils compared to their sterilized counterpart. Additionally, the polyamine biosynthesis pathway (POLYAMINSYN3-PWY), and the pathway for chitin derivatives degradation (PWY-6906) were significantly enriched in Katani plants grown in untreated soil. Katani plants grown in untreated Okanagan soil were significantly enriched in the super pathway of L-threonine metabolism (THREOCAT-PWY) when compared to all other soil treatments. X59 plants grown in sterilized Okanagan soil were significantly enriched in the aerobic thiazole biosynthesis II pathway (PWY-6891), which has been well studied in Bacillus species. Plants grown in untreated Innotech soil were significantly enriched with the peptidoglycan biosynthesis II pathway (PWY-5265) compared to other soil treatments. 90 Table 4. 2. Selected Significantly Differentially Abundant MetaCyc Pathways for Genotypes in Soil Treatments. Each genotype was analyzed separately. Pathways shown were determined to be significantly different between treatments as determined by the W statistic. Selected pathways had highest percent abundance values of at least 400. The full table can be seen in Supplemental File S3. 4.4 Discussion 4.4.1 Seed and Soil Bacteria are both Sources of Inoculum for Germinating Cannabis Seedling Endospheres The effect of growing BC Big Bud, Katani, and Alyssa (one marijuana and two hemp cultivars) in autoclave sterilized and untreated Innotech soil, taken from a hemp research station in Vegreville Alberta, and Okanagan soil from a vineyard in Kelowna BC was examined using DNA amplicon sequencing. Cannabis genotype, soil type, and soil status (autoclaved/untreated) all influence the endophyte community structure in germinating Cannabis seedlings, suggesting inoculum from both seed and soil are used to shape this ecosystem (Figure 4.2, Figure 4.3, Figure 4.5). There was however a core microbiome of bacterial endophytes conserved across genotypes, soil types, and soil sterilization. This suggests that seed-borne endophytes inhertited Innotech Soil Sterile Innotech Soil Okanagan Soil Sterile Okanagan Soil BC Big Bud METHANOGENESIS-PWY CODH-PWY Katani 3-HYDROXYPHENYLACETATE-DEGRADATION-PWY 3-HYDROXYPHENYLACETATE-DEGRADATION-PWY POLYAMINSYN3-PWY POLYAMINSYN3-PWY Chitin Degradation PWY-6906 Chitin Degradation PWY-6906 THREOCAT-PWY X59 Peptidoglycan Biosynthesis II Pathway (PWY-5265) Aerobic Thiazole Biosynthesis II Pathway (PWY-6891) 91 from the mother plant are maintained when seedlings are germinated in the pressence and absence of soil microorganisms. However, it should be noted that soil treatment had a significant effect on the relative abundances of these conserved bacteria in this experiment. Gammaproteobacteria and Bacilli the most abundant taxonomies, were found in all treatments. Furthermore Betaproteobacteria, Flavobacteriia, and Alphaproteobacteria were found in all treatments at typically at lower abundance (Figure 4.2, Figure 4.3). The role of soil and seed-borne inoculum in shaping Cannabis microbiomes is poorly understood. Previous research suggested that Cannabis genotype plays a significant role in shaping microbial communities (Comeau et al., 2020), supporting the genotype effect on beta diversity seen in this experiment. The experiment looked at the microbiomes of vegetatively propagated Cannabis plants at various stages of plant development and found significant dissimilarity in beta-diversity. Additionally alpha diversity was shown to increase over time (Comeau et al., 2020). This is contrasting what other research has seen during seedling emergence, where there is a marked decline in alpha-diversity during the first 96 hours of seed emergence (Barret et al., 2015). This suggests that germinating seedlings place strong selective pressure on certain microbes which appear to quickly grow and out-compete others in this rapidly changing environment. These contrasting observations likely stems from a bottleneck effect on microbial diversity derived from nodal propagation of Cannabis cuttings which may lack a diverse microbiome. This leads to questions about how the microbiome of seed-derived and clonally propagated Cannabis plants differ when the latter does not obtain a founding population of microorganisms from seed. Cannabis seedlings in this experiment showed intra-genotype sample variation, and there were fluctuations in relative abundances between samples and genotypes. Variation between individual organisms in common in microbiome studies (Mohammed et al., 2018). Timing of exposure to soil microorganisms has been suggested to be critical for successful plant 92 colonization (Aleklett et al., 2022). There are major shifts in microbial communities in plants in correlation with plant developmental stage (Liechty et al., 2018), and some research has suggested early colonizers are thought to have a priority effect (Santoyo et al., 2016). However, other research has shown that soil bacteria quickly dominate seedling microbiomes when root surface tissue is also analyzed (Rochefort et al., 2021). Soil sterilization resulted in a significant reduction in Shannon alpha diversity when pooling genotypes and soils (Figure 4.4A). This is unsurprising as soil introduces an additional source of microorganisms that can colonize seedlings to the seed inoculum present. However, when looking at genotypes and soils individually, only Katani plants grown in Okanagan showed this significant reduction in alpha diversity (Figure 4.4D). This may be a result of a genotype specific colonization event to soil microganisms present in this vinyard soil. Other genotypes in this soil showed non-significant increases and decreases. Alpha diversity of Cannabis genotypes grown in sterilized and untreated Innotech also showed non-significant increases and decreases. Interestingly, when pooling soil treatments for each genotype, the marijuana genotype BC Big Bud had significantly higher alpha diversity as compared to the two hemp genotypes: Katani and X59 (Figure 4.4A). Further testing would need to be conducted with more genotypes of both marijuana and hemp to see if this is not a one-off effect. The relative abundances of the bacteria dramically shifted in response to soil sterilization to remove soil microbes. (Figure 4.5, Supplementary Table 2). Genotype had clear and significant effect on Bray-Curtis distances, however, Cannabis genotypes as a whole clustered into their soil treatment groups on the PCoA (Figure 4.5). Genotypes planted in untreated Okanagan and Innotech soils where dramatically different from one another, while the genotypes grown in the sterilized soils clustered far closer together (Figure 9). This is unsurprising as soil microorganisms act as an additional source of inoculum to affect diversity in genotypes. Juvenile maize rhizospheres have been shown to obtain inoculum from both seed 93 and soil (Johnston-Monje et al., 2016). Other research has suggested that soil bacteria are the primary source of inoculum for seedling rhizospheres (Rochefort et al., 2021), an so it is unsurprising that they would have an effect on endosphere communities. Meanwhile, more close clustering of Cannabis genotypes grown in both sterilized Okanagan and Innotech soil is likely a result of seed-borne endophytes being the sole colonizers of niche (Figure 4.5). Abiotic soil characteristics have an effect on microbiome composition (Figure 4.5, Supplementary Table 2). This was shown in the experiment by the same Cannabis genotype grown in different sterilized soil displayed differences in beta-diversity. The physical properties such as the sand, silt, and clay content may influence this, as well as the differences in organic matter, pH, and nutrient content in the Okanagan vineyard soil versus the Innotech hemp farm soil. 4.4.2 Shifts in Bacterial Relative Abundance from Soil Sterilization There were clear shifts in relative abundances of certain bacterial taxonomies when Cannabis genotypes were grown with and without soil microorganisms. In the absence of soil bacteria, Cannabis genotypes were significantly enriched in Betaproteobacteria (1.42x) (Supplemental File S2). Betaproteobacteria are a class of gram-negative bacteria known to play important roles in nitrogen, sulfer, and other nutrient cycling to contribute to the life-support function of soil (Falca et al., 2010; Kowalchuk et al., 1997; Schmalenberger and Kertesz, 2007). They are known to have plant growth-promiting properties, and their abundance in potato rhizoshere has been shown to be cultivar dependent and plant growth stage dependent (Falca et al., 2010) This study showed that in high-starch potato cultivars, there was an increase in Betaproteobacteria rhizosphere abundance towards the flowering stage of the plant life cycle. Perhaps Cannabis-cultivars show a similar effect which leads to an enrichment of these bacteria in flowering tissue, allowing them to be vectored to seedlings, as was observed in this study. 94 Culmulativey, genotypes grown in sterilized soil with only seed-borne inoculum were typically enriched in Bacilli (49x). This large increase was not significant when grouping genotypes as there was relatively equal abundances in Bacilli between sterilized and unsterilized Innotech soil, contrasting what was seen in Okanagan soil where they were significantly enriched in all genotypes except BC Big Bud which saw a non-significant 1.81x increase. Bacilli are a class of economically and agriculturally relevant bacteria known promote plant growth and to create a large variety of antimicrobial secondary metabolites (Cochrane and Verderas, 2014; Hashem et al., 2019; Ongena and Jacques, 2008). Additionally, they form robust endospores capable of surviving harsh environental conditions for long periods of time, and reviving when conditions are favoriable (Hashem et al., 2019; Nicholson, 2002). Interestingly, Bacilli spores have been claimed to be revived from 25-40 million year old amber (Raul and Monica, 1995). It is therefore unsurprising that this class of bacteria would be enriched with seeds (which can be dormant for several years) and be vectored across plant generations in Cannabis. Previous research demostrated Cannabis plants vector culturable Bacillus species to seedlings that antagonize seed-borne pathogens (Dumigan and Deyholos, 2022). The most dramtic shift in relative abundance when growing genotypes in untreated soil with intact microbiomes was the clear shift in abundance towards Gammaproteobacteria (329.4x) when pooling genotypes and soils. Gammaproteobacteria class of bacteria commonly associated with plants, and has even been identified as an indicator species of a healthy microbiome against Fusarium wilt in banana plants (Köberl et al., 2017). Pseudomonas is a genera of within this class that have been identified as having biocontrol activity against Cannabis fungal pathogens (Balthazar et al., 2022).Interestingly, in an innoculation experiment, Pseusdomonas was found to have plant growth promotion activity, but only when inoculated with a consortia of Bacillus and Pseudomonas species (Comeau et al., 2021). It is difficult to 95 study the activity of these-non cultured Gammaproteobacteria (Dumigan and Deyholos, 2022), however, this previous research leads to questions about potential evolution of plant growth promoting and antifungal consortia that may be vectored on seed or obtained from soil to protect emerging Cannabis seedlings. Flavobacteriia and Sphingobacteriia were differentially abundant in Cannabis cultivars grown in the presence of Okanagan soil microorganisms (untreated Okanagan soil). Flavobacteriia are often found in root-associated microbiomes, but research is still unclear about factors influencing their abundance. A recent study found that Flavobacterium (genus of Flavobacteriia) abundance is associated with pectin availability with regards to plant associated communities (Kraut-cohen et al., 2021). These are known to be lower in abundance in soil communities in comparison to plant surfaces (Kraut-cohen et al., 2021). Cannabis fiber is composed of 1-17% pectin (Liu et al., 2017), perhaps from soil Flavobacteriia species have evolved to associate with these plants for their pectin content. The increase Sphingobacteriia in the genus Sphingobium have been well studied for the biodegradative properties and for bioremediation (Boss et al., 2022; Kunihiro et al., 2013).They are also known antagonize pathogenic Pseudomonas (Takeuchi et al., 2001). 4.4.3 Differential Enrichment of Functional Gene Pathways from Seed and Soil Inoculum The observation octane oxidation (P221-PWY) pathway enrichment in untreated soil when pooling genotypes and soils suggests soil is contributing Pseudomonas species with the capacity to degrade this hydrocarbon (Chakrabarty et al., 1973). It is difficult to known whether this enrichment is a product of co-incidence (preferential colonization of these bacteria who happen to have this pathway), or a plant adaptation to a soil environment where octane oxidation is beneficial. Plants grown with only seed-inoculum were enriched in the pathways: L-lysine biosynthesis II (PWY-2941), lactose degradation I (LACTOSECAT-PWY), and S-methyl- 96 5-thio-α-D-ribose 1-phosphate degradation I (PWY-4361). L-lysine is an essential amino acid, and the enrichment of this pathway suggests Cannabis plants could be vectoring bacteria that can produce this amino acid to help the next generation of plants deal with nutrient poor soils that may have low amounts of L-lysine, or there could be some direct benefit to Cannabis plants in hosting bacteria that can produce and share a supplemental source of L-lysine. The enrichment of the lactose degradation I pathway is likely a result of the increased abundance of Bacilli in plants grown in sterile soil, as this pathway is well characterized and commonly found in this taxonomic class (Anderson, 1973). Finally, PWY-4361 was 93-fold more abundant in plants grown in sterile soil compared to untreated soil (Table 4.1). This pathway functions to salvage the amino acid methionine from sulfur containing by-products of polyamine synthesis (Avila et al., 2004), or in some organisms, by-products from the synthesis of the phytohormone ethylene (Albers, 2009; Yang and Hoffman, 1984). Interruption of the this methionine salvaging pathway in Arabidopsis has a negative effect on ethylene production (Bürstenbinder et al., 2007), likely due to a buildup of the inhibitory compounds that are not being recycled (Pattyn et al., 2021). Given the critical role of ethylene in seed dormancy, and the observation that ethylene can induce germination under certain conditions (Wang et al., 2020; Yang and Hoffman, 1984), there is a possible function of these bacterial genes in regulating germination of Cannabis seedlings. Perhaps the enrichment of this pathway in seed-borne Cannabis endophytes has evolved to assist in seedling germination. To get specific information about how gene pathway abundance shifts in response to soil and soil sterilization in each genotype, PiCRUST2 analysis was done individually for each genotype. BC Big Bud plants in sterilized Innotech soil were enriched with the methanogenesis pathway to produce methane from H2 and CO2 (METHANOGENESIS-PWY), indicating the presence of methanogenic archaea being vectored to seedlings via seed. Methanogens grow by utilizing H2 and CO2 conversion to methane to create energy (Buan, 2018). Methanogens have 97 been found in rice endospheres (Edwards et al., 2015), and have been suggested to contribute the methane greenhouse gas emissions (Neue, 1993). The role of these methanogens in germinating Cannabis seedling endospheres needs further examination. Additionally, the reductive acetyl coenzyme A pathway I (CODH-PWY) was enriched in this environment, suggesting autotrophic bacteria that can fix CO2 are being vectored from BC Big Bud seeds to germinating seedlings. Katani plants showed many differentially abundant gene pathways with significant W statistics (Supplemental File S3), however a select few with highest percent abundances greater than 400 were mentioned in the text. Untreated Innotech and Okanagan soil were significantly enriched with the pathway for 4-hydroxyphenylacetate degradation (3-HYDROXYPHENYLACETATE-DEGRADATION-PWY) compared to their sterilized counterpart. This pathway is found in a number of bacteria, including denitrifying bacteria (Seyfried et al., 1991), suggesting that denitrifying bacteria are present in soil and colonizing plant endospheres. In Katani plants, both soils contributed bacteria with the gene pathways for polyamine biosynthesis and chitin degradation (Table 4.2). Polyamines are known act as signaling molecules and play an important role in triggering plant defense and symbiotic plant-microbe interactions (Jiménez-Bremont et al., 2014). Chitin is a major structural compound of fungal cell walls (Beier and Bertilsson, 2013), and the presence of chitin degrading bacteria is not surprising as untreated soil would have an abundance of fungi. It is interesting that these chitin degrading bacteria are colonizing Cannabis seedling endospheres. Available data suggests there are shifts in bacterial community structure towards chitinolytic bacteria in response to chitin availability (Kielak et al., 2013). It could be the germinating Katani plants are selecting for chitin degrading bacteria from soil when grown in an environment with abundant fungi. Finally, in Katani plants grown in untreated Okanagan soil, the super pathway for L-threonine metabolism was significantly enriched compared to other soil treatments. Bacterial amino acid 98 metabolism has recently been shown to be important to synthesize signaling molecules to associate with host plants (Moormann et al., 2022). Perhaps species of Okanagan soil bacteria using L-threonine to create signaling molecules to associate with Katani seedlings. X59 plants grown in sterilized Okanagan soil were significantly enriched with the aerobic thiazole biosynthesis II pathway (PWY-6891). This pathway has been well studied in Bacillus species and is likely a result of the dominance of seed-borne bacilli in X59 endosphere grown in these conditions (Moormann et al., 2022). The role of bacteria synthesized thiazole in germinating Cannabis seedlings seeds further study. X59 plants grown in sterilized Innotech soil were enriched peptidoglycan biosynthesis II pathway (PWY-5265). Peptidoglycan is a major feature of bacterial cell walls and crucial for biofilm formation (Garde et al., 2021). It is essential for adhesion to substrates, and this MetaCyc PWY-5265 pathway has the expected taxonomic range of the phylum Bacillota. The enrichment of this pathway again likely stems from the dominance of seed-borne Bacilli when X59 plants are grown in the absence of soil microorganisms. 4.5 Conclusion and Future Directions Unraveling the complexities of plant-microbe interactions and their effect on plant health is a major research focus for modern plant science. Angiosperms have evolved to vector bioactive microorganisms across plant generations to assist in seedling establishment and plant health. This phenomenon and the benefits provided is poorly understood in cannabis. This research documents for the first time the effects of both seed and soil in shaping microbial communities in this economically relevant crop. The study demonstrates that cannabis seedlings obtain a conserved community of seed-borne endophytes across soil types and treatments, and that the abundances of these bacteria shift dramatically when exposed to soil inoculum. Additionally, soil inoculum contributes a significant amount of endosphere diversity to 99 germinating seedlings. Metagenomic functional gene prediction suggests that these seedling endophytes have enzymatic pathways potentially useful to establishing healthy cannabis plants. Future work should further investigate these activities and subsequent benefit to cannabis plants. 100 Chapter 5 Conclusion This thesis was motivated by the lack of research in the areas of Cannabis pathology and Cannabis microbial ecology. At a high level, this research sought to answer a few simple questions relevant to Cannabis as a modern agricultural crop. What microorganisms are transmitted across plant generations via Cannabis seeds? What do these seed-borne microorganisms do for plant health? Can these seed-borne microorganisms be used to fight Cannabis fungal pathogens? Cannabis production at the time of writing this thesis is plagued by a plethora of fungal disease, and a new fungal disease in Cannabis is even detailed in this dissertation. Growers of this crop in the California and in Canada are severely limited in the tools available to control fungal disease in Cannabis, as conventional synthetic fungicides are prohibited in these jurisdictions. Angiosperms are known to vector seed-borne microorganisms to provide a selective advantage to the next generation of plants, especially during the early and delicate seedling stage of their life cycle. This thesis documents for the first time the process of vertical-transmission of seed-borne bacterial seedling endophytes in Cannabis. Cannabis is known to vector seed-borne fungal disease, and this thesis shows that Cannabis plants also vector beneficial seedling endophytes that can antagonize these pathogens. In the first research chapter, the study demonstrates for the first-time vertical transmission of seed-borne bacterial seedling endophytes in Cannabis. 136 endophytes were isolated in culture from 14 Cannabis cultivars, phenotyped for bioactivities relevant to plant growth promotion. Surprisingly, despite the different geographies where the mother plants were grown to seed, the Cannabis genotypes vectored related microorganisms in the class Bacilli, including phosphate solubilizing Paenibacillus mobilis, which was found in all Cannabis cultivars. Additionally, several Cannabis fungal pathogens were vectored via seed to 101 germinating seedlings – something that has been well known and documented in the field of Cannabis production. An interesting observation from this chapter was the fact that 13 of the seed-borne seedling endophytes isolated in this experiment were found to have antagonistic activity against four seed-borne fungal pathogens: Fusarium, Penicillium, Alternaria, and Aspergillus. Cannabis pathogens such as these can have negative effects on plant health, seed germination, and seedling establishment. This is biologically interesting as it suggests that Cannabis plants have evolved to vector bacterial endophytes to their progeny to protect them against these fungal pathogens during this critical and vulnerable stage in their lifecycle. The strict regulation around fungicide use in Cannabis production means that growers are limited in the methods available to control outbreaks of fungal pathogens. This observation is therefore relevant to industry as there is a demand for safe and effective methods to control fungal pathogens that fit in with the current regulation. The use of Bacilli species is currently permitted in Cannabis production; therefore, these bacterial species may be useful as biocontrol agents. Furthermore, the widespread use of clonal propagation from nodal tissue to obtain genetically identical plants in the industry may inadvertently have selected for plants that are missing these bioactive seed-borne endophytes. The study also observed large variation in seed-germination and fungal disease pressure between the 14 genotypes of Cannabis. The age of the seed likely plays a role in this, as well as the genetics of each individual genotype. However, the microbiome of the seed and subsequent seedling likely plays an additional role in whether a seed germinates and establishes as a seedling, or whether it is dormant or is engulfed by opportunistic seed-borne fungal pathogens. There were several cultivars in this study that when germinated in the absence of environmental microorganisms were quickly engulfed by seed-borne fungi. This observation is particularly applicable to growers of hemp – which is typically seed derived and in the field struggles with low germination rates. Microbiome transfer from the seed of high- 102 germination cultivars to low germination cultivars may provide a microbiome that is more conducive to seed germination and seedling establishment. This could be from phytohormonal regulation, enzymatic stimulation, antagonism of fungal disease, or some other unknown method. Similar to production of marijuana cultivars, hemp cultivation is limited in the seed-treatment options available under current regulation to control fungal disease from seed and soil. Much is still unknown in the areas of Cannabis production, pathology, and disease control. In the second research chapter, the study documents the discovery of a new to drug-type Cannabis fungal pathogens effecting roots in an aeroponic production facility. The study was motivated by an unknown root disease rapidly spreading through a licensed production facility located in Cranbrook BC that tested negative for all commercially available PCR tests. Additionally, the root-disease was not culturable on conventional microbiology media (PDA, TSA, and R2A), further complicating its study. Microscopic imaging showed segmented chlamydospores indicative of fungi in the Thielavopisis/Berkeleyomyces genera. ITS sequencing of fungal tissue scraped from roots confirmed the root-rot pathogen as a member of the Berkeleyomyces genus, something that was not documented in Cannabis in February 2022. The fact that a previously undocumented fungal pathogen in Cannabis can be discovered in 2022 highlights how behind research in Cannabis pathology and microbiology is compared to crops of similar market value. Before this study, there was no genome sequence available for the species of this Cannabis fungal pathogen, Berkeleyomyces rouxiae. This chapter utilized hybrid genome sequencing and assembly with Oxford Nanopore long-read and Illumina short-read sequencing to sequence the first genome of the economically relevant black root-rot pathogen also known to affect a wide variety of crops. DNA isolation was particularly difficult for this fungal species, with a half-dozen different methods tried before enough DNA could be produced for sequencing, 103 something that may have contributed to the lack of a previous genome. The study also satisfied Koch’s postulate by re-infecting Cannabis plants with this pathogen and confirming it was the agent of disease. Future work should use the genome sequence to develop species specific primers to allow rapid detection of this pathogen from Cannabis production facilities. Commercial labs should then be able to diagnose plants infected with black root-rot so that producers of Cannabis can react quickly and quarantine infected plants before the disease spreads across the facility, as was the case in Cranbrook BC which resulted in destruction of an entire crop. Additionally, a seed-borne endophyte from chapter one was shown to have antagonism against this black root-rot pathogen in vitro, and so has potential as a biocontrol agent against this emerging Cannabis disease. With the current regulation surrounding fungicides, a grower’s best means on control is rapid detection and quarantine of this pathogen to avoid total crop destruction. In the third and final research chapter, a metagenomic approach was taken to better understand the role of seed and soil in shaping Cannabis seedling endosphere microbiomes. The majority of bacteria across many studied environments are not easily cultured on conventional microbiology media, and so to get a more holistic view of the microbial ecology of Cannabis seedling endospheres, 16S amplicon sequencing was performed in a multifactorial experiment. Three Cannabis cultivars were selected (BC Big Bud, X59, and Katani) and were each grown in untreated and autoclave sterilized soil from two regions: Innotech soil from a hemp research farm in Vegreville Alberta, and Okanagan soil from a vineyard in Kelowna. This experiment showed both soil and seed microorganisms colonize germinating Cannabis seedling endospheres and effect the bacterial diversity present. Cannabis genotype and soil type affected the beta diversity of germinating Cannabis seedling endospheres. Additionally, abiotic characteristics of the soil affect bacterial diversity. There was however a core microbiome of bacterial endophytes conserved across genotypes, soil types, and soil 104 sterilization, further supporting the hypothesis of a core microbiome that is present in Cannabis seedlings across the environements they were grown in. Soil treatments significantly effected the relative abundances of these conserved bacteria, and soil sterilization shifted the microbial communities compared to Cannabis genotypes germinated in untreated soil. When pooling genotypes, soil sterilization resulted in a significant reduction in Shannon alpha diversity. Differential abundance testing with ANCOM showed Gammaproteobacteria and Bacilli were the two most abundant taxonomic classes across treatments. In the absence of soil bacteria, Cannabis genotypes were significantly enriched in Betaproteobacteria (1.42x), and non-significantly enriched in Bacilli (49x) over plants germinated in untreated soil. There was a dramatic shift in relative abundance of Gammaproteobacteria (329.4x) when Cannabis genotypes were grown in untreated soil. Metagenome functional prediction showed that Cannabis seedling endophytes hosted a number of pathways that have activities relevant to plant growth including denitirfication, amino acid biosynthesis, chitin degradation, and regulation of ethylene production in plants. Future worth should further investigvate the activities of these endophytes on Cannabis seedling germination and health. The historical prohibition, subsequent legalization, and immense interest from eager investors in this space has created a somewhat unique environment for plant research. This is one of the core drivers of this thesis. It is a rare opportunity to study an economically relevant plant that is still at its infancy in terms of understanding and research. This thesis only scratches the surface of what is known about Cannabis microbiology and pathology and leads to many more questions and directions for further research. Future studies will investigate the biocontrol potential of the seed-borne endophytes identified in Chapter 2.1 on Cannabis seedlings and plants. Additionally, the ability of these antifungal seed-borne endophytes to control the emerging fungal Cannabis pathogen identified in Chapter 2.2, Berkeleyomyces rouxiae, should be investigated. Finally, having identified the shifts in endosphere bacterial relative abundance 105 in response to soil microorganisms, the resulting changes in Cannabis seedling germination should be investigated for correlations with different community structure to attempt to identify a “healthy” Cannabis seedling microbiome that is primed for germination and successful plant establishment. 106 Bibliography Abbasi, P. A., and Weselowski, B. (2014). 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Biol. 16, 1–8. doi:10.1371/journal.pcbi.1007981. 125 Appendices Appendix A Chapter 2.1 Supplementary Materials Supplemental Figure S1. 16S Phylogenetic Tree of Culturable Seed-borne Cannabis Seedling Endophytes Across Hemp X59 Generations. Strains of Bacillus megaterium and Paenibacillus mobilis were cultured from two successive generations of Hemp X59 grown to seed. Generation-1 was grown outdoors in soil in Vegreville, AB, Generation-2 was grown indoors in Sunshine Mix in Kelowna, BC. P. mobilis isolates are outlined in teal. B. megaterium isolates are outlined in orange. Amplicons were aligned using MUSCLE algorithm prior to nearest-neighbor phylogenetic tree was construction using Tamura-Nei model on MEGA software (Version 11.0.11) 126 Supplemental Figure S2. Altair Seed-Associated Fungi. Alternaria and Penicillium growth on germinating Altair hemp seeds 4 days after planting on PDA Supplemental File S1.CSV List of Endophytes in this Chapter 127 Appendix B: Chapter 2.2 Supplementary Materials Supplemental Table S1. Genome assembly methods tested in this paper. Assembly Name Methods canu_PM34_PM34 Canu assembly on ONT long-reads flye_assembly Flye assembly on ONT long-reads hybrid_assembly_v1 Masurca assembly with ONT long-reads and Illumina short reads – final assembly with Celera hybrid_assembly_v2 Masurca assembly with ONT long-reads and Illumina short reads – final assembly with Flye illumina_assembly_v1 Masurca assembly with Illumina short reads polca_canu_PM34 Canu assembly on ONT long-reads error corrected with polka using Illumina short reads polca_flye Flye assembly on ONT long-reads error corrected with polka using Illumina short reads samba_assembly Masurca assembly with Illumina short reads then gap-filled with ONT long reads flye_medaka_polca Flye assembly polished with Medaka using ONT long-reads, then error corrected using Illumina short reads 128 Supplemental Figure S3. Genome assembly method scorings. Supplemental Figure S4. Genome assembly k-mer spectrum plot from Merqury with short reads. 129 Supplemental Figure S5. Trial 2 of Cannabis plant inoculated with Berkeleyomyces rouxiae PM34. 130 Appendix C: Chapter 2.3 Supplementary Materials Supplemental Table S2. PERMANOVA Bray-Curtis Beta-diversity distance matrix significant results (bray_curtis_distance_matrix-significance.qzv). (A) Innotech Soil PERMANOVA, (B) Okanagan Soil PERMANOVA, (C) Genotype PERMANOVA A) Group 1 Group 2 Sample size Permutations pseudo-F p-value q-value BC Big Bud Innotech X59 Innotech 15 999 1.18438 0.189 0.189 Katani Innotech X59 Innotech 16 999 1.406435 0.14 0.142154 BC Big Bud Innotech Katani Innotech 15 999 1.355348 0.124 0.127875 131 Supplemental Table S3. PERMANOVA Bray-Curtis Beta-diversity distance matrix significant results (bray_curtis_distance_matrix-significance.qzv). (A) Innotech Soil PERMANOVA, (B) Okanagan Soil PERMANOVA, (C) Genotype PERMANOVA (B) Bray-Curtis Permanova Sample size Permutations pseudo-F p-value q-value BC Big Bud Sterilie-Innotech vs BC Big Bud Sterilie-Okanagan 16 999 1.860815 0.005 0.006346 Katani Sterilie-Innotech vs Katani Sterilie-Okanagan 15 999 3.331946 0.004 0.005280 X59 Sterilie-Innotech X59 Sterilie-Okanagan 15 999 3.684158 0.006 0.007472 (C) Group 1 Group 2 Sample size Permutations pseudo-F p-value q-value BC Big Bud Katani 62 999 2.338869149 0.002 0.003 BC Big Bud X59 62 999 1.972871311 0.005 0.005 Katani X59 62 999 2.69872317 0.002 0.003 132 Supplemental File S2 ANCOM TABLES.zip Supplemental File S3 PICRUST ANCOM TABLE.CSV Supplemental File S4 qiime2 metadata.xlsx
Thesis/Dissertation
eng
Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Deyholos, Michael
Exploring the cannabis microbiome to develop safe and sustainable bio-fungicide
Text
Dataset