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
1 231 phosphate (HPO4)2 was assayed on agar medium containing inorganic phosphate (g.L : glucose,
232 10; NH4Cl, 5.0; NaCl, 1.0; MgSO4*7H2O, 1.0; Ca3(HPO4)2, 0.8; agar, 15, pH 7.2) according to
233 Verma et al. (2001). Development of clear halo zones after 48 h around the strains exhibited their
234 positive phosphate solubilization activity.
235 Siderophore production: Bacteria were assessed for siderophore activity by growing
236 cultures on Chrome azurol S (CAS) media,Draft a complex mixture of Fe CAS (Sigma Aldrich)
237 indicator, piperazine N,N′ bis(2 ethanesulfonic acid) (PIPES) (Sigma Aldrich), and 10 % sterile
238 casamino acid (Difco) solutions as outlined in Alexander & Zuberer (1991). Development of
239 yellow orange halo zone after 24 48 h around the bacterial spot is considered as positive
240 indication for siderophore production.
241 Hydrogen cyanide: (HCN): HCN gas production was assessed based upon a change from
242 bright yellow to orange colour in picric acid (0.5 %, RICCA Chemicals) soaked Whatman filter,
243 lining the lid of a sealed petri dish (Bakker and Schippers 1987).
244 Organic acid production (OAP): OAP was determined using a modified methyl red test
245 (Kumar et al. 2012). Briefly, bacteria were grown in 5 mL of glucose phosphate broth at room
246 temperature with agitation (175 rpm) for 4 days. A small amount (3 5 drops) of methyl red
247 solution was added to the culture and mixed gently. Color change to deep red was scored as
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248 positive, deep orange was scored as a weak reaction and maintenance of yellow coloration was
249 scored negatively. Pure LB and HCL were used as negative and positive controls, respectively.
250 Hormone Production: Indole Acetic Acid (IAA) production was assessed as described by
251 Husen (2003) using L tryptophan (final concentration: 5mM, Sigma Aldrich) amended LB
252 media, shaken for 4 days (23 °C, 175 RPM). The culture supernatant was mixed with FeCl3
253 HClO4 reagent (1:2 ratio) and permitted to react in the dark for 30 min. prior to being
254 spectrometric scoring at 530 nm. Biological replicates and the IAA chemical standards were
255 done in triplicate to provide accurate quantification. Production of IAA was confirmed by the
256 development of pink colour.
257 Antibiotic sensitivity: Bacterial endophytes were tested individually on agar plates
258 containing antibiotics following the procedureDraft of Gagné Bourgue et al. (2013) at either 100
259 µl.mL 1: kanamycin, rifampin, (Sigma Aldrich) streptomycin (Bioshop, ON, Canada) and
260 tetracycline (Fisher Chemicals, ON, Canada); or 125 µl /mL: ampicillin, gentamicin, (Sigma
261 Aldrich Co.) chloramphenicol, (ICN Biomedicals, OH, USA) and hygromycin (Santa Cruz; TX,
262 USA). Bacteria were considered sensitive to an antibiotic at the concentration tested if no visible
263 growth was observed on treatment plates, when there was visible growth on control plates after
264 48 h of incubation at 27°C.
265 Antifungal activity: The ability of endophytes to restrict the radial growth of seven fungi:
266 Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium graminearum, F. solani, Helminthosporium
267 solani, Rhizoctonia solani and binucleate Rhizoctonia was performed as previously described
268 (Gagné Bourgue et al. 2013). The experiment was performed at three separate time points and in
269 triplicate for each bacterium. Reduction in fungal growth was measured as percent of inhibition
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270 ratios and using the formula: (C T)/C X 100; where C is the average radial of control growth and
271 where T is bacterial treatment radius.
272 Fungi: Cellulases and ligninases: Cellulose amended MEA, and indulin AT
273 (MeadWestvaco, VA, USA) amended water agar were used for detecting cellulose and ligninase
274 activities, respectively as described (Gupta et al. 2012; Sundman and Nase 1974). Fungi were
275 allowed to grow for an additional week prior to ligninase testing. For both tests, indicator dye
276 solutions were applied to assess activities at 4 8 days (cellulase) or 11 15 days (ligninase).
277 Assays were performed in biological triplicates and repeated at least once to obtain a consensus.
278 Colonization and tissue internalization of bacterial endophytes in tomato seedlings 279 As proof of concept, we assessedDraft whether representative bacterial strains, that scored 280 positive in at least 6 of the biochemical tests, and exhibited a significant antifungal activity in
281 confrontation assays with pathogenic fungi, are able to colonize and internalize tissues of a dicot
282 plant. The re introduction of bacterial endophytes in the absence of a competing microbial flora
283 is deemed promising for further greenhouse studies. Thus, we selected tomato (solanum
284 lycopersicum L.) as the model dicot plant instead of hemp seeds because hemp seeds could not
285 be rendered completely sterile without significant loss of viability.
286 Single cell colonies of bacterial strains of BTG8 5 and BTC8 1, putatively identified as P.
287 orientalis and P. fulva, respectively were isolated from the petiole tissue of field grown hemp,
288 and were grown in liquid culture in overnight growth in 5 mL of LB (23°C, 175 RPM). These
289 strains exhibited the best biochemical attributes. Three to 4 seeds of S. lycopersicum cv.
290 Beefsteak, surface sterilized as previously described, were planted in 10 g (3 cm deep) of
291 sterilized Agromix® potting soil G12 (Fafard et Frères LTD., QC, Canada) in magenta boxes.
292 Following seeding, 5 mL of 1 X Hoagland solution (Hoagland and Arnon 1950) was added per
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293 box. Seedlings were grown for one week at 16/8 h, day/night cycles and 25/20 °C before
294 thinning to a single, healthy plant per box. Plants were soil drenched with 5 mL of 105 CFU/mL
295 of freshly grown BTG8 5 or BTC8 1 in LB broth at 14 days post seeding (DPS). Control plants
296 were drenched in 5 mL of sterile LB broth. There were 5 replicates per treatment and the
297 experiment was repeated once in a temporally separated trial. At 28 DPS, seedlings were
298 harvested, separated into root, petiole or leaf tissues and surface sterilized as previously
299 described under isolation of microbial endophytes section.
300
301 Confirmation of bacterial endophytes via culture-independent method and PCR assays:
302 A subset of sterile tissue was retained for PCR reaction using the primer set BT14
303 4F1/BT14 4R1 specific for P. orientalisDraft and P. fulva (Table S1) in order to detect and confirm
304 colonization of the target endophytes in plant tissues. Amplification was conducted using HF
305 Bio Rad PCR and similar conditions as described in the molecular identification and
306 characterization of endophytes section with an annealing temperature of 67.5 ºC. Tissue samples
307 were ground to a fine powder in liquid nitrogen prior to DNA extraction. Between 100 and 400
308 mg of samples were used in a modified CTAB method of DNA extraction (Carrigg et al. 2008).
309 Briefly, the sample was incubated with lysis buffer [100 mM Tris HCl; 2 M NaCl; 25mM EDTA,
310 pH 8.0; 5 % polyvinylpyrrolidine; 3 % Cetyl trimethylammonium bromide (CTAB)], 2
311 mercaptoethanol, and 1µl RNAse A (Bioshop, ON, Canada) for 1 h at 70 ºC. Samples then
312 underwent one phenoly:chloroform:isoamyl alcohol (25:24:1) extraction; 2 rounds of
313 chloroform:isoamyl alcohol (24:1) extraction and overnight precipitation in 2/3 volume
314 isopropanol and 1/10 volume 3% sodium acetate (pH 5.2) at room temperature (23 ºC).
315 Precipitated DNA was collected, washed in 70% ethanol and eluted into pure dH2O (pH 7.0).
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316 DNA was visualized on a 1 % agarose gel to confirm quality prior to amplification using the
317 Pseudomonas specific primers BT14 4F1/BT14 4R1 (Table S1).
318 The remaining tissues were ground with 5 mL of sterile water, serially diluted. One 100 µl
319 was spread on non amended LB and incubated at 23°C for 2 days prior to colony forming unit
320 (CFU) scoring. Each dilution point was done in triplicates for each tissue and each treatment for
321 each trial.
322
323 Results
324 Distribution of microbial endophytes
325 Over the course of the growing season of 2013, 1000 tissue segments (leaves, petioles and
326 seeds), collected from hemp cultivars wereDraft screened for culturable endophytes. A total of 134
327 bacterial and 53 fungal strains were isolated and identified to the genus level (Figs 1 and 2).
328 The distribution of bacterial isolates was generally similar among the three hemp cultivars (Fig.
329 1A) with the majority isolated from petiole tissues (67%), followed by leaf (19%) and seed
330 tissues (14%) (Fig. 1B). On the other hand, the majority of the fungal strains (45%) originated
331 from the cultivar Anka followed by cultivar CRS 1 (36%) and cultivar Yvonne (19%) (Fig. 1C).
332 More than half of the fungal isolates originated from leave tissue (Fig. 1D).
333 Irrespective of cultivar or tissue, more bacterial isolates were collected at early and mid
334 late growing periods compared to late growing period (Fig. 1E). There was no particular trend in
335 fungal isolate incidence with consistent numbers at each time point and a slight decrease at later
336 growth periods (Fig. 1E).
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337 Isolate Identification
338 All 134 bacterial strains were partially identified using 16S rRNA primers 27F/534R, and
339 in many cases to the species level. Additional cloning using M13 vector and sequencing was
340 done for some isolates of increased interest to confirm species designation (Table S1). The
341 strains were grouped into 18 different taxa that shared high homology with known sequences and
342 data for endophytic strains have been deposited under the following accession numbers
343 Kx430321 KX430455 with the most frequently isolated endophytes belonging to the gram
344 negative genera, Pseudomonas (35%), and Pantoea (17%), and the gram positive genera,
345 Staphylococcus (16%), and Bacillus (9%) (Fig. 2A).
346 The highest abundance of bacterial strains isolated from leaves were isolates of
347 Pseudomonas (11/25) and Bacillus (4/25).Draft These genera constituted more than half (60%) of the
348 strains isolated. From petioles, strains belonging to Pseudomonas (35/90), Pantoea (14/90),
349 Staphylococcus (14/90) and Bacillus (5/90) were the most abundant. The highest genera
350 associated with the seed were Pantoea (7/19) Staphylococcus (4/19), Bacillus (3/19) and
351 Enterobacter (3/19) (Fig. 3). The distribution of certain taxonomic groups of endophytes was
352 genotype dependent (Table S3). Acinetobacter, Agrobacterium and Enterococcus were
353 recovered from petiole tissues of cultivar Anka. Isolates belonging to Erwinia, Paenibacillus
354 and Rhizobium were recovered from cultivar CRS 1 (Table S3), while the genus
355 Microbacterium was recovered from petioles of cultivar Yvonne.
356 All 53 fungal strains were putatively identified by sequencing the amplified fragments
357 using the ITS1F/LR3 primer pair (Table S1) and grouped into 13 genera with deposited
358 accession numbers KX64193 KX641987 (Fig. 2B). All fungal isolates were members of the
359 dikaryota, with the majority (96 %) belong to the Ascomycota. Only two of the 53 isolates were
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360 members of Basidiomycota, namely: Irpex, and Cryptococcus (Fig. 2B). The most commonly
361 isolated fungal endophytes belonged to the genera Aureobasidium (24%), Alternaria (19%),
362 Cochliobolus (19%), and Cladosporium (15%). Hemp leaves harboured in high abundance
363 isolates belonging to Cochliobolus (10/37) and Aureobasidium (9/37). The highest number of
364 genera associated with petioles was Alternaria (7/14) and Cryptococcus (4/14). Only strains
365 belonging to Aureobasidium (1/2) and Cladosporium (1/2) originated from seeds (Fig. 3B).
366 Certain fungal groups were cultivar specific (Table S4). Isolates belonging to the taxonomic
367 groups Dreschlera and Irpex and Sordariomycetes were Anka specific (Table S4). Fungi
368 belonging to Pleosporales were recovered only from cultivar Yvonne, while Eutypella,
369 Pezizomycetes and Stagonosporpsis were recovered from cultivar CRS 1 (Table S4).
370 The presence of EHB using the genomicDraft DNA of a representative fungal strain from each
371 taxon revealed 6 putative host candidates (Table 1). BLAST search results suggest that putative
372 EHB matched primarily uncultured and often unnamed bacteria. Among named hits were
373 Acinetobacter and Staphylococcus. Of those fungi showing putative EHBs, 4 showed
374 Mycoplasma infection (Table 1).
375 Phylogenetic trees constructed for bacterial and fungal isolates clearly support the
376 BLASTn sequence identification of large groups of bacteria and fungi (Figs 4 and 5). The
377 percentage of replicate trees in which the associated taxa clustered together in the bootstrap test
378 (1000 replicates) is shown next to the figures with bootstraps > 50%. The dominant isolate
379 groups (Pseudomonas, Pantoea and Staphylococcus) remain reasonably well delineated (Fig. 4).
380 The dominant isolate groups for fungi that were well delineated belonged to the Ascomycota
381 (Fig. 5).
382 Biochemical characterization and antagonistic property of hemp endophytes
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383 Initial screening showed the production of siderophore (13/18) and cellulases (11/18) is the
384 most commonly occurring traits found across genera, followed by phosphate solubilization
385 (6/18), and HCN and organic acid production (5/18) (Table 2). All genera except Brevibacterium,
386 Curtobacterium, Microbacterium, Paenibacillus and Staphylococcus were siderophore
387 producers.
388 The top performing strains were all members of the Pseudomonas genus: BTC6 3, BTC8 1,
389 BTG8 5, and BT14 4. All four strains showed varying levels of solubilization of inorganic
390 phosphate, production of siderophores, and all except BTC6 3 demonstrated cellulytic activity
391 (Table 3). HCN production was noted for only BTC6 3 and BTC8 1, and organic acid
392 production was positive in BTC6 3 and BTG8 5. These strains were additionally tested for IAA
393 production, ACC deaminase activity,Draft antibiotic resistance, and fungal inhibition in direct
394 confrontation assays. BTC6 3 and BT14 4 were found to produce the greatest amounts of IAA
395 (10.96 and 9.46 g.mL 1, respectively).
396 Analysis of resistance to various antibiotics showed that all strains belonging to gram
397 positive and gram negative genera were sensitive to rifampin, chloramphenicol and gentamicin
398 at the concentration tested. For gram negative bacteria, resistance to hygromycin (82%) and to
399 ampicillin (64%) was most common. Gram negative strains demonstrated resistance to
400 hygromycin (74%) and ampicillin (43%) (Data not shown)
401 All 53 identified fungal strains were assessed for the production of cellulases and
402 ligninases. Cellulase activity was found to be widespread among the genera (39/53) with 6% of
403 the strains belonging to Cladosporium and Aureobasidium producing lytic zones greater than 10
404 mm. Only 4 strains (2/53) showed ligninase activity: FL11 3, a Pleosporales sp. that showed
405 moderate strength, followed by FL12 3, a Stagonosporopsis sp. (Data not shown).
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406
407 Antifungal activity
408 Seven fungi, covering a wide range of lifestyles were chosen for confrontation
409 assessment against BTC6 3, BTC8 1, BTG8 5, and BT14 4 (Table 4). Botrytis cinerea,
410 Sclerotinia sclerotiorum and Rhizoctonia solani, are pathogens of hemp (McPartland et al. 2000)
411 and were of particular interest. S. sclerotiorum was significantly affected, displaying reductions
412 in radial growth ranging from 17 to 54% against BTC6 3 and BTC8 1, respectively. Growth of B.
413 cinerea was significantly reduced by BTC8 1 and BTG8 5 (19% and 22% reduction).
414 Rhizoctonia solani was significantly reduced by BT14 4 (28%, P=0.0141), and radial growth of
415 binucleate Rhizoctonia was significantly affected in confrontation assays with BTC8 1 and
416 BTG8 5 strains (23.4 and 31.3% reduction).Draft However no other tested fungi were significantly
417 affected (P =0.05).
418
419 Recolonization, internalization and detection of bacterial endophytes in plant tissues
420 The surface sterilization method combined with the imprint technique ensured that the
421 endophytic colonization cell numbers reflect only the number of cells within the interior of the
422 plant tissues. This method is sufficient to kill and/or wash away the surface bacteria while
423 maintaining the survival of the interior bacteria. Bacterial colonies that matched the
424 Pseudomonas phenotype in treated tomato plants were successfully detected in colonized plant
425 homogenates while surface sterilized tissues of control non colonized plants did not yield
426 culturable bacterial colonies. Re isolation and quantification of P. fulva (BTC8 1) and P.
427 orientalis BTG8 5 by the plating method in different tissues of tomato seedlings after soil drench
428 with BTG8 5 and BTC8 1, respectively clearly demonstrate that both strains can form sustaining
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429 endophytic populations in roots, shoots and leaves of tomato (Table 5). However, population
430 numbers in roots were more variable than in petioles and leaves in both trials. Amplification of
431 genomic DNA extracted from colonized tissue samples using designed Pseudomonas specific
432 primers BT14 4F1/BT14 4R1 gave the expected band size (230 bp) in all treated samples.
433 Absence of endophytes was confirmed in tissues of non inoculated tomato seedlings (data not
434 shown).
435
436 Discussion
437 The main focus of this study is to examine varieties of hemp grown in Quebec for the
438 presence of endophytic bacteria and fungi, and to determine their taxon level. Strategically, we
439 were interested in examining the maximumDraft diversity of culturable microbes isolated from above
440 ground plant tissues grown on different microbiological culture media with different sources of
441 carbon. Such an approach may not favour the isolation of viable microbial endophytes that are
442 slow growing or unable to grow. Full assessment of the abundance of unculturable endophytes
443 isolated from hemp is now possible by DNA finger printing and pyrosequencing (Sun et al.
444 2008; Lundberg et al. 2012).
445 This study demonstrated that hemp cultivars harbour diverse assemblage of culturable
446 bacteria and fungi that exist as endophytes and belong to 18 and 13 diverse taxonomic groups,
447 respectively. All strains identified in this work were related to previously identified species with
448 plant growth promoting or biological control activity (Rosenblueth & Martinez Romero, 2006).
449 To the best of knowledge, this is the first report describing the diversity and the distribution of
450 indigenous bacteria and fungi harbouring hemp cultivars, Anka, CRS 1 and Yvonne that are
451 widely used by the local agricultural community for seed and oil production.
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452 Several factors including plant genotype variation, soil type and varying environmental
453 conditions are thought to promote shifts in the structural and functional characteristics of
454 endophyte communities (Dalmastri et al. 1999; Sessitsch et al. 2002; Berg et al. 2002). In this
455 study, the distribution of certain taxonomic groups of endophytes was genotype dependent.
456 Cultivar specificity to bacteria and fungal endophytes has been reported in several crops
457 including Cannabis (Bailey et al. 2005; Rasche et al. 2006; Rosenblueth et al. 2012; Wearn et al.
458 2012; Winston et al. 2014). Hence, studies on biodiversity and cultivar specificity of cultured
459 endophytic microorganisms associated with hemp merit further research.
460 Tissue type is an important factor for endophyte colonization (Compant et al. 2008;
461 Massimo et al. 2015). In this study, the most heavily colonized tissue differed between bacterial
462 and fungal isolates in C. sativa. This distributionDraft pattern of bacterial endophytes in petiole tissue
463 as opposed to leaf and seed tissues is corroborated in studies of other plants (Elvira Recuenco
464 and Vuurde 2000; Kuklinsky Sorbel et al. 2004; Trotel Aziz et al. 2008; Abraham et al. 2013).
465 In contrast, fungal endophytes were isolated with higher frequencies from hemp leaves compared
466 to petiole and seed tissues. Similar patterns of fungal frequency were also reported in medicinal
467 Cannabis (Guatam et al. 2013), and in other plants such as Antarctic grass (Rosa et al. 2009),
468 loblolly pine (Arnold et al. 2007), cyress (Soltani and Moghaddam 2015), and ginseng (Park et al.
469 2012).
470 Seed embryos harboured the lowest numbers of bacterial (19) or fungal isolates (4) of all
471 isolated endophytes. This observation is consistent with similar trends reported elsewhere
472 (Ganley and Newcombe 2006;Truyens et al. 2015). It is reported that the majority of seed
473 associated bacterial endophytes belong to Proteobacteria such as Bacillus, Staphylococcus,
474 Pseudomonas and Pantoea (Rosenblueth et al. 2012; Truyens et al. 2015). The seed associated
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475 endophytes reported in this study belonged to the above taxa. Only two fungal endophytes
476 (Aureobasidium sp. and Cladosporium sp.) were recovered from seeds, and both are reported
477 elsewhere as seed endophytes (Hodgson et al. 2014; Geisen et al. 2017).
478 Many plant associated fungi harbour endosymbiotic bacteria (EHB) that are capable of
479 altering plant fungus interactions (Shaffer et al. 2016). Recent evidence indicates that EHB occur
480 often in diverse leaf endophytic Ascomycota (Hoffman and Arnold 2010), and they can confer
481 plant growth promoting traits when present within their fungal host (Hoffman et al. 2013). This
482 relationship is easily lost through sub culturing of fungal culture on antibiotic amended media
483 (Hoffman and Arnold 2010). In this study, isolation of fungal endophytes was performed on
484 antibiotic amended PDA or MEA, and is most likely that many EHB were lost as a result of
485 antibiotic treatment, despite the fact thatDraft EHB are believed to be widespread (Sharma et al. 2008;
486 Hoffman and Arnold 2010).
487 Endophytic microbes that possess the ability to degrade the plant polymer cellulose have
488 been suspected of colonizing internal tissues of the plants (Compant et al. 2010; Reinhold Hurek
489 and Hurek 2011). Additionally, cellulase producing strains are also able to enhance phosphorus
490 availability to plants through mineralization of organic P by acid phosphatases or by
491 solubilization of mineral phosphate via the production of organic acids (Richardson et al. 2009;
492 Khan et al. 2014). Bacillus, Pseudomonas, Cedecea, and Enterobacter strains, isolated from
493 hemp (this study) and from different crops are reported to solubilize/mineralize minerals such as
494 phosphorus making them more readily available for plant growth (Hameeda et al. 2008; Gagné
495 Bourgue et al. 2013; Akinsanya et al. 2015). This attribute along with the ability of producing
496 cellulases is indicative of their nutrient delivering capacity while interacting with plant hosts
497 (Glick et al. 2007).
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498 The ability to produce siderophores and hydrogen cyanide represents some of the traits that
499 make microorganisms successful competitors in several environments (Compant et al. 2005;
500 Loaces et al. 2011). HCN is a powerful biocontrol agent may protect plants from biotic stresses;
501 bacteria with this property are known to inhibit several plant pathogens and can indirectly
502 enhance plant growth (Santoyo et al. 2012). In our study, the majority of the taxonomic groups,
503 produced siderophores, while 38% of the genera were HCN producers. Pseudomonas, the largest
504 taxon with the highest abundance of strains isolated from hemp, produced HCN, siderophore,
505 and IAA. These traits provide Pseudomonas strains a competitive advantage to colonize plant
506 tissues, contribute to disease suppression by the production of antibiotics or HCN, and exclude
507 other microorganisms from the same ecological niche (Reiter et al. 2002; Leong 1986).
508 The siderophore producing PseudomonasDraft strains in this study, P. fulva (strains BTC6 3
509 and BTC8 1) and P. orientalis (BTG8 5 and BT14 4), exhibited antifungal activities against
510 several plant pathogenic fungi including B. cinerea, a known pathogen of hemp and medicinal
511 Cannabis. Direct evidence to support siderophore production of Pseudomonas strains with
512 antifungal activity has been found during studies on the biocontrol of fungal wilt diseases
513 (Kloepper et al. 1980; Lim et al. 1999) and damping off diseases (Weller 1988)
514 One of the aims of this study was to confirm internal colonization and systemic spread of
515 selected Pseudomonas strains in tomato seedlings under gnotobiotic conditions. Pseudomonas
516 orientalis strain BTG8 5 and P. fulva strain BTC8 1 were detected in roots, petioles and leaves
517 of young tomato seedlings after reintroducing them as soil drench application. These results
518 confirm that the endophytes have moved from the roots upward to the petiole and leaves and
519 maintained reasonable population densities. The exact location of Pseudomonas strains in root
520 and aerial tissues remains to be investigated. Our results concur with previous reports that some
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521 endophytes are able to migrate from root tissue to the upper parts of the plant (Zakria et al. 2007;
522 Lin et al. 2009; Gagné Bourgue et al. 2013). Variation in bacterial population number inside
523 roots and petioles was noticed in our trials. It has been reported that under gnotobiotic and field
524 conditions, the root system is not colonized in a uniform manner, leading to variation in bacterial
525 colonization (Gamalero et al. 2005). The contribution and functionality to plant growth of
526 endophytes localized in aerial parts was not attempted in this study. Future studies are necessary
527 to correlate colonization of the strains, specifically those with specific functions that are
528 important for plant health and growth.
529 Taken together and based on the results obtained in this study, the genus Pseudomonas has a
530 wide spread distribution in the above ground tissues of hemp cultivars. In this work, the
531 antagonistic activity of P. orientalis andDraft P. fulva strains along with their biochemical abilities of
532 siderophore, HCN, IAA production and P solubulization, make these strains excellent candidates
533 as bioresource for the production of the above bioactive compounds. Additionally, research
534 aimed at using these strains to increase plant fitness, suppress disease or augment production of
535 secondary compounds in medicinal marijuana varieties, the closest members of the Cannabis
536 sativa, is highly desirable.
537
538 Acknowledgements
539 The authors are indebted to the Ministère de l’Agriculture, des Pecheries et de
540 l’Alimentation du Québec (MAPAQ), PSIA research grant no. 811025.
541
542
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829 cultivated rice. Microbes Environ. 22(3): 197 206. doi: 10.1264/jsme2.22.197
830 831
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832 833 Legend 834
835 Figure 1. Distribution, frequency of recovered strains of endophytes in Industrial hemp. (A)
836 Bacteria endophytes in hemp cultivars and (B) in different tissues. (C) Fungal endophytes in
837 hemp cultivars and (D) in different tissues. (E) Number of bacteria and fungal strains isolated
838 from industrial hemp over the growing season of summer 2013.
839 Figure 2. Taxonomic groups of culturable endophytes (A) Bacteria and (B) Fungi based on
840 partial sequencing based on 16S rRNA and rDNA ITS analysis, respectively.
841 Figure 3. Distribution of taxonomic taxa of culturable bacteria (A,B,C) and fungal (D,E,F)
842 endophytes isolated from leaves (A,D), petioles (B,E) and seeds (C,F).
843 Figure 4. Maximum parsimony PhylogenyDraft of DNA of endophytic bacteria isolated from
844 industrial hemp. The nodes are supported by 1,000 bootstrap replications. Bootstraps above 50%
845 and the genetic scale are shown. Sequences were obtained and the NCBI accession numbers are
846 listed after each strain.
847 Figure 5. Maximum parsimony Phylogeny of DNA of endophytic fungi isolated from industrial
848 hemp. The nodes are supported by 1,000 bootstrap replications. Bootstraps above 50% and the
849 genetic scale are shown. Sequences were obtained and the NCBI accession numbers are listed
850 after each strain
851 852 853
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Table 1. List of fungal endophytes of hemp that possess positive sequences for endohyphal bacteria Fungal Genus of Fungus Putative Endohyphal Bacteria Support Value# Host (%) strain (95 % OTU Group Level) * (Top BLAST hits from multiple clones)
FLC6-1 Pezizomycetes Novosphingobium 99
Uncultured alpha proteobacterium 99
Acinetobacter 99
FLC7-7 Sordariomycetes Uncultured rumen bacterium 95
Uncultured bacterium partial 16S rRNA 99
Mycoplasma 98
FTG8-1 Aureobasidium Uncultured bacterium partial 16S rRNA gene 99
DraftAcinetobacter 99
Staphylococcus 99
FS9-1 Cladosporium Acinetobacter 99
Uncultured bacterium clone 99
Mycoplasma 99
Staphylococcus 99
FL11-8 Cryptococcus Mycoplasma 98
Uncultured Propionibacterium clone 99
FT13-2 Dothideomycetes Mycoplasma 98
*A Single representative fungal isolate was chosen from 17 different OTUs at the 95 % similarity level. An average of 4 separate clones were sequenced per queried OTU. BLASTn searches were conducted April 2015 # BLASTn support for the identified Putative endohyphal bacteria
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------5 + + + ++ ++ Production Production Organic Acid Organic
- - - - - + + + + + + + + + + + + 13 13 Siderophore Production Siderophore
------HCN
- - - - - + - - - - 6 5 + + + - + - + + + + ++ - Phosphatase
Draft Canadian Journal of Microbiology ------+ + + + + + - - + + - + + + + + + 11 11 https://mc06.manuscriptcentral.com/cjm-pubs Cellulase 11 11 Erwinia Bacillus Cedecea Rhizobium. Pantoea sp. Enterobacter Paenibacillus Enterococcus Pseudomonas Acinetobacter Xanthomonas Ochrobactrum Agrobacterium Staphylococcus Brevibacterium Curtobacterium Microbacterium Genus Genus Stentrophomonas
1 2 3 4 5 6 7 8 9 17 17 18 18 16 16 15 15 14 14 13 13 12 12 11 11 10 10 Total Biochemical attributes of bacterial genera associated with industrial hemp tissues tissues hemp industrial with associated bacterial genera attributes of Biochemical Number of Genera Number Note: - , negative reaction; +, positive reaction showing a clearing zone of 1-3 mm; ++, positive reaction showing a clearing zone zone a clearing showing reaction positive mm; zone ++, 1-3 of a clearing showing reaction positive reaction; , negative +, - Note: mm 4-8 of Table 2.
γ
+ ++ +++ +++ Siderophore Production γ
+ ) HCN 1
3.39±0.59 + 3.36±0.045 10.96±0.31 9.46±0.101 produced IAA (µg.ml γ
+ + Organic Acid γ
+ + ++ ++ Draft Canadian Journal of Microbiology Phosphatase
γ https://mc06.manuscriptcentral.com/cjm-pubs
+ + + strains strains . All tests . replicatedAll were three times and the averages are presented. Pseudomonas
Pseudomonas sp Genus and speciesGenus Activity Cellulase Pseudomonasorientalis Pseudomonasorientalis
Pseudomonas fulva Pseudomonas fulva
. Attributes .of Attributes top the 4 $
Strain BT14 4 BTC6 3 BTC8 1 BTG8 5 Table 3 Table strains are$ All γ Halo ratedsize +, follows: as was Positive (halo <5 mm); ++, Positive and (5<10 +++, mm< Positive (halomm); >10 mm) Page 41 of 49 Canadian Journal of Microbiology Page 42 of 49
Table 4. Inhibition of radial growth of test fungi in confrontation assays Fungus Characteristic Treatment$ Average % Inhibition Significance* Radius (cm)# (P value) Sclerotinia sclerotiorum Broad Pathogen Control 3.44 - - BTC6-3 2.84 17.4 0.1712 BTC8-1 1.59 53.8 <0.0001 BTG8-5 1.79 47.8 <0.0001 BT14-4 1.69 50.7 <0.0001 Botrytis cinerea Broad Pathogen Control 2.96 - - BTC6-3 2.79 5.8 0.9369 BTC8-1 2.53 18.6 0.0248 BTG8-5 2.31 22.0 0.0242 BT14-4 2.56 13.4 0.3321 Rhizoctonia solani Broad Pathogen Control 3.29 - - BTC6-3 3.30 -0.3 1.0000 BTC8-1 2.56 22.3 0.0717 BTG8-5 2.45 25.6 0.1279 BT14-4 2.38 27.6 0.0141 Binucleate Rhizoctonia Biocontrol Control 3.00 - - BTC6-3 2.89 3.8 0.9908 BTC8-1 2.30 23.4 0.0373 BTG8-5 2.06 31.3 0.0065 BT14-4 2.39 20.6 0.0881 Trichoderma virens Plant Growth Control 2.88 - - Promoting BTC6-3 2.89 -0.2 1.0000 Fungi BTC8-1Draft 2.66 7.6 0.8168 BTG8-5 2.40 16.8 0.1468 BT14-4 2.61 9.3 0.6763 Colletotrichum Broad Pathogen Control 2.61 - - gloeosporioides BTC6-3 2.74 -5.1 0.9937 BTC8-1 2.45 6.4 0.9734 BTG8-5 1.90 27.2 0.1740 BT14-4 2.27 13.2 0.7484 Stachybotrys elegans Biocontrol Control 2.64 - - BTC6-3 2.87 -8.7 0.8651 BTC8-1 2.16 18.3 0.2165 BTG8-5 2.03 23.2 0.2216 BT14-4 2.24 15.3 0.3814 Helminthosporium Limited Control 1.87 - - solani Pathogen BTC6-3 2.04 -9.4 0.3993 BTC8-1 2.11 -13.2 0.3805 BTG8-5 2.10 -12.5 0.0951 BT14-4 1.85 1.2 0.9999 Fusarium solani Broad Pathogen Control 2.52 - - BTC6-3 2.31 8.5 0.9931 BTC8-1 2.27 9.8 0.8605 BTG8-5 2.10 16.6 0.6985 BT14-4 2.30 8.7 0.9848 F. graminearum Broad Pathogen Control 1.98 - - BTC6-3 2.04 -3.1 0.8926 BTC8-1 2.11 -6.7 0.8014 BTG8-5 1.81 8.8 0.4330 BT14-4 2.05 -3.5 0.8605 $ All strains are endophytes belong to Pseudomonas sp. # Mean of three replicates of the radial growth of fungi measured in cardinal points surrounding the colony. *Values shown are the results of a Tukey’s Honest Significant Difference test of means using p=0.05 comparison between treatment and control levels. Values are rounded to the fourth decimal and are indicated when less than 0.0001 as <0.0001.
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Table 5. Dynamics of Pseudomonas strains in a dicot model host plant
Treatment Trial Tissue Log cfu/ml± S.D.*
BTC8-1 1 Leaf 2.80 ± 0.14 Stem 3.20 ± 0.19 Root 2.83 ± 0.08 2 Leaf 2.02 ± 0.41 Stem 1.70 ± 0.00 Root 4.13 ± 0.19
BTG8-5 1 Leaf 2.99 ± 0.07 Stem 0.82 ± 0.34 Root 1.30 ± 0.49 2 Leaf 3.24 ± 0.18 Stem 2.18 ± 0.23 Root 6.42 ± 0.14
*Values represent the average of three replicates + standard deviation (S.D.) Colony forming unit (CFU/ml) abundance was determined using sterilized tissuesDraft of bacterized tomato seedlings grown under sterile conditions. CFU values are significantly different between trials and are presented separately as averaged values. Sterilized tissues of non-inoculated control plants did not show colony growth
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. Distribution, frequency of recovered strains of endophytes in Industrial hemp. (A) Bacteria endophytes in hemp cultivars and (B) in different tissues. (C) Fungal endophytes in hemp cultivars and (D) in different tissues. (E) Number of bacteria and fungal strains isolated from industrial hemp over the growing season of summer 2013
180x180mm (300 x 300 DPI)
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Taxonomic groups of culturable endophytes (A) Bacteria and (B) Fungi based on partial sequencing based on 16S rRNA and rDNA ITS analysis, respectively
160x128mm (300 x 300 DPI)
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Distribution of taxonomic taxa of culturable bacteria (A,B,C) and fungal (D,E,F) endophytes isolated from leaves (A,D), petioles (B,E) and seeds (C,F).
160x195mm (300 x 300 DPI)
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Maximum parsimony Phylogeny of DNA of endophytic bacteria isolated from industrial hemp. The nodes are supported by 1,000 bootstrap replications. Bootstraps above 50% and the genetic scale are shown. Sequences were obtained and the NCBI accession numbers are listed after each strain.
119x119mm (600 x 600 DPI)
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Maximum parsimony Phylogeny of DNA of endophytic fungi isolated from industrial hemp. The nodes are supported by 1,000 bootstrap replications. Bootstraps above 50% and the genetic scale are shown. Sequences were obtained and the NCBI accession numbers are listed after each strain
117x114mm (300 x 300 DPI)
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