Canadian Journal of Microbiology Endophytes of industrial hemp (Cannabis sativa L.) cultivars: identification of culturable bacteria and fungi in leaves, petioles and seeds Journal: Canadian Journal of Microbiology Manuscript ID cjm-2018-0108.R2 Manuscript Type: Article Date Submitted by the Author: 03-May-2018 Complete List of Authors: Scott, Maryanne; McGill University Faculty of Agriculture and Environment, Plant Science Rani, Mamta;Draft McGill University Faculty of Agriculture and Environment, Plant Science Samsatly, Jamil; McGill University Faculty of Agriculture and Environment, Plant Science Charron, Jean-Benoit; McGill University Faculty of Agriculture and Environment, Plant Science Jabaji, Suha; McGill University Faculty of Agriculture and Environment, Plant Science; Keyword: Hemp, endophytes, siderophore, Pseudomonas, molecular detection Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/cjm-pubs Page 1 of 49 Canadian Journal of Microbiology Scott et al. Revised- CJM-2018-0108 1 2 Endophytes of industrial hemp (Cannabis sativa L.) cultivars: identification of culturable 3 bacteria and fungi in leaves, petioles and seeds 4 5 Maryann Scott, Mamta Rani, Jamil Samsatly, Jean-Benoit Charron, Suha Jabaji* 6 Plant Science Department, MacDonald Campus of McGill University, 21,111 Lakeshore, Ste. 7 Anne-de-Bellevue, Québec, Canada, H9X3V9 8 9 10 *Corresponding Author 11 Suha Jabaji Draft 12 Phone: 514-398-7561 13 Email: [email protected] 14 15 16 17 18 19 20 1 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 2 of 49 Scott et al. Revised- CJM-2018-0108 21 Abstract 22 23 Plant endophytes are a group of microorganisms that reside asymptomatically within the healthy 24 living tissue. The diversity, molecular and biochemical characterization of industrial hemp- 25 associated endophytes have not been previously studied. This study explored the abundance and 26 diversity of culturable endophytes residing in petioles, leaves and seeds of three industrial hemp 27 cultivars, and examined their biochemical attributes and antifungal potential. A total of 134 28 bacterial and 53 fungal strains were isolated from cultivars Anka, CRS-1 and Yvonne. The 29 number of bacterial isolates was similarly distributed among the cultivars with the majority 30 recovered from petiole tissue. Most fungal strains originated from leaf tissue of cultivar Anka. 31 Molecular and phylogenetic analyses grouped the endophytes into 18 bacterial and 13 fungal 32 taxa, respectively. The most abundant bacterialDraft genera were Pseudomonas, Pantoea and Bacillus, 33 and the fungal genera were Aureobasidium, Alternaria and Cochliobolus. Siderophore, cellulase 34 production, and phosphorus solubilization were the main biochemical traits. In proof-of-concept 35 experiments, re-inoculation of tomato roots with some endophytes confirmed their migration to 36 aerial tissues of the plant. Taken together, this study demonstrates that industrial hemp harbours 37 a diversity of microbial endophytes some of which could be used in growth promotion and/or in 38 biological control designed experiments. 39 40 Key words: Hemp, endophytes, siderophore, Pseudomonas, molecular detection, antifungal 41 activity. 42 2 https://mc06.manuscriptcentral.com/cjm-pubs Page 3 of 49 Canadian Journal of Microbiology Scott et al. Revised- CJM-2018-0108 43 Introduction 44 Originating from the Himalayas, industrial hemp (Cannabis sativa L.) is the most ancient 45 domesticated crop. In Canada, under the Canadian Controlled Drugs and Substances Act, 46 commercial cultivation of industrial hemp as a field crop began in 1998 (Cherney and Small 47 2016). 48 Industrial hemp is a high-growing plant, typically bred for seed and fibre, and also for 49 multipurpose industrial uses such as oils and topical ointments, as well as fibre for clothing, and 50 construction material for homes and building of electric car components (Callaway and Pate 51 2009; Domke and Mude 2015; Yallew et al. 2015). Since its legalization in Canada, the total 52 land area for industrial hemp production has grown steadily, increasing from 3,200 ha to 53 approximately 44,000 ha between 2008 Draftand 2014 (Alberta Agriculture and Forestry, 2015). Both 54 hemp and marijuana varieties are members of the C. sativa species, however industrial hemp 55 cultivars have long been bred for their fibre, oil and seed aspects, and possess very low narcotic 56 value. In Canada, 45 industrial hemp cultivars are approved by Health Canada (Health Canada, 57 2016), and are mandated to have under 0.3 % of ∆-9-tetrahydrocannabinol (THC), the principal 58 intoxicant cannabinoid (van Bakel et al. 2011). These varieties are generally grain or multi-use; 59 where both the seeds and stalk find an end market (Cherney and Small 2016). 60 In Québec, farmers produced hemp on 290 hectares in 2011; a fairly small commitment in 61 comparison to the prairie provinces, which had planted 15,056 hectares in the same year 62 (Government of Alberta, 2012). This means that within the Québec market there is a great 63 potential for growth and profitability. Much of the Québec growing climate is similar to 64 Northern Ontario that has yielded an average stem yield of 6.1 tons per hectare (Ontario Ministry 65 of Agriculture and Food, 2011). Considering that demand appears to be continuing on a positive 3 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 4 of 49 Scott et al. Revised- CJM-2018-0108 66 trend, knowledge on selection of high-performing cultivars in terms of biomass and seed yield 67 (Aubin et al. 2016) and agronomical recommendations and guidelines (Aubin et al. 2015) has 68 been documented. 69 The phytochemical composition of industrial hemp and the marijuana varieties of 70 Cannabis does not make hemp immune to attacks by pathogens. On the contrary, Cannabis is 71 susceptible to many phytopathogens leading to a number of diseases (McPartland et al. 2000) 72 dominant at all growth stages of hemp. Several important diseases have been shown to be caused 73 by fungal pathogens including Botrytis cinerea, the causal agent of grey mold, Rhizoctonia 74 solani, the causal agent of root rot and stem canker, and Sclerotinia sclerotiorum, the causal 75 agent of hemp canker (McPartland et al. 2000). Thus, it is desirable to prevent the loss of 76 industrial hemp as well as medicinal CannabisDraft to opportunistic phytopathogens. 77 Microbial endophytes are beneficial plant-associated bacteria and fungi that are widespread 78 inhabitants inside different plant tissues and organs, without causing harm or exhibiting 79 symptomatic behaviour or visible manifestation of disease. They have been shown to assist plant 80 growth by producing plant hormones, increasing nutrient availability (Compant et al. 2010; 81 Hamilton et al. 2012; Radhakrishnan, et al. 2014; Hardoim et al. 2015; Malhadas et al. 2017), 82 and protecting plants from diseases and abiotic factors through their demonstrated capacity to 83 produce biologically active secondary metabolites (Aly et al. 2010; Kharwar et al. 2011; Gagné- 84 Bourgue et al. 2013; Brader et al. 2014), and establishing a sustainable system (Rodriguez et al. 85 2009; Santoyo et al. 2016). These attributes make microbial endophytes a target for 86 biotechnological and commercial exploitation (Staniek et al. 2008; Li et al. 2012). 87 Medicinal and wild Cannabis has been reported to harbour competent fungal and bacterial 88 endophytes that are capable of providing different forms of fitness benefits to their associated 4 https://mc06.manuscriptcentral.com/cjm-pubs Page 5 of 49 Canadian Journal of Microbiology Scott et al. Revised- CJM-2018-0108 89 host plants, and yielded insights into plant-microbe and microbe-microbe interactions (Kusari et 90 al. 2013; Gautam et al. 2013). However, no knowledge on the diversity, distribution and 91 biochemical attributes of microbial endophytes recovered from industrial hemp cultivars grown 92 in Quebec exists. 93 Our research goal was to determine the prevalence and types of endophytic bacteria and 94 fungi residing in the above-ground tissue of three industrial hemp cultivars grown under field 95 conditions, and evaluate their biochemical attributes. We then proceeded to identify 96 taxonomically the culturable bacteria and fungal endophytes by 16S rRNA and the internal 97 transcribed spacer (ITS) rDNA gene sequence analysis, respectively, and evaluated their 98 substrate utilization patterns. A few bacterial endophytes were selected to determine their 99 antimicrobial properties against economicallyDraft important fungal pathogens including those that 100 cause disease in industrial hemp and medicinal Cannabis, and we confirmed their internalization 101 and systemic spread of the endophytes in plant tissues of a horticultural plant. 102 103 Materials and methods 104 Farm site, cultivars and sample collection 105 In this study, the term hemp will be used hereafter to refer to industrial hemp. Seeds of 106 three multi-use hemp (Cannabis sativa L.) cultivars approved for production in Canada were 107 used for endophyte discovery. CRS-1 (seed use), and Anka and Yvonne (dual use; seed and 108 biomass) under the terms specified in licenses delivered to Jean-Benoit Charron by Health 109 Canada #12-C0142-R-01, 13-C0142-R-01 and 14-C0142-R-01 (Mayer et al. 2015) were grown 110 at the Emile A. Lods Agronomy Research Centre field site (N45°26´N,W73°55´), of Macdonald 111 Campus of McGill University. Detailed information on soil types and fertilization is fully 5 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 6 of 49 Scott et al. Revised- CJM-2018-0108 112 described in Aubin et al. (2015, 2016). Cultivars were seeded in mid-May of 2013 and grown in 113 plots 1.3 m x 5 m, containing 7 rows, spaced at 18 cm apart. No herbicide was applied, and 114 manual weeding was done on all plots throughout the growing season. Only naturally occurring 115 precipitation and light exposure were used throughout the plants’ growth (Aubin et al.