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? :
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2 Endophytes of industrial hemp (Cannabis sativa L.) cultivars: identification of culturable
3 bacteria and fungi in leaves, petioles and seeds
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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
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10 *Corresponding Author
11 Suha Jabaji Draft
12 Phone: 514 398 7561
13 Email: [email protected]
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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.
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40 Key words: Hemp, endophytes, siderophore, Pseudomonas, molecular detection, antifungal
41 activity.
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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
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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
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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.
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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
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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. 2016).
116 Compound leaves and petioles were collected from plants grown in cultivar trial
117 experiments and also from nitrogen fertility trials. Collection began in mid June of 2013 and
118 continued biweekly from mid June until mid August of 2013. Approximately two dozen leaves
119 including the vascular petiole tissue of each cultivar were collected from multiple plants within
120 the plot at each time point. At maturity, seeds were thrashed from the rest of the biomass using a
121 stationary combine (Aubin et al. 2015). Field samples were placed in labelled Ziplock® bags on
122 ice for transportation, stored in a 4°C walk inDraft cold room, and processed within 48 h of collection.
123 All seeds were stored at 4°C in 50 mL falcon™ tubes until further use.
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125 Isolation of microbial endophytes
126 All plant tissues (leaves, petioles and seed embryos) were surface sterilized following
127 stepwise, agitated immersion in ethanol (70%), sodium hypochlorite (3.5% available Cl ), and
128 sterile deionized water (dH2O) according to Schulz (1993). To maximize the isolation of
129 culturable organisms from different tissues following sterilization, leaves were sectioned (0.5
130 cm) using a sterile scalpel, and placed directly onto the surface of different selective media ideal
131 for bacteria and fungal growth. Petiole sections (a dozen pieces of ~10 mm each), and seed
132 embryos (~approximately 40) were placed in a Waring blender and homogenized in 5 mL of
133 sterile water, and 150 µl of petiole or embryo macerates were spread plated onto selective
134 culture media that are described below. The effectiveness of the sterilization procedure was
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135 tested using both imprinting, and wash plating methods depending upon the material used
136 (Schulz et al. 1993; Ji et al. 2008; Kaga et al. 2009).
137 Bacterial isolates: To ensure the purity of the emergent microorganisms, bacterial colonies
138 from sterilized tissues were passed through four rounds of single colony isolation via streaking
139 on Lysogeny broth (LB; 1.0% tryptone, 0.5% yeast extract, 1.0% NaCl, 1.5 % agar; Difco,
140 Lawrence, KS, USA) or nutrient agar plates (NA; 0.5% peptone, 0.3% yeast extract, 0.5% NaCl,
141 1.5% agar, Difco) amended with filter sterilized, antifungal agent benomyl (10 mg/ L; Sigma
142 Aldrich, ON, Canada). Stock cultures of pure bacteria were prepared from overnight LB broth
143 mixed with 25 % glycerol stock at 1:1 ratio, and stored at 80 °C.
144 Fungal isolates: Emergent fungal mycelia were isolated on potato dextrose agar (PDA; 0.2%
145 dextrose, 0.04% potato starch, 1.5% agar;Draft Difco) or malt extract agar (MEA; malt extract 0.3%,
146 peptone 0.05%, 1.5% agar; Difco) amended with 100 mg.L 1 penicillin and 100 mg.L 1
147 streptomycin (Sigma Aldrich). Fungal isolates were purified through 4 rounds of re isolation by
148 carefully removing a 3 mm section from the edge of the growing colony, transferring to fresh,
149 amended PDA plates and being incubated for an additional 24 48 h before subsequent re
150 isolation. Pure fungal cultures were stored at 80 °C in 25 % glycerol.
151 Molecular identification and characterization of endophytes
152 Bacteria: One hundred and thirty four (134) bacterial strains were grown on LB at room
153 temperature for 16 18 h with agitation (175 RPM) to achieve concentrations between 108 1010
154 CFU.mL 1. Cells were collected for Gram reaction staining (Steinbach and Shetty 2001), and
155 DNA extraction using the PrestoTM Mini gDNA Bacteria Kit (FroggaBio, ON, Canada)
156 according to the manufacturer’s instructions. DNA quality was confirmed on 1 % agarose gel
157 prior to subsequent reactions.
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158 Bacterial primer pairs (27F/534R) amplifying the positions of 27 and 534 of bacterial 16S
159 rRNA genes (Table S1) were used according to previously published protocols (Gagné Bourgue
160 et al. 2013) to identify over 130 bacterial endophytes. The iProofTM High Fidelity (HF) PCR kit
161 (Bio Rad, ON, Canada) and 40 ng of genomic DNA were used per 50 µl reaction. A positive,
162 amplified genomic DNA sample, and negative control without DNA were run concurrently with
163 each reaction. An annealing temperature of 63 °C and 35 cycles were used. The amplified PCR
164 product was cleaned using a Gel/PCR DNA Fragments Extraction Kit (FroggaBio, ON, Canada)
165 as instructed, and sequenced at Genome Québec (Montreal, QC, Canada), or in some cases the
166 PCR product was cloned using the StartClone PCR cloning kit (Agilent Technoliogies, CA,
167 USA) following manufacturer’s recommendations. Purified plasmid DNA was sent for
168 sequencing. Results were queried betweenDraft December 2013 and March 2014 using NCBI’s
169 BLASTn software program (Altschul et al. 1990). The top, named search results were used to
170 identify isolates to the genus level where possible.
171 Sequences of endophytic bacteria were aligned using ClustalW software in SDSC Biology
172 Workbench, and the non conserved regions were used to design primers for selected bacteria
173 (Table S1). Specific primers were synthesized by Integrated DNA Technologies (Corlville, IA,
174 USA) and were tested against all bacteria to ensure specificity.
175 Fungi: Fifty three (53) fungal isolates were grown on MEA culture medium for one week
176 prior to extraction of total genomic DNA using the Extract N Amp Plant DNA extraction kit
177 (Sigma Aldrich). The primers ITS1F and LR3 (Table S1), corresponding to the first ITS of the
178 small subunit, and the large subunit sections of ribosomal DNA were used to amplify a fragment
179 of 1 to 1.2 kbs in length using previously described cycle conditions (Hoffman and Arnold 2010).
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180 The PCR product was cleaned with Exo SapIT reagents (Affymetrix) as directed, and Sanger
181 sequenced at the University of Arizona Genetics core (Tucson, AZ, USA).
182 Sequences were examined and trimmed manually using Sequencer 4.5 (GeneCodes Corp.,
183 MI, USA) to obtain a high quality consensus read. In cases of discrepancies in the consensus
184 read (5.7 % of isolates), the ITS1F and LR3 sequences were assessed separately and search
185 results were compared. A single consensus sequence was also generated in Mesquite version
186 3.02 (Maddison and Maddison 2015) from the ITS1F and LR3 sequences. The consensus
187 identity within the top named hits was used to identify isolates to the family level, and where
188 possible, to the genus level. Fungal sequences were clustered into 90, 95 and 100 % OTU groups
189 based upon sequence similarity using the phylogenetic tool Mesquite. Isolate identity was
190 conducted using the 95% similarity group,Draft which has been found to roughly correspond to the
191 species level in other endophytic fungi (U’ren et al. 2009).
192 Some plant associated fungi harbour bacteria within their hyphae (Hoffman and Arnold,
193 2010). These bacteria reside in living hyphae of fungal endophytes and are known as endohyphal
194 bacteria (EHB). To confirm the presence of EHB within the living hyphae of hemp fungal
195 endophytes, a representative fungal isolate was chosen from 13 different OTUs ( 95 % similarity
196 level), and was queried according to the protocol by Hoffman and Arnold (2010). Briefly, total
197 genomic DNA of identified fungal isolates was amplified with the primers (27F and 1429R) of
198 16S rRNA gene. PCR products of insufficient concentration for sequencing were cloned using
199 Agilent Technologies StraClone kit (Agilent) following the manufacturer’s recommendations.
200 Amplification and sequencing of the positive clones was accomplished using M13F and M13R.
201 Products were cleaned using the ExoSap IT kit (ThermoFisher) as described by the manufacturer
202 and sent for Sanger sequencing at the University of Arizona Genomics Analysis and Technology
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203 Core Facility. An average of 4 separate clones was sequenced per queried OTU. Returned
204 sequences were assessed using the BLASTn program April 2015, and results of E. coli, or fungal
205 species were dismissed as cloning artifacts.
206 Microbial phylogenetic tree generation
207 Reverse sequences of 534R amplified sequences from DNA of bacterial endophytes were
208 generated using the Reverse Sequence tool from Bioinformatic Organization (MA, USA) and
209 aligned into a single sequence in BioEdit v7.2.5 (Ibis Biosciences; CA, USA). Sequences were
210 aligned using the CLUSTAL tool to be used in tree generation.
211 Phylogenetic trees for bacterial and fungal isolates were constructed separately from 212 aligned DNA sequence files using the freewareDraft program MEGA version 6.0 (Tamura et al. 2013). 213 Trees were generated using Maximum Parsimony methods and used a Subtree Pruning Re
214 grafting algorithm (Nei and Kumar; 2000). Positions with fewer than 95% site coverage were
215 eliminated. The bootstrap consensus tree was inferred from 1000 replicates, and only branches
216 above 50% bootstrap score were displayed. The analysis for bacteria involved 134 nucleotide
217 sequences, and 53 nucleotide sequences for fungi. All positions containing gaps and missing data
218 were eliminated. In the bacterial tree there were a total of 310 positions in the final dataset and
219 for fungi there were 676 positions.
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221 Phenotypic and chemical characterization of endophytes
222 Several chemical tests listed in Table S2 were conducted for the characterization of
223 biochemical traits of bacterial and fungal isolates.
224 Bacteria: The enzymatic assays and biochemical tests were performed in triplicates to
225 characterize the bacterial endophytes. Single bacterial colonies were grown in 4 mL of LB broth
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226 overnight (16 18 h, 175 RPM). Following appropriate dilution in LB broth, 25 µl of 105
227 CFU.mL 1 were used in all tests unless otherwise stated.
228 Cellulase and phosphatase solubilization: Cellulases were assayed on indicator plates with
229 carboxymethyl cellulose (Sigma Aldrich) amendment, and stained in Congo red solution (0.2 %)
230 as outlined by Gupta et al. (2012). The ability of endophytic bacteria to solubilize inorganic