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Plant growth-promoting rhizobacteria (PGPR) in Cannabis sativa ‘Finola’

cultivation: An alternative fertilization strategy to improve plant growth and
quality characteristics

Article in Industrial Crops and Products · June 2018

DOI: 10.1016/j.indcrop.2018.06.033

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 Industrial Crops & Products 123 (2018) 75–83

Contents lists available at ScienceDirect

Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

Plant growth-promoting rhizobacteria (PGPR) in Cannabis sativa ‘Finola’ T

cultivation: An alternative fertilization strategy to improve plant growth and
quality characteristics
Giancarlo Pagnania,1, Marika Pellegrinia,1, Angelica Galienia,b, Sara D’Egidioa,
⁎
Federica Matteuccic, Antonella Riccia, Fabio Stagnaria, , Manuel Sergia, Claudio Lo Sterzoa,
Michele Pisantea, Maddalena Del Galloc
a
Faculty of Bioscience and Technologies for Food, Agriculture and Environment, University of Teramo, Italy
b
Council for Agricultural Research and Economics, Research Centre for Vegetable and Ornamental Crops, Italy
c
Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: The massive employment of chemical fertilizers entails substantial costs for agriculture and leads to significant
Antioxidant environmental pollution, soils depletion and crop productivity declines. The aim of this preliminary study was to
Cannabinoids evaluate the suitability of plant growth-promoting rhizobacteria (PGPR) as an alternative fertilization approach
Cannabis sativa L. ‘Finola’ in Cannabis sativa L. ‘Finola’, one of the low-psychoactive substances industrial hemp varieties cultivated in the
PGPR
Abruzzo territory. The PGPR inoculum was first studied in a model system by monitoring the colonization and
Phenolic compounds
survival of bacteria in roots of hemp seedling grown in vitro. Following a complete randomized block design with
SEM
three replicates, female plants were also cultivated in greenhouse and subjected to different cultivation condi-
tions: (i) two different PGPR inoculum concentrations, (ii) nitrogen fertilization, and (iii) unfertilized control. At
the flowering stage, plant growth parameters, main cannabinoid content, antioxidant, and total phenolic con-
tent, were assessed. In the model system experiment, scanning electron microscope (SEM) imaging revealed an
excellent ability of bacteria to adhere to the surface of roots, and to colonize root vascular tissues of hemp
seedlings. Under greenhouse conditions PGPR favored plant growth and development as well as plant secondary
metabolites accumulation and, consequently, antioxidant capacity. In particular, the lowest PGPR concentration
allowed obtaining results comparable with those induced by the recommended nitrogen fertilization. These
results underline the potentiality of PGPR application in hemp plants in terms of both higher biomass accu-
mulation and chemical composition, also meeting environmental goals such as an increase in soil biodiversity
and a reduction in chemical inputs. This study represents the first step toward the potential application of PGPR
in hemp cultivation and could be the base for future extensive evaluations.

1. Introduction
In order to increase crop yields and quality, the application of
chemical fertilizers in agriculture has increased over time with im-
plications in terms of higher production costs as well as of environ-
mental threats regarding water pollution (ground water, lakes, rivers),
air (GHG emission) and soil (reduction of pH and exchangeable bases,
low organic matter content) (Savci, 2012). A promising alternative to
chemical fertilizers is represented by plant growth-promoting
rhizobacteria (PGPR) which, since they firstly definition (Kloepper and
Schroth (1978), have been efficiently applied in agriculture due to their
positive effect on crop productivity and ecosystem functioning (Vejan
et al., 2016). These bacteria are able to promote plant growth and
development through several mechanisms, such as: atmospheric ni-
trogen fixation, siderophores production (siderophores chelate iron to
be available to the plant root), minerals solubilization (particularly
phosphorus), production of plant-growth promoting substances (such as
auxins, cytokinins, gibberellins), and the synthesis of several other

⁎
Corresponding author.
E-mail addresses: gpagnani@unite.it (G. Pagnani), mpellegrini@unite.it (M. Pellegrini), angelica.galieni@crea.gov.it (A. Galieni), sdegidio@unite.it (S. D’Egidio),
federica.matteucci@univaq.it (F. Matteucci), aricci@unite.it (A. Ricci), fstagnari@unite.it (F. Stagnari), msergi@unite.it (M. Sergi), closterzo@unite.it (C. Lo Sterzo),
mpisante@unite.it (M. Pisante), maddalena.delgallo@univaq.it (M. Del Gallo).
1
These authors contributed equally to this article.

https://doi.org/10.1016/j.indcrop.2018.06.033
Received 27 February 2018; Received in revised form 17 May 2018; Accepted 9 June 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
G. Pagnani et al.
growth-promoting compounds (e.g. enzymes) (Glick and Bashan, 1997;
Pérez-Montaño et al., 2014). These microorganisms, which associate
with almost all plant species (Rosenblueth and Martínez-Romero,
2006), belong to the genera Acinetobacter, Alcaligenes, Arthrobacter,
Azospirillium, Azotobacter, Bacillus, Beijerinckia, Burkholderia, En-
terobacter, Erwinia, Flavobacterium, Gluconacetobacter, Herbaspirillum,
Rhizobium, Serratia, and others still unknown (James et al., 2001;
Roncato-Maccari et al., 2003; Sturz and Nowak, 2000). In particular
Azospirillum sp., by stimulating elongation of root systems (Dobbelaere
et al., 1999), formation of lateral and adventitious roots (Molina-Favero
et al., 2008), root hairs (Fulchieri et al., 1993), and root hairs branching
(Jain and Patriquin, 1985), plays a major role in the improvement of
crop growth and yield under several environmental and soil conditions
(Bashan and de-Bashan, 2010; Pereg et al., 2016). Azospirillum species
have demonstrated their potentialities as biofertilizers applied both
alone (Mehnaz, 2015), or in consortium with other microbial species
(Raja et al., 2006; Rajasekar and Elango, 2011; Sarma et al., 2015;
Shahzad et al., 2017): Azospirillum brasilense with Gluconacetobacter
diazotrophicus, Herbaspirillum seropedicae, and Burkholderia ambifaria,
demonstrated its beneficial effects on Solanum lycopersicum L. plant
growth and development (Botta et al., 2013). Similar benefits have
been recorded with the employment of Gluconacetobacter diazotrophicus
on Saccharum officinarum L. and Sorghum bicolor L. (Muthukumarasamy
et al., 2002; Yoon et al., 2016), of Herbaspirillum seropedicae on Oryza
sativa L., Sorghum bicolor L., and Zea mays L. (James et al., 2001;
Gyaneshwar et al., 2002; Roncato-Maccari et al., 2003; Canellas et al.,
2013) and of Burkholderia ambifaria on Sorghum bicolor L. and Zea mays
L. (Di Cello et al., 1997; Chiarini et al., 1998, 2006; Compant et al.,
2008;).
It is known PGPR positive influence on secondary plant metabolism
(Jain et al., 2014; Khalid et al., 2017; Kilam et al., 2015; Kiprovski
et al., 2016) although the mechanisms behind this effect has not been
completely clarified.
In Italy the cultivation of industrial hemp, (Cannabis sativa L.) in the
past disappeared both for economic and administrative reasons
(Cappelletto et al., 2001), has been recently recovered in several re-
gions, i.e. Abruzzo principally with cv. ‘Finola’ due to its short growth
cycle and height. Nowadays the plant offers several potential utiliza-
tions in biological, alimentary, and industrial fields (Fike, 2016), mostly
thanks to its secondary metabolites with different biological properties,
including cannabinoids - mainly Δ9-tetrahydrocannabinol (THC), can-
nabinol (CBN), and cannabidiol (CBD) - as well as terpenoid-like and
phenolic compounds (Flores-Sanchez and Verpoorte, 2008; Matteucci
et al., 2016). In particular, in the past two decades, hemp female in-
florescences and their cannabinoids have played an increasing role in
medicine for their therapeutic effects towards the treatment of a large
number of disorders (Grotenhermen and Müller-Vahl, 2016). At the
same time, the antioxidant activity associated to polyphenolic com-
pounds is usually exploited in the pharmaceutical and food industry
(Abootalebian et al., 2016; Pellegrini et al., 2018). In this context, a key
role is recovered by the analytical techniques that allows the identifi-
cation and quantification of these bioactive compounds (e.g. liquid
chromatography tandem-mass spectometry) as well as the widely
adopted in vitro assays for evaluating the antioxidant capacity with easy
and low-cost procedures (e.g. radical scavenging and total phenolic
content assays) (Sun et al., 2018).
To date, there are not available data on the potential benefits of root
inoculation with PGPR on biomass yield, growth, as well as on sec-
ondary metabolites production/accumulation in industrial hemp.
Besides, mineral nutrition represents a critical issue, in particular N
supply which, although essential, seems to exert positive effects only at
limited range of application doses on biomass growth and development
(Finnan and Burke, 2013; Amaducci et al., 2015; Tang et al., 2017):
Campiglia et al. (2017) recently assessed that rates from 50 to 100 Kg N
ha−1 are adequate under several farm conditions. Hence, the present
work represents a first attempt aimed at investigating the effects of a
76
Industrial Crops & Products 123 (2018) 75–83
consortium of PGPR (Azospirillum brasilense, Gluconacetobacter diazo-
trophicus, Burkholderia ambifaria, and Herbaspirillum seropedicae) on fe-
male plants of an industrial dioecious hemp variety (cv. ‘Finola’), on
morphological and physiological traits, and especially on precise
quality characteristics. To estimate the effects of such consortium, a
comparison with a nitrogen mineral nutrition was imposed. To test our
hypothesis, a two-phase experiment, under controlled conditions, was
carried out: one preliminary in vitro, to study seed inoculum growth and
hemp root colonization, and one under greenhouse to test PGPR bio-
fertilization potential, on plant growth parameters, cannabinoid con-
tent, total phenolic accumulation, and antioxidant capacity.
2. Materials and methods
2.1. Finola
Cannabis sativa ‘Finola’ (previously “FIN-314”) is a dioecious cul-
tivar characterized by early sowing and flowering, low branching and
short length, with smaller seeds and lower levels of biomass recovery
than other hemp varieties; Finola was first accepted in Finland in the
list of official plant cultivars, in 2003 and was published in the EU
Common Catalogue (Callaway, 2004). Finola, as well as the other hemp
cultivars, is a low-maintenance crop, which needs no herbicides or
other pesticides; best cultivation results are usually obtained from
warm, moist and well-drained soils, rich in organic matter and nutrient
availability (Callaway, 2004). The seeds utilized in the trial belonged to
a Canadian Finola cultivar 1st generation (Ref. no. 9-107075 – January
2016), sampled and analyzed according to ISTA rules for orange cer-
tificates by 20/20 seedlabs EC rules and standards, and obtained by a
local certified dealer (Hemp Farm Italia, Tortoreto (TE), Italy).
2.2. Plant material and growth conditions
Two homologous experiments were carried out simultaneously at
the greenhouse of Agronomy and Crop Sciences Research and
Education Center, Department of Food Science, University of Teramo,
Italy, (elevation 15 m above sea level; 42°42′40.0″ N,13°54′10.0″ E)
from September to October 2016. The greenhouse was covered with a
single layer of ethylene-vinyl acetate film (PATILUX, P.A.T.I. S.p.A., San
Zenone degli Ezzelini, TV, Italy) and characterized by a natural venti-
lation system. Seeds of C. sativa ‘Finola’, were sown in a nursery sub-
strate media (Huminsubstrat N3, Neuhaus, Klasmann-Deilmann,
Geeste, Germany) with the following characteristics: 90% peat, 10%
clay; pH 6; NPK 14:16:1; density 1.3 kg m−3; conductivity 35 mS m−1,
and kept in a growth chamber for 15 days. Hemp phenological stages
were scored following the Decimal Code presented by Mediavilla et al.
(1998). After 15 days from sowing, uniform seedlings (second leaf pair,
corresponding to the vegetative stage 1004 - 3 leaflets) were trans-
planted into 20 cm (5 L) diameter plastic pots, 1 per pot. The growth
substrate consisted of a mixture of peat-based compost potting soil
(Terriccio Universale di Qualità®, COMPO SANA Italia S.r.l., Cesano
Maderno (MB), Italy), vermiculite and perlite at a ratio of 2:1:1 (v/v).
Starting from transplanting, environmental conditions were constantly
monitored through sensors of temperature, humidity, total solar ra-
diation (pyranometer, PYR), and photosynthetically active radiation
(PAR), connected to a data logger system (EM50 Data Collection
System, Decagon Devices Inc., Pullman, WA, USA). Pots were manually
re-watered with tap water approximately every 4 days, in order to keep
the volumetric soil content (monitored with moisture sensors connected
to the data logger system) at 0.180 m3m−3, on average. No fungicide,
insecticide, or additional fertilizers, were applied.
2.3. Experimental design, data collection, and growth analysis
The two experiments were arranged on a completely randomized
block design with three replicates, where the treatments under
G. Pagnani et al.
comparison were three different fertilization strategies: (i) mineral
fertilization at the rate of 80 kg N ha−1 (Nitrogen), (ii) biofertilization
with the PGPR consortium at an inoculum density of 106 cells mL−1
(Inoculum 1) and, (iii) 107 cells mL−1 (Inoculum 2), (iv) an unfertilized
control (Control). For each experiment, the single experimental unit
consisted in 12 pots replicated three times with, as consequence, the
single Treatment amounting for 36 pots. Nitrogen was supplied as
ammonium nitrate. To eliminate the possible variability due to the
bacterial cultural medium, Control and Nitrogen plants were treated
with the same amount of autoclaved bacterial solutions. The different
fertilization protocols were imposed starting from 7 days after trans-
planting (DAT) following the selections of female plants on stage 2001
(flower primordia) as proposed by Mediavilla et al. (1998).
At the flowering stage (2202 – Mediavilla et al., 1998), three plants
per each experimental unit were sampled and separated into stem and
leaves (the inflorescences were added to the leaves) weighted for fresh
(FW) and dry weight (DW) determinations after oven-drying at 80 °C to
assure constant weight. Before drying, stem length (cm) was recorded
and leaf area (LA cm2) was measured with an image analysis software
(ImageJ, National Institutes of Health, Bethesda, MD, USA) after pho-
tocopying the leaves of each plant; specific leaf area (SLA, cm2 g−1) was
then calculated.
In our experimental conditions, the flowering stage was reached at
41 DAT, corresponding to 848.45 growing degree days (GDD, °C). GDD
were calculated as the accumulation of mean air temperature exceeding
the base temperature of 1 °C (Van der Werf et al., 1995).
2.4. Bacterial strains, culture conditions, media, and treatments
The single strains of the PGPR consortium, previously described in
Botta et al. (2013), were routinely grown at 30 °C on their specific
cultural medium. For the inoculum, all bacteria were separately grown
on a common medium T4, with fructose as the carbon source since it
was the only sugar utilized by all the strains. Each PGPR strain was
grown aerobically on a rotating shaker (150 rpm) at 27 °C, collected at
the beginning of the stationary phase, washed, and diluted 108 cells
mL−1 with sterile physiological solution. The inoculum was obtained
by mixing together the four strains at two final concentrations. A
concentration of 106 cells mL−1 was utilized to assess the capacity to
colonize roots of the hemp seedlings (in vitro), while the 106 and 107
cells mL−1 working inoculum solutions were utilized in the hemp
greenhouse experiment to test biofertilizer potentiality (in vivo).
2.5. Seed inoculation with the PGPR consortium and scanning electron
microscopy (SEM) analysis of bacteria-root colonization
Three pools of 25 seeds of Finola were sterilized with an initial
shaking step for 15 min in a 20% (v/v) NaClO solution; the seeds were
washed with sterilized distilled water, treated for 3 min in 70% (v/v)
ethanol, and final rinsed three times in distilled water for 15 min. Seed
sterility was ascertained by incubating 10 seeds on tryptone soya agar
(TSA) plates at 30 °C for 4 days. Seeds were inoculated by immersion for
1 h in the inoculum solution of 106 cell mL−1 and then washed in
sterilized water. They were transferred to glass tubes containing 10 mL
of a sterile Murashige e Skoog agar medium (Murashige and Skoog,
1962) and incubated in an environmentally controlled room (22 °C;
16 h/8 h light/dark; 120 μmol m−2 s−1 photosynthetically active ra-
diation, 65% relative humidity (RH)). After 8 days, fresh roots from
inoculated and non-inoculated seedlings were collected from the tubes,
washed briefly in sterile water, and excised with a sterile lancet in 5 mm
sections. The sections were fixed in 2.5% glutaraldehyde in 0.2 M ca-
codylate buffer, pH 7.2–7.4 at 4 °C for 24–48 h; the excised sections
were then washed in the same buffer for 20 min and dehydrated in
alcoholic series 30, 50, 70, 96%, and then in absolute acetone. After
dehydration, root samples were cut into thin sections and covered with
gold. All the micrographs were acquired using an accelerating voltage
77
Industrial Crops & Products 123 (2018) 75–83
of 20 kV. The observations were carried out with a XL30 CP scanning
electron microscope (SEM) (Philips, Eindhoven, The Netherlands).
2.6. SPAD readings
The estimation of chlorophyll (Chl) content in hemp leaves was
obtained with the SPAD (soil-plant analysis development) 502 plus
portable chlorophyll meter (Konica Minolta, Inc., Tokyo, Japan).
Measurements were carried out at flowering, in the mid-section of five
fully expanded and same sun-oriented leaves per plant (three randomly
selected plants per experimental unit).
2.7. Samples extraction
Dried inflorescences (see paragraph 2.3) were homogenized in an
agate mortar until the whole sample took uniform color and con-
sistency. 10 mg of each sample was transferred in a 2 mL test tube and
extracted with 1 mL of methanol, for 30 min in ultrasonic bath at 20 °C.
After the extraction the tubes were centrifuged at 10,000 rpm for
10 min and the supernatants were collected; after a 0.2 μm filtration
(1 mL Minisart® PTFE syringe filters - Sartorius, Göttingen, Germany)
the extracts were stored at −20 °C until analyzed for their main can-
nabinoid contents, antioxidant capacities, and total phenolic contents.
2.8. Chemical standards and reagents
Cannabinoids THC, CBN, and CBD standard solutions (1 mg mL−1,
purity ≥99%) were purchased from LGC standard (Sesto San Giovanni,
Milan, Italy). Working standard mixtures were prepared by appropriate
dilution in methanol and stored at −20 °C. Ultrapure water, formic
acid, methanol, and acetonitrile, all UPLC-MS grade, were purchased
from Carlo Erba (Milan, Italy).
2.9. LC–MS/MS analysis
An UHPLC system Nexera XR (Shimadzu, Tokyo, Japan) coupled to
a Qtrap 4500 (Sciex, Toronto, Canada) was utilized for the LC–MS/MS
analysis. The compounds were separated using a Kinetex C18-XB
column (100 × 2.1 mm ID, Phenomenex, Torrance, CA, USA) packed
with core-shell particles of 1.7 μm A security Guard Ultra Cartridge
packed with C18 particles was also employed to avoid column damages.
The mobile phases were: (A) water and (B) methanol both containing
1.25 mM ammonium acetate. The flow rate was 0.3 mL min−1 entirely
driven into the ion source. The gradient elution program was: phase B
was increased from 60 to 100% in 2 min, kept for 2 min, and then
switched back to the initial 60% in 0.5 min. The complete separation of
all substances occurred in 6 min.
The mass spectrometer was equipped with a heated electrospray
ionization source (V-source) and operated in the positive ionization
mode. The ion spray voltage was −4.5 kV, nebulizer gas (air) at 60 psi,
turbo gas (nitrogen) at 40 psi and temperature 500 °C. The acquisition
was performed in Multi Reaction Monitoring (MRM) mode with two
precursor ion/fragment transitions for each analyte. All source and
instrument parameters for the monitored analytes were tuned by in-
jecting each single standard solution at a concentration of 0.1 ng L−1 at
10 L min−1 by a syringe pump. All the source parameters were checked
in flow injection analysis with the same chromatographic conditions
(flow and solvent composition). Peak areas for the selected ions were
determined using MultiQuant software from Sciex and quantitation was
performed versus a calibration curves prepared in 5 points between
0.1–150 ng mL−1. The selected transitions, together with the main
LC–MS/MS parameters, are shown in Table 1, an example of extracted
ion currents (XICs) of target analytes is in Fig. 1.
Intra-day precision (RSD% = Standard deviation / mean × 100)
and accuracy (A% = calculated concentration / theoretical con-
centration × 100) were calculated on 3 replicates of each sample
G. Pagnani et al. Industrial Crops & Products 123 (2018) 75–83

Table 1
UHPLC-ESI-MS/MS parameters for the selected analytes: cannabidiol (CBD),
cannabinol (CBN) and Δ9-tetrahydrocannabinol (THC).
Analyte Rt Q1 DP EP Q3 CE CXP

CBD 3.96 315.1 20 5 193.2 27 6

135.1 25 9

CBN 4.27 311.1 20 12 241.2 28 9

223.1 28 9

THC 4.42 315.0 20 5 193.0 29 10

123.0 41 10

CBD – cannabidiol; THC - delta-9-tetrahydrocannabinol; CBN – cannabinol; Rt -

retention time (min); Q1 - precursor ion mass (amu); DP - declustering potential
(amu); EP - entrance potential (V); Q3 - product ion mass (amu); CE - collision
energy; and CXP - cell exit potential.

spiked after extraction at three concentration levels (CL - 1, 10,

100 ng mL −1). LOD and LOQ were calculated for each analyte on the
less intense transition according to LOD = μB + 3 σB and LOQ = μB +
10 σB equations, where μB and σB were the mean of blank signal and
the standard deviation, respectively. The main validation data of the
method are in Table S1 (see Supplementary material).

2.10. Antioxidant activity and total phenolic content

For the antioxidant capacity (AOC) and total phenolic content (TPC)
assessments, 0.1–10 mg mL−1 methanolic solutions were subjected to
the different spectrophotometric assays by means of Lambda Bio 20
UV/Vis spectrophotometer (Perkin Elmer, Waltham, Massachusetts,
USA).
For AOC determination, Trolox equivalent antioxidant capacity,
with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) - TEAC/
ABTS, ferric reducing antioxidant power - FRAP, and 2,2-diphenyl-1-
picrylhydrazyl – DPPH methods were utilized. The TEAC/ABTS assay
was determined as described by Masaldan and Iyer (2011); FRAP was
detected by the potassium ferricyanide-ferric chloride method de-
scribed by Oyaizu (1986) and the DPPH antioxidant potential was
measured according to the method described by Brand-Williams et al.
(1995). In TEAC/ABTS, FRAP, and DPPH, Trolox was employed as re-
ference standard and results were expressed as mg Trolox equivalent
(TE) g−1 DW as mean of three replicates.
The TPC was measured by the Folin-Ciocâlteu method (Singleton
and Rossi, 1965). TPC results were expressed as mg Gallic acid
equivalents (GAE) g−1 DW as mean of three replicates.

2.11. Statistical analysis

A two-way analysis of variance (ANOVA) was applied to test (F-test)

the effects of experiment and treatment on all the investigated vari-
ables. Before the ANOVA, the data were analyzed to test for normality
and homoschedasticity assumptions, through graphical methods. Since
no significant effects of “Experiment” or “Experiment × Treatment”
interactions were detected, only the main effect of Treatment is re-
ported in Tables and Figures. When significant differences emerged,
means separation was performed by the Least Significant Difference test
(LSD), at 1% and 5% (p < 0.01 and p < 0.05, respectively) levels of
significance. Statistical analyses were performed utilizing the R soft-
ware (R package; Fox, 2015; De Mendiburu, 2016; R Core Team, 2017).
A correlation test (Pearson’s) was applied to AOC and TPC results by
means of XLSTAT 2016 (Addinsoft, Paris, France). The positive/nega-
tive strength of correlation was considered low for +/−0.1 < r < +/
−0.3, moderate for +/−0.3 < r < +/−0.7, and strong for r > +/
−0.7; for values of r < +/− 0.1 the variables were considered not
correlated.
Fig. 1. Extracted ion currents (XICs) of the Multiple Reaction Monitoring
(MRM) transitions (only quantifiers) obtained from cannabidiol (CBD), Δ9-
tetrahydrocannabinol (THC) and cannabinol (CBN) at a concentration of
10 ng mL −1.
3. Results and discussion
3.1. PGPR colonization of Hemp seedling roots
The PGPR consortium ability to colonize root tissues was in-
vestigated in 8 days-old seedlings of hemp by scanning electron mi-
croscopy (SEM). The obtained SEM images are reported in Fig. 2. From
the images is clearly visible that: (i) the bacteria actively stimulate root
hair proliferation (Fig. 2A), (ii) adhere to root surface (Fig. 2B), and (iii)

78
G. Pagnani et al.
Fig. 2. SEM images of fresh roots obtained from hemp seedlings inoculated
with the PGPR consortium. Samples were collected after 8 days of in vitro
aseptic growth conditions. The figure shows a hemp seedling root in which
hairy roots are visible (A). The four bacteria actively colonize the plant on the
root surface (B) and penetrate inside the root tissues (C).
penetrate inside the inner root tissues (Fig. 2C). A comparison between
treated and control root surface is shown in Fig. 3. In this Figure the
bacteria strictly adhere to the root surface of the inoculated seedling
(Fig. 3A) while they are not present in the not inoculated Control
(Fig. 3B); the absence of bacteria in the latter confirms the aseptic ex-
perimental conditions.
These bacteria, indeed, adhere to the root surface and are capable of
colonizing the internal tissues, crucial abilities for plant growth pro-
motion. They are also mandatory for successful applications in con-
trolled environment and open field, where increase resilience against
79
Industrial Crops & Products 123 (2018) 75–83
biotic and abiotic stresses (Botta et al., 2013).
3.2. Plant growth parameters
As shown in Fig. 4, the average temperature and humidity detected
were 21.4 °C and 71%, respectively; the crop cycle reached 41 DAT.
Regarding day-length, PYR and PAR availability during the daylight
hours were registered, 13 h of light per day, 198.18 W m−2, and of
438.99 μmol m−2s−2, respectively.
Morphological and physiological traits of hemp plants at flowering
were significantly affected by the different fertilization strategies
(Table 2). Nutrients availability from the substrates is necessary for
plant growth and development, and Nitrogen and Inoculum 1 condi-
tions demonstrated to promote significantly (p < 0.01) stem length
with respect to Control, by 27.0% and 32.3%, respectively, while the
effect of Inoculum 2 resulted not significant (+2.6% with respect to
Control) (Table 2).
Stem dry weight registered similar response, but in this case
Inoculum 2 showed higher significant values than Control as much as
all other fertilization treatments (p < 0.01) (+62.8%, averaged over
fertilization strategies) (Table 2). Similar results were recorded for leaf
dry weight, with an average increase of 46.7% from the fertilization
treatments with respect to Control plants (Table 2).
Our results demonstrate that the application of PGPR at the lowest
inoculum density (i.e. 106 cells mL−1) and its stimulation in terms of
plant growth, is comparable with that obtained with fertilization by a
conventional N mineral fertilizer. These results are in accordance with
the findings of several authors (Banchio et al., 2009, 2005; Cappellari
et al., 2013; Gray and Smith, 2005; Van Loon, 2007) who indicate
significant increasing in growth and related growth parameters due to
the inoculation with similar PGPR consortia, although in different plant
species. The four bacteria consortium employed in our inoculum have
already demonstrated interesting stimulation of plant growth and de-
velopment on Lycopersicon esculentum L. (Botta et al., 2013).
Our results indicate also positive effects on leaf area expansion from
Inoculum 1, Nitrogen, and Inoculum 2 with respect to the Control, with
increasing values ranging from 54.5%, 52.2% to 46.7%, respectively;
besides, no significant influences from fertilization was observed with
regard to SLA (Table 2). Previous studies, based on the application of
PGPR strains, have already highlighted interesting increases of leaf
surface in Arabidopsis, corn (Zea mays L.), and tomatoes (Lycopersicon
esculentum L.) (Gholami et al., 2009; Olivares et al., 2015; Rangel de
Souza et al., 2015; Romero et al., 2014). Such a wider photosynthetic
apparatus translates into a higher CO2 fixation as well as biomass ac-
cumulation. The positive effects on photosynthetic activity were esti-
mated also through physiological assessments. Indeed, the observed
behavior in terms of crop growth was significantly correlated (data not
shown) to an ameliorated hemp physiological status (see the estimated
content of chlorophyll in leaves (SPAD)). Inoculum 1 and Nitrogen
conditions exhibited the highest SPAD values (59.92 and 60.04, for
Inoculum 1 and Nitrogen, respectively), followed by Inoculum 2
(54.30), and Control (49.69) (Table 2). Besides, it is important to point
out that SPAD is considered also an indicator N concentration in plant
tissues (Evans, 1983; Yildirim et al., 2011): higher SPAD values regis-
tered in fertilized plants should be also ascribed to the improved N
availability thanks to both N mineral fertilization (Nitrogen) as well as
to the efficiency of PGPR to promote N2-fixation (Inoculum 1 and In-
oculum 2) (Boddey et al., 2003; Prabha et al., 2017).
In summary, our results underlined that Inoculum 1 biofertilization
allowed better plant physiological status and growth performances,
comparable with those obtained or obtainable with ordinary mineral
fertilization practices, estimated at rate of 80 Kg N ha−1. Such N dose
fall within 50–120 Kg N ha−1, and it is suggested as the optimal N rate
for several hemp varieties (Callaway, 2004). This means that mineral
fertilizers could be usefully replaced with more “environmental
friendly” formulates without compromising yields, even if particular
G. Pagnani et al.
Fig. 3. Inoculated (A) and non-inoculated (B) fresh roots obtained from hemp seedlings collected after 8 days of in vitro aseptic growth conditions at the SEM.
Fig. 4. Patterns of mean temperature (Tmean, °C), relative humidity (RH, %),
total solar radiation (PYR, W m−2) and photosynthetically active radiation
(PAR, μmol m−2 s−1) as recorded during hemp growing cycle, starting from
transplanting. Each data point represents the daily mean of 24 hourly collected
values; for PYR and PAR values only the daylight hours are considered. Time is
expressed in days after transplanting (DAT). For each variable, the dashed line
represents the overall average value.
attention must be given to the inoculum concentration. Significant
plant responses in fact, are already appreciable at low inoculum density
(106 cells mL−1, Inoculum 1) while slightly higher values tend to de-
crease hemp growth and development as already described by Fallik
et al., (1988) in maize crop. It is already known that biostimulants have
a growth-promoting activity when applied at low quantities (du Jardin,
2015) while high density could led to a decreasing of plant growth and
development (Dobbelaere et al., 1999).
80
Industrial Crops & Products 123 (2018) 75–83
3.3. LC–MS/MS main cannabinoids content
The results obtained from main cannabinoid profile analysis by
UHPLC-ESI-MS/MS are shown in Table 3. From the correlation of CBD
to THC (CBD:THC) it is interesting to observe that all treatments ex-
hibited a 12:1 CBD:THC ratio. Such a cannabinoid profile is typical of
hemp varieties characterized by high contents of CBD and low of THC
(Stout et al., 2012). Both THC content and CBD:THC ratio are similar to
those found by Callaway (Callaway, 2002) for the same variety. Call-
away stated that Finnish Finola mature females were characterized by a
THC content that ranges between 0.04–0.16 g 100g−1 DW and a CBD/
THC ratio higher than 10:1. The high amounts of neutral cannabinoids
forms, can be due to the 80 °C oven-drying treatment of the plant
samples. Cannabinoids, in fact, are usually present in their acid forms in
plants; treatment at high temperatures leads to the production of their
neutral forms by decarboxylation (Andre et al., 2016; Turner and
Mahlberg, 1984). A recent study on the degradation of THCA (THC acid
form) to THC (Taschwer and Schmid, 2015) determined that the ex-
position of the plants to temperatures higher than 50 °C led to an ac-
celerated decarboxylation of THCA.
In Table 3 cannabinoid concentrations from the different conditions
are shown. Nitrogen and both PGPR inocula affected the cannabinoids
contents, while Control showed the lowest concentrations (p < 0.01)
of THC, CBN, and CBD. According to these results, PGPR fertilizations
had a positive effect on CBD and THC contents, with no significant
(p > 0.05) difference with Nitrogen. Although several studies have
demonstrated a main role of plant nutrition in the production of sec-
ondary metabolites (Pacifico et al., 2008), its role in cannabinoid pro-
duction is still uncertain (Gorelick and Bernstein, 2017). While some
literature report that an increased mineral content of macronutrients
(e.g. calcium, iron, magnesium, nitrogen) lead to an increase of THC
content (Gorelick and Bernstein, 2017), high nitrogen availability
seems to lead to a consistent THC decrease (Bócsa et al., 1997). In
addition, Mechtler et al. (2004) reported that too small or too expanded
hemp leaves were lower in THC than medium ones.
3.4. Antioxidant activity and Total phenolic content
All AOC assay methods utilized for the extracts of the inflorescences
revealed that Inoculum 1 and Nitrogen fertilizations are able to sig-
nificantly enhance antioxidant potentials (p < 0.01) (Table 4). Besides,
Inoculum 2 performed worse than Inoculum 1 and Nitrogen, although
its induced antioxidant potential was higher (p < 0.01) than Control
according to FRAP assay; however, no significant differences emerged
when DPPH (p > 0.05) and TEAC/ABTS (p > 0.05) methods were
employed. The obtained results were in accordance with those in the
literature for different plant species: Ordookhani et al. (2010) assessed
that the use of the PGPR in combination with arbuscular mycorrhizal
G. Pagnani et al. Industrial Crops & Products 123 (2018) 75–83

Table 2
Plant growth parameters. The table shows the effects of bacterial and nitrogen applications on stem length, stem dry weight, leaf dry weight, SPAD (soil plant analysis
development), leaf area (LA) and specific leaf area (SLA) of Cannabis sativa ‘Finola’. For each column, means labeled with the same letter did not differ significantly at
p < 0.05 (Fisher’s LSD test).
Treatment Stem Length Stem Dry Weight Leaf Dry Weight SPAD LA SLA
(cm plant−1) (g plant−1) (g plant−1) (cm2 plant−1) (cm2 g−1)

Control 42.79 b 0.60 b 2.04 b 49.69 c 283.41 b 139.45

Inoculum 1 56.60 a 0.94 a 3.08 a 59.92 a 437.96 a 142.83
Inoculum 2 43.91 b 0.99 a 2.87 a 54.30 b 415.74 a 146.20
Nitrogen 54.36 a 1.03 a 3.03 a 60.04 a 431.42 a 142.93
** ** ** * **
F-test NS
LSD 6.08 0.22 0.53 3.69 96.44 –

NS = not significant.
* p < 0.05.
** p < 0.01.

Table 3 (TPC) was assessed (Table 4), ranging from 82.8 to 102.7 mg GAE g−1
Contents of main cannabinoids expressed as g 100g−1 dry weight. For each DW for Control and Inoculum 1, respectively. PGPR and Nitrogen fer-
column, means labeled with the same letter did not differ significantly at tilizations were particularly effective also to stimulate a significant
p < 0.05 (Fisher’s LSD test). enhancement of such trait. These values were higher than those ob-
Treatment THC CBN CBD tained by Vonapartis et al. (2015) for the same variety (15.47 mg GAE
g−1 DW), probably due to their cultivation in open field. In this study
Control 0.159 b 0.017 b 1.899 b
an average phenolic content from 10 industrial hemp cultivars was
Inoculum 1 0.175 a 0.020 a 2.080 a
Inoculum 2 0.171 a 0.020 a 2.059 a
obtained (22.24 mg GAE g−1 DW), with a wide variability with values
Nitrogen 0.176 a 0.022 a 2.065 a ranging from 13.68 mg GAE g−1 DW to 51.60 mg GAE g−1 DW. In
** ** **
F-Test addition, different studies have already highlighted the antioxidant
LSD 6.73 1.78 8.36 potential of products obtained from Finola variety. Among them, the
recent study by Smeriglio et al. (2016) highlighted an interesting an-
THC - delta-9-tetrahydrocannabinol; CBN – cannabinol; CBD – cannabidiol.
tioxidant activity of cold-pressed Finola seed oil due to the presence of
** p < 0.01.
phenolic compounds. The significant correlation between phenolic
compounds and antioxidant capacity was already reported for several
Table 4
plant species (Piluzza and Bullitta, 2011). We also calculated the cor-
Antioxidant activity obtained from the three different assays Trolox Equivalent
Antioxidant Capacity with 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid relation coefficients (Pearson’s correlation) between TPC and AOC,
(TEAC/ABTS), Ferric Reducing Antioxidant Power (FRAP) and 2,2-diphenyl-1- obtaining a moderate positive correlation between TPC and FRAP,
picrylhydrazyl (DPPH). For each column, means labeled with the same letter ABTS and DPPH assays (r values of 0.652, 0.582, and 0.438, respec-
did not differ significantly at p < 0.05 (Fisher’s LSD test). tively), possibly indicating that antioxidant activity is mostly due to
TPC.
Treatment ABTS FRAP DPPH TPC
(mg TE g−1 (mg TE g−1 (mg TE g−1 (mg GAE g−1
DW) DW) DW) DW)
4. Summary/conclusions
Control 118.41 b 107.11 c 11.92 c 82.77 b
Inoculum 1 154.52 a 202.49 a 18.98 b 102.68 a
Inoculum 2 130.01 b 141.19 b 11.94 c 101.86 a This preliminary study allowed obtaining information on the utili-
Nitrogen 166.53 a 199.52 a 20.09 a 99.78 a zation of PGPR as biofertilizer for C. sativa ‘Finola’. In vitro study re-
* ** ** **
F-Test vealed an excellent ability of the bacteria to adhere to the roots surface,
LSD 18.44 14.33 0.65 11.27 and to colonize root vascular tissues of hemp seedling. From our in vivo
−1 results it emerges that the application of PGPR could be very interesting
mg TE g DW = mg Trolox Equivalents per g of dry weight matrix.; mg GAE
g−1 DW = mg Gallic Acid Equivalents per g of dry weight matrix.
since it improves not only growth and plant physiological status, but
* p < 0.05. also plant secondary metabolites accumulation and antioxidant ac-
** p < 0.01. tivity. The spread of Inoculum 1 is comparable with N fertilization at
the recommended nitrogen rate (50–120 Kg N ha−1), with the ad-
fungi (AMD) increased significantly the antioxidant activity of tomato vantages of better sustainability as well as an increase on soil biodi-
fruits (Lycopersicon esculentum L.), stating that PGPR and AMD possibly versity, threatened by chemical fertilizers.
reduce negative effects of environmental stress trough the increasing of Further studies should be undertaken to validate the biofertilizer
antioxidant capacity. In other works an increasing in antioxidant ca- potentialities in the field, as well as to better characterize the phenolics
pacity was observed in pea pods (Pisum sativum L.) (Jain et al., 2014), and secondary metabolites profile in relation to PGPR application. Our
Stevia rebaudiana (Kilam et al., 2015), soybean (Glycine max L.) findings represent a valid basis for future extensive evaluations, parti-
(Kiprovski et al., 2016) and spinach (Spinacia oleracea L.) (Khalid et al., cularly on PGPR effectiveness on hemp, on Finola variety, in particular,
2017). The high overall ability to scavenge different reactive species, as and its antioxidant potential.
observed also in unfertilized plants, demonstrates that this hemp
variety is, in any case, an important source of antioxidant compounds.
Scientific works on different C. sativa varieties have already identified Authors contribution
the presence of some secondary metabolites responsible for the anti-
oxidant properties (Andre et al., 2016; Brenneisen, 2007; Flores- The manuscript was written through contributions of all authors. All
Sanchez and Verpoorte, 2008). authors have given approval to the final version of the manuscript.
In our work an interesting accumulation of phenolic compounds

81
G. Pagnani et al.
Declaration of interest
None.
Acknowledgments
The Authors are greatly thankful to Alessandro Palumbo of “Hemp
Farm Italia” for providing plant seeds material and to Maria Di
Giammatteo and Lorenzo Arrizza of “Centro di Microscopie – Università
degli dell’Aquila” for their support with the Scanning Electron
Microscopy.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2018.06.033.
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