ARTICLE

Endophytes of industrial hemp (Cannabis sativa L.) cultivars:identification of culturable bacteria and fungi in leaves,petioles, and seedsMaryanne Scott, Mamta Rani, Jamil Samsatly, Jean-Benoit Charron, and Suha Jabaji

Abstract: Plant endophytes are a group of microorganisms that reside asymptomatically within the healthy livingtissue. The diversity and molecular and biochemical characterization of industrial hemp-associated endophyteshave not been previously studied. This study explored the abundance and diversity of culturable endophytesresiding in petioles, leaves, and seeds of three industrial hemp cultivars, and examined their biochemical attri-butes and antifungal potential. A total of 134 bacterial and 53 fungal strains were isolated from cultivars Anka,CRS-1, and Yvonne. The number of bacterial isolates was similarly distributed among the cultivars, with themajority recovered from petiole tissue. Most fungal strains originated from leaf tissue of cultivar Anka. Molecularand phylogenetic analyses grouped the endophytes into 18 bacterial and 13 fungal taxa, respectively. The mostabundant bacterial genera were Pseudomonas, Pantoea, and Bacillus, and the fungal genera were Aureobasidium,Alternaria, and Cochliobolus. The presence of siderophores, cellulase production, and phosphorus solubilizationwere the main biochemical traits. In proof-of-concept experiments, re-inoculation of tomato roots with someendophytes confirmed their migration to aerial tissues of the plant. Taken together, this study demonstrates thatindustrial hemp harbours a diversity of microbial endophytes, some of which could be used in growth promotionand (or) in biological control designed experiments.

Key words: hemp, endophytes, siderophore, Pseudomonas, molecular detection, antifungal activity.

Résumé : Les endophytes des plantes forment un groupe de microorganismes qui résident de manière asymp-tomatique à l’intérieur des tissus vivants sains. La diversité, la caractérisation moléculaire et biochimique desendophytes associés au chanvre industriel n’ont pas été étudiées jusqu’à présent. Cette étude a explorél’abondance et la diversité d’endophytes cultivables qui résident dans les pétioles, les feuilles et les semences detrois cultivars de chanvre industriel, et examiné leurs caractéristiques biochimiques et leur potentiel anti-fongique. Un total de 134 souches bactériennes et 53 souches fongiques ont été isolées à partir des cultivars Anka,CRS-1 et Yvonne. Le nombre d’isolats bactériens était distribué de manière similaire entre les cultivars, la majoritéétant récupérée du tissu des pétioles. La plupart des souches fongiques provenaient du tissu foliaire du cultivarAnka. Les analyses moléculaires et phylogénétiques groupaient les endophytes en 18 taxons bactériens et 13 taxonsfongiques, respectivement. Les genres bactériens Pseudomonas, Pantoea et Bacillus et les genres fongiques Aureobasidium,Alternaria et Cochliobolus étaient les plus abondants. La présence de sidérophores, la production de cellulase et lasolubilisation du phosphore constituaient les principales caractéristiques biochimiques des endophytes. Lorsd’expériences de validation de principe, la réinoculation de racines de tomates avec certains endophytes a con-firmé qu’ils migraient vers les tissus aériens des plants. Dans son ensemble, cette étude démontre que le chanvreindustriel comporte une diversité d’endophytes microbiens dont certains pourraient être utilisés dans des expéri-ences de stimulation de la croissance ou de contrôle biologique. [Traduit par la Rédaction]

Mots-clés : chanvre, endophytes, sidérophore, Pseudomonas, détection moléculaire, activité antifongique.

IntroductionOriginating from the Himalayas, industrial hemp

(Cannabis sativa L.) is the most ancient domesticated crop.In Canada, under the Canadian Controlled Drugs andSubstances Act, commercial cultivation of industrial

hemp as a field crop began in 1998 (Cherney and Small2016).

Industrial hemp is a high-growing plant, typically bredfor seed and fibre, and also for multipurpose industrialuses such as oils and topical ointments, as well as fibre

Received 20 February 2018. Revision received 3 May 2018. Accepted 4 May 2018.

M. Scott, M. Rani, J. Samsatly, J.-B. Charron, and S. Jabaji. Plant Science Department, MacDonald Campus of McGill University,21 111 Lakeshore, Ste. Anne-de-Bellevue, QC H9X 3V9, Canada.Corresponding author: Suha Jabaji (email: suha.jabaji@mcgill.ca).Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.

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Can. J. Microbiol. 64: 1–17 (2018) dx.doi.org/10.1139/cjm-2018-0108 Published at www.nrcresearchpress.com/cjm on 18 June 2018.

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for clothing, and construction material for homes andfor building electric car components (Callaway and Pate2009; Domke and Mude 2015; Yallew et al. 2015). Since itslegalization in Canada, the total land area for industrialhemp production has grown steadily, increasing from3200 ha to approximately 44 000 ha between 2008 and2014 (Alberta Agriculture and Forestry 2015). Both hempand marijuana varieties are members of the C. sativa spe-cies; however, industrial hemp cultivars have long beenbred for their fibre, oil, and seed aspects, and possessvery low narcotic value. In Canada, 45 industrial hempcultivars are approved by Health Canada (Health Canada2018) and are mandated to have under 0.3% of THC (�-9-tetrahydrocannabinol), the principal intoxicant cannabi-noid (van Bakel et al. 2011). These varieties are generallygrain or multi-use; where both the seeds and stalk findan end market (Cherney and Small 2016).

In Quebec, farmers produced hemp on 290 ha in 2011;a fairly small commitment in comparison to the prairieprovinces, which had planted 15 056 ha in the same year(Government of Alberta 2012). This means that withinthe Quebec market there is a great potential for growthand profitability. Much of the Quebec growing climateis similar to Northern Ontario, which has yielded anaverage stem yield of 6.1 tons·ha–1 (Ontario Ministry ofAgriculture and Food 2011). Considering that demandappears to be continuing on a positive trend, knowledgeon selection of high-performing cultivars in terms of bio-mass and seed yield (Aubin et al. 2016) and agronomicalrecommendations and guidelines (Aubin et al. 2015) hasbeen documented.

The phytochemical composition of industrial hempand the marijuana varieties of Cannabis does not makehemp immune to attacks by pathogens. On the contrary,Cannabis is susceptible to many phytopathogens leadingto a number of diseases (McPartland et al. 2000) domi-nant at all growth stages of hemp. Several importantdiseases have been shown to be caused by fungal patho-gens, including Botrytis cinerea, the causal agent of greymold; Rhizoctonia solani, the causal agent of root rot andstem canker; and Sclerotinia sclerotiorum, the causal agentof hemp canker (McPartland et al. 2000). Thus, it is desir-able to prevent the loss of industrial hemp as well asmedicinal Cannabis to opportunistic phytopathogens.

Microbial endophytes are beneficial plant-associatedbacteria and fungi that are widespread inhabitants in-side different plant tissues and organs, without causingharm or exhibiting symptomatic behaviour or visiblemanifestation of disease. They have been shown to assistplant growth by producing plant hormones, increasingnutrient availability (Compant et al. 2010; Hamilton andBauerle 2012; Radhakrishnan et al. 2014; Hardoim et al.2015; Malhadas et al. 2017), and protecting plants fromdiseases and abiotic factors through their demonstratedcapacity to produce biologically active secondary metab-olites (Aly et al. 2010; Kharwar et al. 2011; Gagne-Bourgue

et al. 2013; Brader et al. 2014), and establishing a sustain-able system (Rodriguez et al. 2009; Santoyo et al. 2016).These attributes make microbial endophytes a target forbiotechnological and commercial exploitation (Stanieket al. 2008; Li et al. 2012).

Previous studies have reported that medicinal andwild Cannabis harbour competent fungal and bacterialendophytes that are capable of providing different formsof fitness benefits to their associated host plants, andyielded insights into plant–microbe and microbe–microbe interactions (Gautam et al. 2013; Kusari et al.2013). However, no knowledge on the diversity, distri-bution, and biochemical attributes of microbial endo-phytes recovered from industrial hemp cultivars grownin Quebec exists.

Our research goals were to determine the prevalenceand types of endophytic bacteria and fungi residing inthe aboveground tissue of three industrial hemp culti-vars grown under field conditions and to evaluate theirbiochemical attributes. We identified taxonomically theculturable bacterial and fungal endophytes by gene se-quence analysis of 16S rRNA and internal transcribedspacer (ITS) rDNA, respectively, and evaluated their sub-strate utilization patterns. A few bacterial endophyteswere selected to determine their antimicrobial proper-ties against economically important fungal pathogens,including those that cause disease in industrial hempand medicinal Cannabis, and we confirmed their internal-ization and systemic spread of the endophytes in planttissues of a horticultural plant.

Materials and methods

Farm site, cultivars, and sample collectionIn this study, the term hemp will be used hereafter to

refer to industrial hemp. Seeds of three multi-use hemp(Cannabis sativa L.) cultivars approved for production inCanada were used for endophyte discovery. CRS-1 (seeduse) and Anka and Yvonne (dual use; seed and biomass)under the terms specified in licenses delivered to Jean-Benoit Charron by Health Canada (Nos.12-C0142-R-01, 13-C0142-R-01, and 14-C0142-R-01) (Mayer et al. 2015) weregrown at the Emile A. Lods Agronomy Research Centrefield site (45°26=N, 73°55=W) of Macdonald Campus ofMcGill University. Detailed information on soil types andfertilization is fully described in Aubin et al. (2015, 2016).Cultivars were seeded in mid-May of 2013 and grown inplots 1.3 m × 5 m, containing seven rows, spaced at 18 cmapart. No herbicide was applied, and manual weedingwas done on all plots throughout the growing season.Only naturally occurring precipitation and light expo-sure were used throughout the plants’ growth (Aubinet al. 2016).

Compound leaves and petioles were collected fromplants grown in cultivar trial experiments and also fromnitrogen fertility trials. Collection began in mid-June of2013 and continued biweekly from mid-June until late

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August of 2013. Approximately two dozen leaves includ-ing the vascular petiole tissue of each cultivar were col-lected from multiple plants within the plot at each timepoint. At maturity, seeds were thrashed from the rest ofthe biomass using a stationary combine (Aubin et al.2015). Field samples were placed in labelled Ziplock®bags on ice for transportation, stored in a 4 °C walk-incold room, and processed within 48 h of collection. Allseeds were stored at 4 °C in 50 mL falcon™ tubes untilfurther use.

Isolation of microbial endophytesAll plant tissues (leaves, petioles, and seed embryos)

were surface-sterilized following stepwise, agitated im-mersion in ethanol (70%), sodium hypochlorite (3.5%available Cl–), and sterile deionized water (dH2O) accord-ing to Schulz et al. (1993). To maximize the isolation ofculturable organisms from different tissues followingsterilization, leaves were sectioned (0.5 cm) with a sterilescalpel and placed directly onto the surface of differentselective media ideal for bacterial and fungal growth.Petiole sections (a dozen pieces of �10 mm each) andseed embryos (�approximately 40) were placed sepa-rately in a Waring blender and homogenized in 5 mL ofsterile water, then 150 �L of petiole or embryo macerateswas spread-plated onto selective culture media that aredescribed below. The effectiveness of the sterilizationprocedure was tested using both imprinting, and washplating methods depending upon the material used(Schulz et al. 1993; Ji et al. 2008; Kaga et al. 2009).

Bacterial isolatesTo ensure the purity of the emergent microorganisms,

bacterial colonies from sterilized tissues were passedthrough four rounds of single-colony isolation via streak-ing on lysogeny broth plates (LB; 1.0% tryptone, 0.5%yeast extract, 1.0% NaCl, 1.5% agar; Difco, Lawrence, Kan-sas, USA) or nutrient agar plates (NA; 0.5% peptone, 0.3%yeast extract, 0.5% NaCl, 1.5% agar, Difco) amended withfilter-sterilized, antifungal agent benomyl (10 mg·L–1;Sigma-Aldrich, Ontario, Canada). Stock cultures of purebacteria were prepared from overnight LB broth mixedwith 25% glycerol stock at 1:1 ratio and stored at –80 °C.

Fungal isolatesEmergent fungal mycelia were isolated on potato

dextrose agar (PDA; 0.2% dextrose, 0.04% potato starch,1.5% agar; Difco) or malt extract agar (MEA; malt ex-tract 0.3%, peptone 0.05%, 1.5% agar; Difco) amendedwith 100 mg·L−1 penicillin and 100 mg·L−1 streptomycin(Sigma-Aldrich). Fungal isolates were purified throughfour rounds of re-isolation by carefully removing a 3 mmsection from the edge of the growing colony, transfer-ring to fresh, amended PDA plates, and incubating for an

additional 24–48 h before subsequent re-isolation. Purefungal cultures were stored in 25% glycerol at –80 °C.

Molecular identification and characterization ofendophytes

BacteriaOne hundred and thirty-four bacterial strains were

grown on LB at room temperature for 16–18 h with agi-tation (175 r·min–1) to achieve concentrations between108 and 1010 colony-forming units (CFU)·mL−1. Cells werecollected for Gram reaction staining (Steinbach andShetty 2001), and DNA extraction using the Presto™ MinigDNA Bacteria kit (FroggaBio, Ontario, Canada) accordingto the manufacturer’s instructions. DNA quality was con-firmed on 1% agarose gel prior to subsequent reactions.

The bacterial primer pair 27F–534R, amplifying thepositions of 27 and 534 of bacterial 16S rRNA genes(Table S11), was used according to previously publishedprotocols (Gagne-Bourgue et al. 2013) to identify over130 bacterial endophytes. The iProof™ High-Fidelity (HF)PCR kit (Bio-Rad, Ontario, Canada) and 40 ng of genomicDNA were used per 50 �L reaction. A positive, amplifiedgenomic DNA sample and a negative control withoutDNA were run concurrently with each reaction. An an-nealing temperature of 63 °C and 35 cycles were used.The amplified PCR product was cleaned using a Gel/PCRDNA Fragments Extraction kit (FroggaBio, Ontario, Can-ada) as instructed and was sequenced at Genome Québec(Montréal, Quebec, Canada), or in some cases the PCRproduct was cloned using the StartClone PCR cloning kit(Agilent Technologies, California, USA) following themanufacturer’s recommendations. Purified plasmid DNAwas sent for sequencing. Results were queried betweenDecember 2013 and March 2014 using NCBI’s BLASTnsoftware program (Altschul et al. 1990). The top namedsearch results were used to identify isolates to the genuslevel where possible.

Sequences of endophytic bacteria were aligned usingClustalW software in SDSC Biology Workbench, and thenonconserved regions were used to design primers forselected bacteria (Table S11). Specific primers were syn-thesized by Integrated DNA Technologies (Coralville,Iowa, USA) and were tested against all bacteria to ensurespecificity.

FungiFifty-three fungal isolates were grown on MEA culture

medium for 1 week prior to extraction of total genomicDNA using the Extract-N-Amp Plant DNA extraction kit(Sigma-Aldrich). The primers ITS1F and LR3 (Table S11),corresponding to the first ITS of the small subunit andthe large subunit sections of ribosomal DNA, respec-tively, were used to amplify a fragment of 1 to 1.2 kbin length using previously described cycle conditions

1Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjm-2018-0108.

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(Hoffman and Arnold 2010). The PCR product wascleaned with Exo-SapIT reagents (Affymetrix) as directed,and Sanger sequenced at the University of ArizonaGenetics core (Tucson, Arizona, USA).

Sequences were examined and trimmed manually us-ing Sequencer 4.5 (GeneCodes Corp., Michigan, USA) toobtain a high-quality consensus read. In cases of discrep-ancies in the consensus read (5.7% of isolates), the ITS1Fand LR3 sequences were assessed separately and searchresults were compared. A single consensus sequence wasalso generated in Mesquite version 3.02 (Maddison andMaddison 2015) from the ITS1F and LR3 sequences. Theconsensus identity within the top named hits was usedto identify isolates to the family level and, where possi-ble, to the genus level. Fungal sequences were clusteredinto 90%, 95%, and 100% operational taxonomic unit(OTU) groups based upon sequence similarity using thephylogenetic tool Mesquite. Isolate identity was con-ducted using the 95% similarity group, which has beenfound to roughly correspond to the species level in otherendophytic fungi (U’ren et al. 2009).

Some plant-associated fungi harbour bacteria withintheir hyphae (Hoffman and Arnold 2010). These bacteriareside in living hyphae of fungal endophytes and areknown as endohyphal bacteria (EHB). To confirm thepresence of EHB within the living hyphae of hemp fungalendophytes, a representative fungal isolate was chosenfrom 13 different OTUs (95% similarity level) and wasqueried according to the protocol by Hoffman andArnold (2010). Briefly, total genomic DNA of identifiedfungal isolates was amplified with the primers (27F and1429R) of 16S rRNA gene. PCR products of insufficientconcentration for sequencing were cloned using AgilentTechnologies StraClone kit (Agilent) following the manu-facturer’s recommendations. Amplification and sequenc-ing of the positive clones was accomplished using M13Fand M13R. Products were cleaned using the ExoSap-IT kit(ThermoFisher) as described by the manufacturer andsent for Sanger sequencing at the University of ArizonaGenomics Analysis and Technology Core Facility. An av-erage of four separate clones was sequenced per queriedOTU. Returned sequences were assessed using the BLASTnprogram April 2015.

Microbial phylogenetic tree generationReverse sequences of 534R amplified sequences from

DNA of bacterial endophytes were generated using theReverse Sequence tool from Bioinformatic Organization(Massachusetts, USA) and aligned into a single sequencein BioEdit version 7.2.5 (Ibis Biosciences; California,USA). Sequences were aligned using the CLUSTAL tool tobe used in tree generation.

Phylogenetic trees for bacterial and fungal isolateswere constructed separately from aligned DNA sequencefiles using the freeware program MEGA version 6.0(Tamura et al. 2013). Trees were generated using Maxi-mum Parsimony methods and used a Subtree-Pruning-

Re-grafting algorithm (Nei and Kumar 2000). Positionswith fewer than 95% site coverage were eliminated. Thebootstrap consensus tree was inferred from 1000 replicates,and only branches above a 50% bootstrap score were dis-played. The analysis for bacteria involved 134 nucleotidesequences, and 53 nucleotide sequences for fungi. All posi-tions containing gaps and missing data were eliminated. Inthe bacterial tree there were a total of 310 positions in thefinal data set and for fungi there were 676 positions.

Phenotypic and chemical characterization of endophytesSeveral chemical tests were conducted for the charac-

terization of biochemical traits of bacterial and fungalisolates (see Table S21).

BacteriaThe enzymatic assays and biochemical tests were per-

formed in triplicates to characterize the bacterial endo-phytes. Single bacterial colonies were grown in 4 mL ofLB broth overnight (16–18 h) with agitation at 175 r·min–1.Following appropriate dilution in LB broth, 25 �L of105 CFU·mL−1 were used in all tests unless otherwise stated.

Cellulase and phosphatase solubilizationCellulases were assayed on indicator plates with car-

boxymethyl cellulose (Sigma-Aldrich) amendment, andstained in Congo-red solution (0.2%) as outlined by Guptaet al. (2012). The ability of endophytic bacteria to solubi-lize inorganic phosphate (HPO4)2 was assayed on agarmedium containing inorganic phosphate (g·L−1: glucose,10; NH4Cl, 5.0; NaCl, 1.0; MgSO4·7H2O, 1.0; Ca3(HPO4)2,0.8; agar, 15, pH 7.2) according to Verma et al. (2001).Development of clear halo zones after 48 h around thestrains exhibited their positive phosphate solubilizationactivity.

Siderophore productionBacteria were assessed for siderophore activity by

growing cultures on Chrome azurol S (CAS) media, acomplex mixture of Fe–CAS (Sigma-Aldrich) indicator,piperazine-N,N=-bis(2-ethanesulfonic acid) (PIPES) (Sigma-Aldrich), and 10% sterile casamino acid (Difco) solutionsas outlined in Alexander and Zuberer (1991). Develop-ment of a yellow–orange halo zone after 24–48 h aroundthe bacterial spot is considered as positive indication forsiderophore production.

Hydrogen cyanide (HCN)HCN gas production was assessed based upon a change

from bright yellow to orange colour in picric acid (0.5%,RICCA Chemicals) soaked Whatman filter, lining the lidof a sealed petri dish (Bakker and Schippers 1987).

Organic acid productionOrganic acid production was determined using a mod-

ified methyl red test (Kumar et al. 2012). Briefly, bacteriawere grown in 5 mL of glucose-phosphate broth at roomtemperature with agitation (175 r·min–1) for 4 days. Asmall amount (3–5 drops) of methyl red solution was

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added to the culture and mixed gently. Colour change todeep red was scored as positive, deep orange was scoredas a weak reaction and maintenance of yellow coloura-tion was scored negatively. Pure LB and HCL were used asnegative and positive controls, respectively.

Hormone productionIndole acetic acid (IAA) production was assessed as de-

scribed by Husen (2003) using L-tryptophan (final concen-tration: 5 mmol·L–1, Sigma-Aldrich) amended LB media,and shaken for 4 days (23 °C, 175 r·min–1). The culturesupernatant was mixed with FeCl3–HClO4 reagent (1:2ratio) and permitted to react in the dark for 30 min priorto spectrometric scoring at 530 nm. Biological replicatesand the IAA chemical standards were done in triplicateto provide accurate quantification. Production of IAAwas confirmed by the development of a pink colour.

Antibiotic sensitivityThe antibiotic sensitivity of bacterial endophytes was

tested individually on agar plates containing antibioticsfollowing the procedure of Gagne-Bourgue et al. (2013).The antibiotics tested were kanamycin, rifampicin, (Sigma-Aldrich), streptomycin (Bioshop, Ontario, Canada), andtetracycline (Fisher Chemicals, Ontario, Canada) at100 �L·mL−1, and ampicillin, gentamicin (Sigma-AldrichCo.), chloramphenicol (ICN Biomedicals, Ohio, USA), andhygromycin (Santa Cruz, Texas, USA) at 125 �L·mL–1. Bac-teria were considered sensitive to an antibiotic at theconcentration tested if no visible growth was observedon treatment plates, when there was visible growth oncontrol plates after 48 h of incubation at 27 °C.

Antifungal activityThe ability of endophytes to restrict the radial growth

of seven fungi (Botrytis cinerea, Sclerotinia sclerotiorum,Fusarium graminearum, F. solani, Helminthosporium solani,Rhizoctonia solani, and binucleate Rhizoctonia) was per-formed as previously described (Gagne-Bourgue et al.2013). The experiment was performed at three separatetime points and in triplicate for each bacterium. Reduc-tion in fungal growth was measured as percent of inhi-bition ratios and using the formula: (C – T)/C × 100, whereC is the average radial of control growth and T is bacterialtreatment radius.

Fungi: cellulases and ligninasesCellulose-amended MEA and indulin-AT (MeadWestvaco,

Virginia, USA) amended water agar were used for detect-ing cellulose and ligninase activities, respectively, as de-scribed by Sundman and Nase (1974) and Gupta et al.(2012). Fungi were allowed to grow for an additionalweek prior to ligninase testing. For both tests, indicatordye solutions were applied to assess activities at 4–8 days(cellulase) or 11–15 days (ligninase). Assays were performedin biological triplicates and repeated at least once to obtaina consensus.

Colonization and tissue internalization of bacterialendophytes in tomato seedlings

As proof-of-concept, we assessed whether representa-tive bacterial strains, i.e., scored positive in at least six ofthe biochemical tests and exhibited a significant antifun-gal activity in confrontation assays with pathogenicfungi, are able to colonize and internalize tissues of adicot plant. The re-introduction of bacterial endophytesin the absence of a competing microbial flora is deemedpromising for further greenhouse studies. Thus, we se-lected tomato (Solanum lycopersicum L.) as the model dicotplant instead of hemp seeds because hemp seeds couldnot be rendered completely sterile without significantloss of viability.

Single-cell colonies of bacterial strains of BTG8-5 andBTC8-1, putatively identified as Pseudomonas orientalis andPseudomonas fulva, respectively, were isolated from thepetiole tissue of field-grown hemp and were grown over-night in liquid culture in 5 mL of LB (23 °C, 175 r·min–1).These strains exhibited the best biochemical attributes.Three to four seeds of S. lycopersicum Beefsteak, surface-sterilized as previously described, were planted in 10 g(3 cm deep) of sterilized Agromix® potting soil G12(Fafard et Frères Ltd., Quebec, Canada) in magenta boxes.Following seeding, 5 mL of 1× Hoagland solution(Hoagland and Arnon 1950) was added per box. Seed-lings were grown for 1 week under a photoperiod of16 h (light) : 8 h (dark) cycles at 25 °C : 20 °C beforethinning to a single, healthy plant per box. Plants weresoil drenched with 5 mL of 105 CFU·mL–1 freshly grownBTG8-5 or BTC8-1 in LB broth at 14 days after seeding.Control plants were drenched with 5 mL of sterile LBbroth. There were five replicates per treatment and theexperiment was repeated once in a temporally separatedtrial. At 28 days after seeding, seedlings were harvested,separated into root, petiole, or leaf tissues, and surface-sterilized as previously described under isolation of mi-crobial endophytes section.

Confirmation of bacterial endophytes viaculture-independent method and PCR assays

A subset of sterile tissue was retained for PCR using theprimer set BT14-4F1 and BT14-4R1 specific for P. orientalisand P. fulva (Table S11) to detect and confirm coloniza-tion of the target endophytes in plant tissues. Ampli-fication was conducted using HF Bio-Rad PCR andsimilar conditions as described in the molecular iden-tification and characterization of endophytes sectionwith an annealing temperature of 67.5 °C. Tissue sam-ples were ground to a fine powder in liquid nitrogenprior to DNA extraction. Between 100 and 400 mg ofsamples were used in a modified CTAB (cetyl trimethyl-ammonium bromide) method of DNA extraction (Carrigget al. 2007). Briefly, the sample was incubated with lysisbuffer (100 mmol·L–1 Tris–HCl, 2 mol·L–1 NaCl, 25 mmol·L–1

EDTA, pH 8.0, 5% polyvinylpyrrolidine, 3% CTAB),2-mercaptoethanol, and 1 �L of RNAse A (Bioshop, On-

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tario, Canada) for 1 h at 70 °C. Samples then underwentone round of phenolchloroform:isoamyl alcohol (25:24:1)extraction, two rounds of chloroform:isoamyl alcohol(24:1) extraction, and overnight precipitation in 2/3 vol-ume of isopropanol and 1/10 volume of 3% sodium ace-tate (pH 5.2) at room temperature (23 °C). PrecipitatedDNA was collected, washed in 70% ethanol, and elutedinto pure dH2O (pH 7.0). DNA was visualized on a 1%agarose gel to confirm quality prior to amplification us-ing the Pseudomonas specific primers BT14-4F1 and BT14-4R1 (Table S11).

The remaining tissues were ground with 5 mL of ster-ile water, serially diluted. A volume of 100 �L was spreadon nonamended LB and incubated at 23 °C for 2 daysprior to CFU scoring. Each dilution point was done intriplicates for each tissue and each treatment for eachtrial.

Results

Distribution of microbial endophytesOver the course of the growing season of 2013, 1000

tissue segments (leaves, petioles, and seeds) collectedfrom hemp cultivars were screened for culturable endo-phytes. A total of 134 bacterial and 53 fungal strains wereisolated and identified to the genus level (Figs. 1 and 2).The distribution of bacterial isolates was generally simi-lar among the three hemp cultivars (Fig. 1A) with themajority isolated from petiole tissues (67%), followed byleaf (19%), and seed tissues (14%) (Fig. 1B). On the otherhand, the majority of the fungal strains (45%) originatedfrom the cultivar Anka followed by cultivar CRS-1 (36%)and cultivar Yvonne (19%) (Fig. 1C). More than half of thefungal isolates originated from leave tissue (Fig. 1D).

Irrespective of cultivar or tissue, more bacterial iso-lates were collected at early and mid growing periods

Fig. 1. Distribution, frequency of recovered strains of endophytes in 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 bacterial and fungalstrains isolated from hemp over the growing season of summer 2013.

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than late growing periods (Fig. 1E). There was no partic-ular trend in fungal isolate incidence with consistentnumbers at each time point and a slight decrease at latergrowth periods (Fig. 1E).

Isolate identificationAll 134 bacterial strains were partially identified using

16S rRNA primers 27F and 534R and, in many cases, wereidentified to the species level. Additional cloning usingM13 vector and sequencing were done for some isolatesof increased interest to confirm species designation(Table S11). The strains were grouped into 18 differenttaxa that shared high homology with known sequencesand data for endophytic strains have been deposited un-der the following accession Nos. KX430321–KX430455,with the most frequently isolated endophytes belongingto the Gram-negative genera Pseudomonas (35%) andPantoea (17%) and the Gram-positive genera Staphylococcus(16%) and Bacillus (9%) (Fig. 2A).

The highest abundance of bacterial strains isolatedfrom leaves were isolates of Pseudomonas (11/25) andBacillus (4/25). These genera constituted more than half(60%) of the strains isolated. From petioles, strains belong-ing to Pseudomonas (35/90), Pantoea (14/90), Staphylococcus(14/90), and Bacillus (5/90) were the most abundant. The

highest genera associated with the seed were Pantoea(7/19), Staphylococcus (4/19), Bacillus (3/19), and Enterobacter(3/19) (Fig. 3). The distribution of certain taxonomicgroups of endophytes was genotype-dependent (Table S31).Acinetobacter, Agrobacterium, and Enterococcus were recov-ered from petiole tissues of cultivar Anka. Isolates be-longing to Erwinia, Paenibacillus, and Rhizobium wererecovered from cultivar CRS-1 (Table S31), while the ge-nus Microbacterium was recovered from petioles of culti-var Yvonne.

All 53 fungal strains were putatively identified by se-quencing the amplified fragments using the ITS1F–LR3primer pair (Table S11) and grouped into 13 genera, withdeposited accession Nos. KX641935–KX641987 (Fig. 2B).All fungal isolates were members of the Dikaryota, withthe majority (96%) belong to the Ascomycota. Only two ofthe 53 isolates were members of Basidiomycota, namely,Irpex and Cryptococcus (Fig. 2B). The most commonlyisolated fungal endophytes belonged to the generaAureobasidium (24%), Alternaria (19%), Cochliobolus (19%),and Cladosporium (15%). Hemp leaves harboured in highabundance isolates belonging to Cochliobolus (10/37) andAureobasidium (9/37). The highest number of genera asso-ciated with petioles was Alternaria (7/14) and Cryptococcus(4/14). Only strains belonging to Aureobasidium (1/2) andCladosporium (1/2) originated from seeds (Fig. 3F). Certainfungal groups were cultivar-specific (Table S41). Isolatesbelonging to the taxonomic groups Dreschlera and Irpexand Sordariomycetes were specific to Anka (Table S41).Fungi belonging to Pleosporales were recovered onlyfrom cultivar Yvonne, while Eutypella, Pezizomycetes, andStagonosporpsis were recovered from cultivar CRS-1(Table S41).

The presence of EHB using the genomic DNA of a rep-resentative fungal strain from each taxon revealed sixputative host candidates (Table 1). BLAST search resultssuggest that putative EHB matched primarily unculturedand often unnamed bacteria. Among named hits wereAcinetobacter and Staphylococcus. Of those fungi showingputative EHBs, four showed Mycoplasma infection (Table 1).

Phylogenetic trees constructed for bacterial and fun-gal isolates clearly support the BLASTn sequence identi-fication of large groups of bacteria and fungi (Figs. 4and 5). The percentage of replicate trees in which theassociated taxa clustered together in the bootstrap test(1000 replicates) is shown next to the figures with boot-straps ≥50%. The dominant isolate groups for bacteria(Pseudomonas, Pantoea, and Staphylococcus) remain reason-ably well delineated (Fig. 4). The dominant isolate groupsfor fungi that were well delineated belonged to theAscomycota (Fig. 5).

Biochemical characterization and antagonistic propertyof hemp endophytes

Initial screening showed that the production of sidero-phore (13/18) and cellulases (11/18) are the most com-monly occurring traits found across genera, followed by

Fig. 2. Taxonomic groups of culturable endophytic(A) bacteria and (B) fungi based on partial sequencinganalysis of 16S rRNA and rDNA ITS, respectively. [Colouronline.]

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phosphate solubilization (6/18) and HCN and organic acidproduction (5/18) (Table 2). All genera except Brevibacterium,Curtobacterium, Microbacterium, Paenibacillus, and Staphylococcuswere siderophore producers.

The top performing strains were all members of thePseudomonas genus: BTC6-3, BTC8-1, BTG8-5, and BT14-4.

All four strains showed varying levels of solubilizationof inorganic phosphate, production of siderophores,and all except BTC6-3 demonstrated cellulytic activity(Table 3). HCN production was noted for only BTC6-3 andBTC8-1, and organic acid production was positive inBTC6-3 and BTG8-5. These strains were additionally

Fig. 3. Distribution of taxa of culturable bacterial (A, B, C) and fungal (D, E, F) endophytes isolated from leaves (A, D), petioles (B, E),and seeds (C, F) of hemp. [Colour online.]

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tested for IAA production, ACC deaminase activity, anti-biotic resistance, and fungal inhibition in direct confron-tation assays. BTC6-3 and BT14-4 were found to producethe greatest amounts of IAA (10.96 and 9.46 �g·mL−1,respectively).

Analysis of resistance to various antibiotics showedthat all strains belonging to Gram-positive and Gram-negative genera were sensitive to rifampicin, chloram-phenicol, and gentamicin at the concentration tested.For Gram-negative bacteria, resistance to hygromycin(82%) and ampicillin (64%) was most common. Gram-negative strains demonstrated resistance to hygromycin(74%) and ampicillin (43%) (data not shown).

All 53 identified fungal strains were assessed for theproduction of cellulases and ligninases. Cellulase activitywas found to be widespread among the genera (39/53),with 6% of the strains belonging to Cladosporium andAureobasidium producing lytic zones greater than 10 mm.Only four strains (2/53) showed ligninase activity: FL11-3,a Pleosporales sp. that showed moderate strength, fol-lowed by FL12-3, a Stagonosporopsis sp. (data not shown).

Antifungal activitySeven fungi, covering a wide range of lifestyles, were

chosen for confrontation assessment against BTC6-3,BTC8-1, BTG8-5, and BT14-4 (Table 4). Botrytis cinerea,Sclerotinia sclerotiorum, and Rhizoctonia solani are patho-gens of hemp (McPartland et al. 2000) and were of partic-ular interest. Sclerotinia sclerotiorum was significantlyaffected, displaying reductions in radial growth rangingfrom 17% to 54% against BTC6-3 and BTC8-1, respectively.Growth of B. cinerea was significantly reduced by BTC8-1and BTG8-5 (19% and 22% reduction). Rhizoctonia solani wassignificantly reduced by BT14-4 (28%, p = 0.0141), and ra-dial growth of binucleate Rhizoctonia was significantly

affected in confrontation assays with BTC8-1 and BTG8-5strains (23.4% and 31.3% reduction). However, no othertested fungi were significantly affected (p = 0.05).

Recolonization, internalization, and detection ofbacterial endophytes in plant tissues

The surface-sterilization method combined with theimprint technique ensured that the endophytic coloni-zation cell numbers reflect only the number of cellswithin the interior of the plant tissues. This method issufficient to kill and (or) wash away the surface bacteriawhile maintaining the survival of the interior bacteria.Bacterial colonies that matched the Pseudomonas pheno-type in treated tomato plants were successfully detectedin colonized plant homogenates while surface-sterilizedtissues of control noncolonized plants did not yield cul-turable bacterial colonies. Re-isolation and quantifica-tion of P. fulva BTC8-1 and P. orientalis BTG8-5 by theplating method in different tissues of tomato seedlingsafter soil drenching with BTG8-5 and BTC8-1, respec-tively, clearly demonstrated that both strains can formsustaining endophytic populations in roots, shoots, andleaves of tomato (Table 5). However, population numbersin roots were more variable than in petioles and leaves inboth trials. Amplification of genomic DNA extractedfrom colonized tissue samples using designed Pseudomonas-specific primers BT14-4F1 and BT14-4R1 gave the expectedband size (230 bp) in all treated samples. Absence of en-dophytes was confirmed in tissues of noninoculated to-mato seedlings (data not shown).

DiscussionThe main focus of this study was to examine varieties

of hemp grown in Quebec for the presence of endophyticbacteria and fungi and to determine their taxon level.

Table 1. List of fungal endophytes of hemp that possess positive sequences for endohyphal bacteria.

Fungal hoststrain

Genus of fungus(95% OTU group level)*

Putative endohyphal bacteria(top BLAST hits from multiple clones)

Supportvalue† (%)

FLC6-1 Pezizomycetes Novosphingobium 99Uncultured alpha proteobacterium 99Acinetobacter 99

FLC7-7 Sordariomycetes Uncultured rumen bacterium 95Uncultured bacterium partial 16S rRNA 99Mycoplasma 98

FTG8-1 Aureobasidium Uncultured bacterium partial 16S rRNA gene 99Acinetobacter 99Staphylococcus 99

FS9-1 Cladosporium Acinetobacter 99Uncultured bacterium clone 99Mycoplasma 99Staphylococcus 99

FL11-8 Cryptococcus Mycoplasma 98Uncultured Propionibacterium clone 99

FT13-2 Dothideomycetes Mycoplasma 98*A single representative fungal isolate was chosen from 17 different operational taxonomic units (OTUs) at the

95% similarity level. An average of four separate clones were sequenced per queried OTU. BLASTn searches wereconducted in April 2015.

†BLASTn support for the identified putative endohyphal bacteria.

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Strategically, we were interested in examining the max-imum diversity of culturable microbes isolated fromaboveground plant tissues grown on different microbio-logical culture media with different sources of carbon.Such an approach may not favour the isolation of viablemicrobial endophytes that are slow growing or unable togrow. Full assessment of the abundance of unculturableendophytes isolated from hemp is now possible by DNAfingerprinting and pyrosequencing (Sun et al. 2008;Lundberg et al. 2012).

This study demonstrated that hemp cultivars harbourdiverse assemblage of culturable bacteria and fungi thatexist as endophytes and belong to 18 and 13 diverse tax-onomic groups, respectively. All strains identified in thiswork were related to previously identified species withplant-growth-promoting or biological control activity(Rosenblueth and Martínez-Romero 2006). To the best ofknowledge, this is the first report describing the diversityand the distribution of indigenous bacteria and fungi har-bouring hemp cultivars Anka, CRS-1, and Yvonne that are

Fig. 4. Maximum parsimony phylogeny of DNA of endophytic bacteria isolated from hemp. The nodes are supported by 1000bootstrap replications. Bootstraps above 50% and the genetic scale are shown. Sequences were obtained and the NCBIaccession numbers are listed after each strain.

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widely used by the local agricultural community for seedand oil production.

Several factors including plant genotype variation, soiltype, and varying environmental conditions are thoughtto promote shifts in the structural and functional char-acteristics of endophyte communities (Dalmastri et al.1999; Berg et al. 2002; Sessitsch et al. 2002). In this study,the distribution of certain taxonomic groups of endo-phytes was genotype-dependent. Cultivar specificity tobacterial and fungal endophytes has been reported in

several crops, including Cannabis (Bailey et al. 2005; Rascheet al. 2006; Rosenblueth et al. 2012; Wearn et al. 2012;Winston et al. 2014). Hence, studies on biodiversity andcultivar specificity of cultured endophytic micro-organisms associated with hemp merit further research.

Tissue type is an important factor for endophytecolonization (Compant et al. 2008; Massimo et al.2015). In this study, the most heavily colonized tissuediffered between bacterial and fungal isolates in C. sativa.This distribution pattern of bacterial endophytes in

Fig. 5. Maximum parsimony phylogeny of DNA of endophytic fungi isolated from hemp. The nodes are supported by 1000bootstrap replications. Bootstraps above 50% and the genetic scale are shown. Sequences were obtained and the NCBIaccession numbers are listed after each strain.

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petiole tissue as opposed to leaf and seed tissues iscorroborated in studies of other plants (Elvira-Recuencoand van Vuurde 2000; Kuklinsky-Sobral et al. 2004;Trotel-Aziz et al. 2008; Abraham et al. 2013). In contrast,fungal endophytes were isolated with higher frequenciesfrom hemp leaves compared with petiole and seed tis-sues. Similar patterns of fungal frequency were also re-ported in medicinal Cannabis (Gautam et al. 2013), and inother plants such as Antarctic grass (Rosa et al. 2009),loblolly pine (Arnold et al. 2007), cypress (Soltani andHosseyni Moghaddam 2015), and ginseng (Park et al.2012).

Seed embryos harboured the lowest numbers of bacte-rial (19) or fungal isolates (4) of all isolated endophytes.This observation is consistent with similar trends re-ported elsewhere (Ganley and Newcombe 2006; Truyenset al. 2015). It is reported that the majority of seed-associated bacterial endophytes belong to Proteobacteriasuch as Bacillus, Staphylococcus, Pseudomonas, and Pantoea(Rosenblueth et al. 2012; Truyens et al. 2015). The seed-associated endophytes reported in this study belonged tothe above taxa. Only two fungal endophytes (Aureobasidium sp.and Cladosporium sp.) were recovered from seeds, and

both are reported elsewhere as seed endophytes (Hodgsonet al. 2014; Geisen et al. 2017).

Many plant-associated fungi harbour EHB that are ca-pable of altering plant–fungus interactions (Shaffer et al.2016). Recent evidence indicates that EHB occur often indiverse leaf-endophytic Ascomycota (Hoffman and Arnold2010), and they can confer plant-growth-promoting traitswhen present within their fungal host (Hoffman et al.2013). This relationship is easily lost through subcultur-ing of fungal culture on antibiotic-amended media(Hoffman and Arnold 2010). In this study, isolation offungal endophytes was performed on antibiotic-amended PDA or MEA, and it is most likely that manyEHB were lost as a result of antibiotic treatment, despitethe fact that EHB are believed to be widespread (Sharmaet al. 2008; Hoffman and Arnold 2010).

Endophytic microbes that possess the ability to de-grade the plant polymer cellulose have been suspected ofcolonizing internal tissues of the plants (Compant et al.2010; Reinhold-Hurek and Hurek 2011). Additionally,cellulase-producing strains are also able to enhance phos-phorus availability to plants through mineralization oforganic P by acid phosphatases or through solubilization

Table 2. Biochemical attributes of bacterial genera associated with hemp tissues.

GenusCellulaseproduction

Phosphataseproduction

HCNproduction

Siderophoreproduction

Organic acidproduction

Acinetobacter + – + + –Agrobacterium + – – + –Bacillus + ++ – + +Brevibacterium – – – – –Curtobacterium – + – – –Cedecea + + – + +Enterobacter – + + + +Enterococcus + + + + –Erwinia + – – + –Microbacterium – – – –Ochrobactrum + – – + –Pantoea sp. – – + + ++Paenibacillus – – – – –Pseudomonas + + + + ++Rhizobium + – – + –Staphylococcus – – – – –Stentrophomonas + – – + –Xanthomonas + – – + –

Total 11 6 5 13 5Note: –, negative reaction; +, positive reaction showing a clearing zone of 1–3 mm; ++, positive

reaction showing a clearing zone of 4–8 mm.

Table 3. Attributes of the top four Pseudomonas strains.

Strain* SpeciesCellulaseactivity†

Phosphataseactivity†

Organic acidproduction†

IAA production(�g·mL–1)

HCNproduction†

Siderophoreproduction†

BTC6-3 Pseudomonas fulva – + + 10.96±0.31 + +BTC8-1 Pseudomonas fulva + + – 3.39±0.59 + +++BTG8-5 Pseudomonas orientalis + ++ + 3.36±0.045 – ++BT14-4 Pseudomonas orientalis + ++ – 9.46±0.101 – +++

*All strains are Pseudomonas spp. All tests were replicated three times and the means are presented.†+, appearance of halo; –, no halo. Halo size was rated as follows: +, <5 mm; ++, ≥5 to <10 mm; +++, ≥10 mm.

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of mineral phosphate via the production of organic acids(Richardson et al. 2009; Khan et al. 2014). Bacillus,Pseudomonas, Cedecea, and Enterobacter strains, isolatedfrom hemp (this study) and from different crops are re-

ported to solubilize or mineralize minerals such as phos-phorus, making them more readily available for plantgrowth (Hameeda et al. 2008; Gagne-Bourgue et al. 2013;Akinsanya et al. 2015). This attribute along with the abil-

Table 4. Inhibition of radial growth of test fungi in confrontation assays.

Fungus Characteristic Treatment*Mean radius(cm)† % Inhibition

Significance‡

(p value)

Sclerotinia sclerotiorum Broad pathogen Control 3.44 — —BTC6-3 2.84 17.4 0.1712BTC8-1 1.59 53.8 <0.0001BTG8-5 1.79 47.8 <0.0001BT14-4 1.69 50.7 <0.0001

Botrytis cinerea Broad pathogen Control 2.96 — —BTC6-3 2.79 5.8 0.9369BTC8-1 2.53 18.6 0.0248BTG8-5 2.31 22.0 0.0242BT14-4 2.56 13.4 0.3321

Rhizoctonia solani Broad pathogen Control 3.29 — —BTC6-3 3.30 –0.3 1.0000BTC8-1 2.56 22.3 0.0717BTG8-5 2.45 25.6 0.1279BT14-4 2.38 27.6 0.0141

Binucleate Rhizoctonia Biocontrol Control 3.00 — —BTC6-3 2.89 3.8 0.9908BTC8-1 2.30 23.4 0.0373BTG8-5 2.06 31.3 0.0065BT14-4 2.39 20.6 0.0881

Trichoderma virens Plant growth promoting Control 2.88 — —BTC6-3 2.89 –0.2 1.0000BTC8-1 2.66 7.6 0.8168BTG8-5 2.40 16.8 0.1468BT14-4 2.61 9.3 0.6763

Colletotrichum gloeosporioides Broad pathogen Control 2.61 — —BTC6-3 2.74 –5.1 0.9937BTC8-1 2.45 6.4 0.9734BTG8-5 1.90 27.2 0.1740BT14-4 2.27 13.2 0.7484

Stachybotrys elegans Biocontrol Control 2.64 — —BTC6-3 2.87 –8.7 0.8651BTC8-1 2.16 18.3 0.2165BTG8-5 2.03 23.2 0.2216BT14-4 2.24 15.3 0.3814

Helminthosporium solani Limited pathogen Control 1.87 — —BTC6-3 2.04 –9.4 0.3993BTC8-1 2.11 –13.2 0.3805BTG8-5 2.10 –12.5 0.0951BT14-4 1.85 1.2 0.9999

Fusarium solani Broad pathogen Control 2.52 — —BTC6-3 2.31 8.5 0.9931BTC8-1 2.27 9.8 0.8605BTG8-5 2.10 16.6 0.6985BT14-4 2.30 8.7 0.9848

Fusarium graminearum Broad pathogen Control 1.98 — —BTC6-3 2.04 –3.1 0.8926BTC8-1 2.11 –6.7 0.8014BTG8-5 1.81 8.8 0.4330BT14-4 2.05 –3.5 0.8605

*All strains are endophytes belong to Pseudomonas spp.†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 as <0.0001 when less than 0.0001.

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ity to produce cellulases is indicative of their nutrient-delivering capacity while interacting with plant hosts(Glick et al. 2007).

The ability to produce siderophores and HCN repre-sents some of the traits that make microorganisms suc-cessful competitors in several environments (Compantet al. 2005; Loaces et al. 2011). HCN being a powerfulbiocontrol agent may protect plants from biotic stresses;bacteria with this property are known to inhibit severalplant pathogens and can indirectly enhance plant growth(Santoyo et al. 2012). In our study, the majority of thetaxonomic groups produced siderophores, while 38% ofthe genera were HCN producers. Pseudomonas, the largesttaxon with the highest abundance of strains isolatedfrom hemp, produced HCN, siderophores, and IAA.These traits provide Pseudomonas strains a competitiveadvantage to colonize plant tissues, contribute to diseasesuppression by the production of antibiotics or HCN, andexclude other microorganisms from the same ecologicalniche (Leong 1986; Reiter et al. 2002).

The siderophore-producing Pseudomonas strains in thisstudy, P. fulva (strains BTC6-3 and BTC8-1) and P. orientalis(strains BTG8-5 and BT14-4), exhibited antifungal activitiesagainst several plant pathogenic fungi including B. cinerea, aknown pathogen of hemp and medicinal Cannabis. Directevidence to support siderophore production of Pseudomonasstrains with antifungal activity has been found duringstudies on the biocontrol of fungal wilt diseases(Kloepper et al. 1980; Lim et al. 1999) and damping offdiseases (Weller 1988).

One of the aims of this study was to confirm internalcolonization and systemic spread of selected Pseudomonasstrains in tomato seedlings under gnotobiotic condi-tions. Pseudomonas orientalis BTG8-5 and P. fulva BTC8-1were detected in roots, petioles, and leaves of young to-

mato seedlings after reintroducing them as a soil drenchapplication. These results confirm that the endophytesmoved from the roots upward to the petiole and leavesand maintained reasonable population densities. The ex-act location of Pseudomonas strains in root and aerial tis-sues remains to be investigated. Our results concur withprevious reports that some endophytes are able to mi-grate from root tissue to the upper parts of the plant(Zakria et al. 2007; Lin et al. 2009; Gagne-Bourgue et al.2013). Variation in bacterial population number insideroots and petioles was noticed in our trials. It has beenreported that under gnotobiotic and field conditions, theroot system is not colonized in a uniform manner, lead-ing to variation in bacterial colonization (Gamalero et al.2005). The contribution to plant growth of endophytes lo-calized in aerial parts was not attempted in this study.Future studies are necessary to correlate colonization ofthe strains, specifically those with specific functions thatare important for plant health and growth.

Taken together and based on the results obtained inthis study, the genus Pseudomonas has a wide-spread dis-tribution in the aboveground tissues of hemp cultivars.In this work, the antagonistic activity of P. orientalis andP. fulva strains along with their biochemical abilities ofsiderophore, HCN, IAA production and P solubilizationmake these strains excellent candidates as bioresourcefor the production of the above bioactive compounds.Additionally, research aimed at using these strains toincrease plant fitness, suppress disease, or augment pro-duction of secondary compounds in medicinal mari-juana varieties, the closest members of the Cannabissativa, is highly desirable.

AcknowledgementsThe authors are indebted to the Ministère de l’Agriculture,

des Pecheries et de l’Alimentation du Québec (MAPAQ),and Agri-food Innovation Support Program (PSIA) re-search grant No. 811025.

ReferencesAbraham, A., Philip, S., Jacob, C.K., and Jayachandran, K. 2013.

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Akinsanya, M.A., Goh, J.K., Lim, S.P., and Ting, A.S.Y. 2015. Di-versity, antimicrobial and antioxidant activities of cultur-able bacterial endophyte communities in Aloe vera. FEMSMicrobiol. Lett. 362: fnv184. doi:10.1093/femsle/fnv184.

Alberta Agriculture and Forestry. 2015. Industrial hemp enter-prise [online]. Agdex 153/830-1. Available from http://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/agdex126/$file/153-830-1.pdf [accessed 27 May 2016].

Alexander, D.B., and Zuberer, D.A. 1991. Use of chrome azurolS reagents to evaluate siderophore production by rhizospherebacteria. Biol. Fertil. Soils, 12: 39–45. doi:10.1007/BF00369386.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J.1990. Basic local alignment search tool. J. Mol. Biol. 215(3):403–410. doi:10.1016/S0022-2836(05)80360-2. PMID:2231712.

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Table 5. Dynamics of Pseudomonas strains in a dicotmodel host plant.

Treatment Trial Tissue Log CFU·mL–1*

BTC8-1 1 Leaf 2.80±0.14Stem 3.20±0.19Root 2.83±0.08

2 Leaf 2.02±0.41Stem 1.70±0.00Root 4.13±0.19

BTG8-5 1 Leaf 2.99±0.07Stem 0.82±0.34Root 1.30±0.49

2 Leaf 3.24±0.18Stem 2.18±0.23Root 6.42±0.14

*Values represent the mean ± standard deviation (SD) ofthree replications. Colony-forming unit (CFU·mL–1) abun-dance was determined using sterilized tissues of bacterizedtomato seedlings grown under sterile conditions. CFU valuesare significantly different between trials and are presentedseparately as averaged values. Sterilized tissues of noninocu-lated control plants did not show colony growth.

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