Exploiting Beneficial Pseudomonas spp. for Cannabis Production

Abstract

Among the oldest domesticated crops, cannabis plants ( Cannabis sativa L., marijuana and hemp) have been used to produce food, fiber, and drugs for thousands of years. With the ongoing legalization of cannabis in several jurisdictions worldwide, a new high-value market is emerging for the supply of marijuana and hemp products. This creates unprecedented challenges to achieve better yields and environmental sustainability, while lowering production costs. In this review, we discuss the opportunities and challenges pertaining to the use of beneficial Pseudomonas spp. bacteria as crop inoculants to improve productivity. The prevalence and diversity of naturally occurring Pseudomonas strains within the cannabis microbiome is overviewed, followed by their potential mechanisms involved in plant growth promotion and tolerance to abiotic and biotic stresses. Emphasis is placed on specific aspects relevant for hemp and marijuana crops in various production systems. Finally, factors likely to influence inoculant efficacy are provided, along with strategies to identify promising strains, overcome commercialization bottlenecks, and design adapted formulations. This work aims at supporting the development of the cannabis industry in a sustainable way, by exploiting the many beneficial attributes of Pseudomonas spp.

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REVIEW
published: 14 January 2022
doi: 10.3389/fmicb.2021.833172
Edited by:
Tofazzal Islam,
Bangabandhu Sheikh Mujibur
Rahman Agricultural University,
Bangladesh
Reviewed by:
Puneet Singh Chauhan,
National Botanical Research Institute
(CSIR), India
Jordi Petit Pedró,
Polytechnic University of Valencia,
Spain
Zamir Punja,
Simon Fraser University, Canada
*Correspondence:
Martin Filion
martin.filion@agr.gc.ca
Specialty section:
This article was submitted to
Microbe and Virus Interactions with
Plants,
a section of the journal
Frontiers in Microbiology
Received: 10 December 2021
Accepted: 27 December 2021
Published: 14 January 2022
Citation:
Balthazar C, Joly DL and Filion M
(2022) Exploiting Beneficial
Pseudomonas spp. for Cannabis
Production.
Front. Microbiol. 12:833172.
doi: 10.3389/fmicb.2021.833172
Exploiting Beneficial Pseudomonas
spp. for Cannabis Production
Carole Balthazar1, David L. Joly1and Martin Filion2*
1Department of Biology, Faculty of Sciences, Université de Moncton, Moncton, NB, Canada, 2Saint-Jean-sur-Richelieu
Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC, Canada
Among the oldest domesticated crops, cannabis plants (Cannabis sativa L., marijuana
and hemp) have been used to produce food, fiber, and drugs for thousands of years.
With the ongoing legalization of cannabis in several jurisdictions worldwide, a new high-
value market is emerging for the supply of marijuana and hemp products. This creates
unprecedented challenges to achieve better yields and environmental sustainability,
while lowering production costs. In this review, we discuss the opportunities and
challenges pertaining to the use of beneficial Pseudomonas spp. bacteria as crop
inoculants to improve productivity. The prevalence and diversity of naturally occurring
Pseudomonas strains within the cannabis microbiome is overviewed, followed by their
potential mechanisms involved in plant growth promotion and tolerance to abiotic
and biotic stresses. Emphasis is placed on specific aspects relevant for hemp and
marijuana crops in various production systems. Finally, factors likely to influence
inoculant efficacy are provided, along with strategies to identify promising strains,
overcome commercialization bottlenecks, and design adapted formulations. This work
aims at supporting the development of the cannabis industry in a sustainable way, by
exploiting the many beneficial attributes of Pseudomonas spp.
Keywords: Cannabis sativa, marijuana, hemp (Cannabis sativa L.), Pseudomonas, plant growth-promoting
rhizobacteria (PGPR), biological control, abiotic stress, microbial inoculant
INTRODUCTION
Cannabis (Cannabis sativa L.) is an annual dioecious herbaceous plant from the Cannabaceae
family. Cannabis crops likely originated from the temperate regions of Eurasia where their early
cultivation and domestication began thousands of years ago. Human selection and breeding
resulted in the creation of a plethora of cultivated varieties (cultivars) that are now disseminated
worldwide (Clarke and Merlin, 2016). Following the steps of early farmers who harvested the stalks,
seeds, and inflorescences of wild cannabis plants to produce fiber, oil, and drugs, respectively,
generations of breeders have developed modern cultivars with distinctive traits to efficiently
produce these economically valuable commodities.
While hemp cultivars harvested for fiber production tend to have a tall stature with elongated
stem internodes yielding long bast fibers, oilseed cultivars usually display a short branching
architecture producing lots of nutritious seeds (Small, 2015). Fiber and oilseed hemp cultivars must
also yield low levels of 1-9-tetrahydrocannabinol (THC)—the main psychoactive cannabinoid
compound of cannabis—to comply with laws in North America and Europe that define hemp as
containing up to 0.3% of THC and/or its acidic precursor on a dry weight basis.
On the other hand, marijuana plants have been bred mostly clandestinely for their large female
inflorescences (the “buds”) harboring glandular trichomes that yield high levels of psychoactive
THC. By eliminating all male plants from the crop to prevent pollination, seedless female
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inflorescences yielding more than 20% THC by dry weight can
thus be obtained from the most potent marijuana cultivars
(Clarke and Merlin, 2016). Apart from THC, other molecules
of pharmaceutical interest, like cannabidiol (CBD) and aromatic
terpenes, can also be obtained from the glandular trichomes
of both marijuana and hemp cultivars. These drug-type crops
(e.g., marijuana and hemp cultivars grown for phytochemical
production, including THC and/or CBD) are very lucrative and
are generally reproduced vegetatively by clonal cuttings from
elite female plants with desirable genotypes, then transplanted
in enclosed outdoor plots, secured greenhouses, or indoor
cultivation rooms. Contrastingly, commercial fiber and oilseed
hemp crops are mostly directly seeded in agricultural fields for
cost efficiency (Small, 2015;Thiessen et al., 2020).
Because of the widespread criminal status associated
with some marijuana products, reliable research and
economic development with cannabis plants have been
largely prevented during most of the 20th century. Therefore,
great opportunities and challenges remain to improve cannabis
cultivation in the wake of the worldwide legalization trend
led by progressive countries like Uruguay and Canada
(Taghinasab and Jabaji, 2020).
Pseudomonas is a diversified genus of rod-shaped, motile,
aerobic Gammaproteobacteria. With more than 200 type strains
distributed in intricate lineages and phylogenetic groups, it is
currently the Gram-negative genus with the highest number
of recognized species (Lalucat et al., 2020). These versatile
bacteria are abundant in a wide range of environmental niches,
including soil, water, plants, and animals, demonstrating their
great metabolic flexibility and lifestyle adaptability. Indeed, while
some species are infamous human or plant pathogens, like
Pseudomonas aeruginosa and Pseudomonas syringae, respectively,
others like Pseudomonas putida and Pseudomonas fluorescens
have been exploited as bioremediation agents for polluted soils,
plant growth-promoting rhizobacteria (PGPR), and biocontrol
agents (Sitaraman, 2015). Model organisms for plant-microbe
interactions, beneficial Pseudomonas spp. are ubiquitous in
soils and competitively colonize all compartments of the plant
microbiome, including the soil close to the roots (rhizosphere),
the surface of aerial organs (phyllosphere), and the inner
plant tissues (endosphere). The host plants, in turn, benefit
from growth- and health-promoting effects including improved
nutrient availability, increased tolerance to abiotic stresses, and
repression of pests and diseases by antibiosis, competition, and
elicitation of induced systemic resistance (ISR) (Nadeem et al.,
2016;Backer et al., 2018). This review aims at identifying
important aspects of these beneficial traits to consider when
developing Pseudomonas spp. inoculants tailored for hemp and
marijuana production.
OCCURRENCE AND DIVERSITY OF
Pseudomonas spp. IN THE CANNABIS
MICROBIOME
Many of the numerous studies that started to unravel the
cannabis microbiome in recent years have consistently identified
Pseudomonas spp. as major components of the cannabis
rhizosphere, phyllosphere and endosphere communities (Backer
et al., 2019;Taghinasab and Jabaji, 2020).
Beneficial species such as P. fluorescens,Pseudomonas
protegens, and P. putida seem to be naturally present
throughout all hemp and marijuana tissues and surrounding
soil compartments (Table 1). For instance, Pseudomonas spp.
were the most abundant culturable bacteria recovered from
above-ground endosphere samples of three different hemp
cultivars in Canada, accounting for 44% of all bacterial isolates
in leaves, 39% in petioles, and 5% in seeds (Scott et al., 2018).
Regarding the below-ground compartments, Pseudomonas
spp. and related Proteobacteria are also commonly identified
as part of the core community of cannabis root colonizers,
regardless of the different cropping systems, growing substrates,
climatic conditions and host characteristics surveyed at various
geographic locations (Table 1).
As plant genotype and edaphic factors cooperatively shape
the structure and diversity of microbial communities within
the rhizosphere and root tissues (Bulgarelli et al., 2013),
complex mechanisms like modulation of root exudates, root
morphology, and regulation of the plant immune system drive
the recruitment and proliferation of beneficial microorganisms
from surrounding bulk soil toward the roots (Sasse et al., 2018).
In this context, since cannabis phytochemicals possess well-
documented antimicrobial properties (Ali et al., 2012), they
may act as repellents or chemotaxis molecules that potentially
inhibit or favor the colonization of bacteria in the vicinity of
the roots and/or in the phyllosphere (Comeau et al., 2020).
Accordingly, factors like cannabis cultivar and/or developmental
stage have been reported to affect the microbiome composition
within root tissues (Winston et al., 2014;Comeau et al.,
2020), while soil characteristics and cropping practices also
exert a preponderant influence on rhizosphere communities
(Winston et al., 2014;Perea, 2019;Barnett et al., 2020;Ahmed
et al., 2021;Comeau et al., 2021). Based on the ubiquity of
Pseudomonas spp. in the cannabis microbiome, it is expected that
directed microbial inoculations with beneficial strains should be
applicable consistently under a wide range of commercial and
agricultural conditions.
Phytopathogenic Pseudomonas species are also reported on
cannabis plants, including Pseudomonas cannabina causing
bacterial blight (water soaked leaf spots turning into necrotic
lesions) (McPartland et al., 2000;Bull et al., 2010) and P. syringae
pv. mori causing striatura ulcerosa (elongated stem lesions with
fluid-filled pustules rupturing into ulcers) (McPartland et al.,
2000;McPartland and Hillig, 2004). P. cannabina and P. syringae
are host-specialized phyllosphere pathogens that invade the
intercellular apoplast space and use a type III secretion system
to deliver virulence effectors into the plant cells (Xin et al.,
2018). Even though recent reports of bacterial pathogens are
surprisingly scarce in cannabis, phytopathogenic Pseudomonas
spp. are expected to cause emerging disease problems with the
surge in hemp and marijuana cultivation (Punja, 2021).
Finally, a last group of Pseudomonas strains potentially
relevant in cannabis cultivation concerns P. aeruginosa. Since this
opportunistic human pathogen can cause important infections
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TABLE 1 | Pseudomonas spp. (or higher related taxa) associated with the cannabis microbiome.
Crop type (cultivar) Sample type Environment Reported taxa References
Fiber/oilseed hemp (Anka, CRS-1, Yvonne) Leaves, petioles, seeds
(endosphere)
Outdoor field Pseudomonas spp.,
Pseudomonas fulva and
Pseudomonas orientalis
Scott et al., 2018
Fiber/oilseed hemp (Anka) Bulk soil, rhizosphere, roots
(endosphere), leaves and
flowers (phyllosphere)
Outdoor field Pseudomonas spp. Barnett et al., 2020
Fiber/oilseed hemp (Anka) Bulk soil, rhizosphere Indoor growth
chamber
Pseudomonas spp. Comeau et al.,
2021
Fiber/oilseed/drug hemp (Gansuqingshui, Yunnan
1, Yunmaza 1, and Huoma 1)
Bulk soil, rhizosphere,
roots, leaves, stems,
flowers (endosphere)
Indoor growth
chamber
Pseudomonas spp. Wei et al., 2021
Fiber hemp (Fedora 17) Retting stems Outdoor field Pseudomonas fluorescens,
Pseudomonas psychrotolerans,
Pseudomonas rhizosphaerae,
Pseudomonas graminis,
Pseudomonas fulva, Pseudomonas
viridiflava, and Pseudomonas
syringae
Ribeiro et al., 2015
Fiber hemp (USO-31) Retting stems Outdoor field Pseudomonas argentinensis,
Pseudomonas rhizosphaera, and
Pseudomonas syringae
Liu et al., 2017
Fiber hemp (Futura 75, Felina 32, and SS Alpha) Retting stems Greenhouse Pseudomonas spp. Law et al., 2020
Fiber hemp Diseased stems Greenhouse/field Pseudomonas syringae McPartland and
Hillig, 2004
Hemp Diseased stems or leaves Natural habitat/field Pseudomonas cannabina and
Pseudomonas syringae
McPartland et al.,
2000
Hemp Diseased leaves Outdoor field Pseudomonas cannabina Bull et al., 2010
Hemp Bulk soil, rhizosphere Industrial site Pseudomonas balearica and
Pseudomonas stutzeri
Liste and Prutz,
2006
Wild hemp Roots, shoots (endosphere) Industrial site Pseudomonas sp. Iqbal et al., 2018
Wild hemp Rhizosphere, roots
(endosphere)
Natural habitat Pseudomonas geniculata,
Pseudomonas koreensis,
Pseudomonas plecoglossicida, and
Pseudomonas taiwanensis
Afzal et al., 2015
Drug-type hemp (TJ’s CBD) Rhizosphere, roots Outdoor field Pseudomonadales Ahmed et al., 2021
Drug-type hemp (Tangerine) Rhizosphere, roots, leaves,
flowers (endosphere)
Outdoor field Gammaproteobacteria Willman et al., 2021
Drug-type hemp Diseased leaves Outdoor field Pseudomonas koreensis Thiessen et al.,
2020
Drug-type marijuana Dried inflorescences
(medicinal products)
Indoor commercial
facility
Pseudomonas spp.,
Pseudomonas fluorescens,
Pseudomonas putida,
Pseudomonas stutzeri, and
Pseudomonas aeruginosa
McKernan et al.,
2016
Drug-type marijuana Dried inflorescences
(medicinal products)
Commercial setting Pseudomonas sp.,
Pseudomonas monteilii,
Pseudomonas oryzihabitans,
Pseudomonas putida,
Pseudomonas coleopterorum, and
Pseudomonas fluorescens
McKernan et al.,
2021
Drug-type marijuana Dried inflorescences
(medicinal products)
Commercial setting Pseudomonas fluorescens,
Pseudomonas protegens,
Pseudomonas putida,
Pseudomonas mendocina, and
Pseudomonas aeruginosa
Thompson et al.,
2017
Drug-type marijuana (CBD Yummy, CBD Shark,
and Hash)
Rhizosphere, roots
(endosphere)
Indoor commercial
facility
Proteobacteria Comeau et al.,
2020
Drug-type marijuana (Sour Diesel, Bookoo Kush,
Burmese, Maui Wowie, and White Widow)
Bulk soil, rhizosphere, roots
(endosphere)
Commercial setting Pseudomonas spp. Winston et al.,
2014
Drug-type marijuana (Ghost Train, Afgooey, Dulce,
Caboose, Special Queen, Gila Kush, Golden
Gate, Kandy Kush, Kushy Kush, and Ghost Haze)
Bulk soil, rhizosphere Greenhouse Pseudomonas spp. Perea, 2019
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in immunocompromised and hospitalized patients, screening for
contaminations in marijuana products is usually implemented
during production quality control and may vary according to
local regulations (McPartland and McKernan, 2017). Testing
for P. aeruginosa contaminants is especially important for fresh
raw plant products, since this Gram-negative non-sporulating
bacterium is highly sensitive to heat and desiccation, and
therefore unlikely to survive the processes of marijuana drying,
curing, decarboxylation, extraction and/or smoking (Holmes
et al., 2015). Even though little data is available regarding bacterial
infections in humans caused by contaminated marijuana
(Montoya et al., 2020), P. aeruginosa DNA material has still been
detected in some medicinal products (McKernan et al., 2016;
Thompson et al., 2017) and a severe case of pneumonia has been
associated with inhalation of P. aeruginosa from a contaminated
marijuana smoking device (Kumar et al., 2018). Therefore,
preventive precautions should be taken during production to
avoid any biosafety risk.
MODES OF ACTION OF BENEFICIAL
Pseudomonas spp.
Various strategies can be explored to increase the yield and
quality of cannabis crops. Several recent literature reviews can
be consulted about the current opportunities and challenges
associated with cannabis genetic diversity, cultivar breeding
and agronomic traits improvement (Salentijn et al., 2015;
Clarke and Merlin, 2016;Schluttenhofer and Yuan, 2017;
Hesami et al., 2020), cannabinoid elicitation (Gorelick and
Bernstein, 2017;Backer et al., 2019), disease management
(Punja, 2021), production factors optimization (Backer et al.,
2019;Eichhorn Bilodeau et al., 2019), and biosafety practices
to reduce contaminants (McPartland and McKernan, 2017;
Montoya et al., 2020;Vujanovic et al., 2020). Within all these
promising developments, microbiome engineering and beneficial
microbial inoculants appear as a recurring prospective trend,
potentially promoting plant growth and fitness (Kusari et al.,
2017;Backer et al., 2019;Lyu et al., 2019;Söderström, 2020),
enhancing cannabinoid production (Lyu et al., 2019;Taghinasab
and Jabaji, 2020;Ahmed and Hijri, 2021), controlling diseases
(Kusari et al., 2017;Lyu et al., 2019;Söderström, 2020;Punja,
2021), and improving product biosafety (Vujanovic et al., 2020).
Research studies have thus started to examine the potential
benefits of inoculating beneficial Pseudomonas spp. or other
microorganisms on cannabis plants (Table 2). The following
sections present the modes of action of beneficial Pseudomonas
spp. in terms of plant growth promotion, product quality, and
tolerance to biotic and abiotic stresses, with special emphasis on
their relevance for each cannabis crop type (hemp and marijuana
supplying fibers, oilseeds, and/or phytochemicals) (Figure 1).
Plant Growth Promotion by Beneficial
Pseudomonas spp.
Beneficial Pseudomonas spp. and other PGPR employ a variety
of plant growth-promoting mechanisms, such as enhancing
nutrient availability and modulating phytohormonal balance,
that result in increased biomass yield for many crops (Khan
et al., 2016;Backer et al., 2018). However, as discussed below,
qualitative improvements are equally needed, or even more, by
hemp and marijuana producers.
Fiber Hemp Crops
In hemp, two main kinds of fibers are derived from the plant
stalk, namely the woody xylem fibers (hurd fibers) and the
primary phloem fibers (bast fibers). While hurd fibers are valuable
by-products with diversified industrial and biofuel uses, the
primary bast fibers are the most lucrative commodity and are
separated from the rest of the stem tissues by a process called
retting. Traditional retting is accomplished by exposing the
harvested stems to decaying microorganisms in the field (dew
retting), or by immersing the stems in large water tanks (water
retting), to degrade pectins and other binding components. The
long bast fibers are then extracted from the retted stalks by
a mechanical decortication step, and transformed into textiles
and other applications (Small, 2015). Fiber hemp cultivars have
thus been selected to exhibit key desirable traits, like biomass
yield, ease of retting, and bast fiber content, length, and strength
(Schluttenhofer and Yuan, 2017). “Dual usage” crops are also
commonly employed for both oilseed and fiber production,
even though fiber quality is then reduced because of stem
lignification during seed maturation (Small, 2015). Cultivars
dedicated to specific industrial uses have also been developed
with, for example, chlorophyll-deficient stalks for cheaper dyeing
and paper pulp processing (Clarke and Merlin, 2016).
Pioneering assays of microbial inoculants in cannabis, using
P. putida,P. protegens,Pseudomonas synxantha and Pseudomonas
simiae among other PGPR microorganisms, have already
successfully increased several indicators of biomass and fiber
yield, such as total plant weight and/or stem length, diameter,
and weight (Jin et al., 2014;Conant et al., 2017;Pagnani et al.,
2018;Balthazar et al., 2020;Comeau et al., 2021;Kakabouki
et al., 2021a,b). Further studies are needed to assess the full
agronomic potentials of this promising avenue with indicators
considering fiber quality, such as internode length and bast fiber
content (Jin et al., 2014), fiber width (Müssig and Amaducci,
2018), pectin and lignin content reduction and fiber decortication
efficiency (Petit et al., 2020). Encouragingly, in other crops
like cotton, inoculation with P. fluorescens and other PGPR
strains effectively increased fiber quality properties and yield
(Abdulla and Karademir, 2019). Modulation of phytohormones
implicated in phloem differentiation and fiber formation, like
auxins, gibberellins and ethylene, has also been demonstrated
with Pseudomonas spp. (Backer et al., 2018), but the involvement
of this hypothetical mechanism remains to be validated in
this context. Finally, regarding the retting process, several
studies have established that pectinolytic Pseudomonas strains
are particularly important as natural retting agents for harvested
hemp stalks under aerobic conditions (Ribeiro et al., 2015;Liu
et al., 2017;Law et al., 2020). Dedicated Pseudomonas spp.
retting inoculants could therefore be developed to optimize this
degradation process which is currently the main factor limiting
the quality and production of hemp fibers (Schluttenhofer and
Yuan, 2017), as already demonstrated with other microorganisms
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like fungus Phlebia radiata for hemp dew retting (Liu et al., 2017)
and bacteria Bacillus sp. and Clostridium sp. for hemp water
retting (Di Candilo et al., 2010).
Oilseed Hemp Crops
The oil extracted from hemp seeds is highly nutritious because
it is rich in polyunsaturated fatty acids (mostly linoleic acid,
α-linolenic acid, oleic acid, and γ-linolenic acid), some of which
are essential fatty acids that humans must acquire from their diet.
Edible oil can be obtained by cold-pressing the hemp seeds and is
added to human food, cosmetics, and industrial fluids, while the
residual seed cake can be transformed into protein-rich flour or
livestock feed supplements. In countries like Canada, the oilseed
hemp industry has even better economic prospects than the fiber
market, though it does not currently compete with major oilseed
crops like flax, sunflower or canola (Small, 2015).
Significant advances in hemp oil production could be obtained
by increasing the seed yield per plant, the seed size, weight
and maturity uniformity at harvest, but also its oil and protein
contents, and desirable fatty acid and amino acid profiles
(Schluttenhofer and Yuan, 2017). However, unlike their tall fiber
hemp counterparts, short oilseed cultivars are preferred for an
easier mechanized harvest and efficient allocation of the plant
energy toward seed production (Small, 2017), while flowering
earlier than fiber cultivars allows time for seed maturation
during late season. While these quantitative and qualitative
improvements are part of ongoing cultivar breeding objectives,
the use of Pseudomonas spp. inoculants constitutes a promising
complimentary approach, as demonstrated in other oilseed crops
including sesame (Kumar et al., 2009), sunflower (Majeed et al.,
2018), flax (Rajabi-Khamseh et al., 2020), soybean, canola, and
corn gromwell (Jiménez et al., 2020) where increased seed
yield, oil yield and/or composition in desirable fatty acids were
reported. Additionally, more research is still needed to investigate
the largely unknown mechanisms resulting in these alterations of
seed oil content and composition by PGPR.
Drug-Type Marijuana and Hemp Crops
Cannabis phytochemicals accumulate primarily in the secretory
cavity of glandular trichomes, which are especially abundant on
unpollinated female inflorescences and, to a lesser extent, other
aerial plant parts. Over 90 different cannabinoids have been
discovered, however, THC and CBD are the most studied because
of their psychoactive and therapeutic effects, respectively (Andre
et al., 2016). Whereas marijuana cultivars often yield primarily
THC and less CBD, hemp cultivars primarily yield CBD and very
little THC to comply with legal requirements, as explained above.
Consequently, while marijuana crops have found numerous—
and sometimes illicit—applications in both the recreational and
healthcare markets for a long time, medicinal hemp cultivars
with high CBD contents are now of particular interest to the
healthcare market as well (Schluttenhofer and Yuan, 2017).
Upon harvest, plants are usually dried and cured, then processed
into marketable products, including (non-exhaustively) dried
inflorescences, resinous preparations (e.g., hashish) and solvent-
based extracts, that can either be smoked, inhaled, or ingested
(Holmes et al., 2015). While THC and CBD contents mainly
drive their market value, the synergistic “entourage effect” of the
many other phytochemicals found in plant-based products also
contributes to their pharmacological effectiveness compared to
single molecules produced in bioreactors (Russo, 2019). These
important cannabis secondary metabolites can also alter the
organoleptic and qualitative properties of the marketed products,
including diverse terpenes conferring unique aroma and flavors,
phenolic flavonoids lending antioxidant and anti-inflammatory
properties (Andre et al., 2016), and anthocyanin pigments
responsible for the purple coloration of popular marijuana
cultivars (Small, 2015), among other compounds.
Inoculations with P. putida or other beneficial
microorganisms have already successfully increased several
indicators of phytochemical yield in hemp and marijuana crops,
such as inflorescence yield, inflorescence dry weight, and/or
cannabinoid content (Conant et al., 2017;Pagnani et al., 2018;
Kakabouki et al., 2021b). However, excessive THC elicitation
should be avoided in hemp crops, because of the maximum limit
permitted by laws for these cultivars. Fortunately, the range of
variations in THC content due to environmental factors is quite
limited with hemp plants (Small, 2015) and is also restricted by
competition with CBD biosynthesis pathways (Jalali et al., 2019).
Regarding the qualitative aspects of drug-type crops, increased
levels of terpenes, phenols, flavonoids, alkaloids and anthocyanin
pigments were obtained with Pseudomonas spp. inoculants in
other aromatic plants like peppermint, sage, oregano, sweet
marjoram, marigold, and geranium; medicinal plants like
valerian, datura, black henbane, Madagascar periwinkle, stevia,
black Atractylodes, turmeric, tea plant, bushmint, and Indian
ginseng; and fruit crops like blackberry, strawberry, and pea;
as referenced in previous reviews (Bona et al., 2016;Thakur
et al., 2019;Çakmakçı et al., 2020). Notably, promotion of
phytochemical accumulation by beneficial microorganisms was
associated with increased glandular trichome density and/or size
in basil, tomato, peppermint, geranium and artemisia (Bona
et al., 2016;Çakmakçı et al., 2020;Balestrini et al., 2021).
The mechanisms through which microbial inoculants can
alter the accumulation of phytochemicals in aromatic and
medicinal plants are not fully understood yet. Hypothetically,
beneficial microorganisms may be recognized as a potential
threat by the plant which synthetizes phytochemicals as a defense
response (Thakur et al., 2019;Balestrini et al., 2021). Supporting
this, treatments with stress-related phytohormones significantly
increase THC and CBD contents (Mansouri et al., 2009;Jalali
et al., 2019;Apicella et al., 2021). Alternatively, promoting root
growth and improving nutrient availability and uptake may
allow for greater plant biomass and overall wealth, resulting in
thriving secondary metabolism functions (Çakmakçı et al., 2020)
and early maturation (Conant et al., 2017;Backer et al., 2019).
This is supported by the positive response of cannabinoid and
inflorescence yields to fertilizer applications during marijuana
vegetative growth (Caplan et al., 2017b). Finally, it has been
suggested that some endophytes can partially share secondary
metabolism pathways with their host and intimately interact with
phytochemical production (Ludwig-Müller, 2015), as illustrated
by the modulation of terpene metabolism in Atractylodes
medicinal plants by endophytic P. fluorescens (Zhou et al., 2018).
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TABLE 2 | Studies using microbial inoculants with cannabis plants.
Crop type
(cultivar)
Inoculant type Microorganisms inoculated Experimental
setting
Reported effects References
Fiber/oilseed hemp
(Felina 34)
Roots (soil drench) Pseudomonas sp. DSMZ 13134
(Proradix)
Greenhouse potted
plants
Reduced broomrape weed
infestation
Gonsior et al.,
2004
Fiber/oilseed hemp
(Anka)
Roots (soil drench) Pseudomonas synxantha
LBUM223, Pseudomonas simiae
WCS417r,Bacillus velezensis
LBUM279, Bacillus subtilis LBUM979
(single-strains and consortia)
Indoor growth
chamber
Increased plant weight, no ISR,
no biocontrol against Botrytis
Balthazar et al.,
2020
Fiber/oilseed hemp
(Anka)
Roots (soil drench) Pseudomonas synxantha
LBUM223, Pseudomonas protegens
LBUM825,Bacillus velezensis
LBUM279, LBUM1082, Bacillus subtilis
LBUM979 (single-strains and consortia)
Indoor growth
chamber
Increased plant weight,
modulated rhizosphere
microbiome
Comeau et al.,
2021
Fiber/oilseed hemp
(Carmagnola)
Roots (soil mix) Glomus mosseae BEG 12 Outdoor potted
plants
Root colonization, better heavy
metals translocation, reduced
plant weight
Citterio et al.,
2005
Fiber/oilseed hemp
(Fedora 17 and
Felina)
Roots (irrigation
system)
Trichoderma harzianum T-22
(Trianum-P)
Greenhouse potted
plants
Increased root density, plant
height, weight, inflorescence
yield, CBD content
Kakabouki
et al., 2021b
Fiber hemp
(USO-31)
Roots (irrigation
system)
Rhizophagus irregularis (MycoPlant) Greenhouse
hydroponic system
Increased root length, stem
weight, quality, P content,
seedling survival
Kakabouki
et al., 2021a
Oilseed hemp
(Finola)
Roots (soil drench) Azospirillum brasilense,
Gluconacetobacter diazotrophicus,
Burkholderia ambifaria, Herbaspirillum
seropedicae (consortium)
Greenhouse potted
plants
Root colonization, increased
biomass, stem length and
weight, cannabinoid,
antioxidant, and phenolic
contents
Pagnani et al.,
2018
Fiber/oilseed hemp
(Anka)
Leaves (foliar spray) Pseudomonas synxantha
LBUM223, Pseudomonas protegens
Pf-5,Bacillus velezensis LBUM279,
FZB42, LBUM1082, Bacillus subtilis
LBUM979
Indoor growth
chamber
Biocontrol against Botrytis Balthazar et al.,
2021
Fiber hemp (YunMa
1)
Leaves (foliar spray) Chaetomium sp., Fusarium sp.,
Plectosphaerella sp., Nigrospora sp.,
Graphium sp., Colletotrichum sp.
Outdoor field Increased plant growth,
antioxidant activity, fiber yield,
and/or fiber length
Jin et al., 2014
Fiber hemp
(Fibranova)
Retting stems
(water incubation)
Clostridium sp. L1/6, Bacillus sp.
ROO40B (consortium)
Indoor water-retting
tanks
Increased fiber quality and
retting ease
Di Candilo
et al., 2010
Fiber hemp
(USO-31)
Retting stems
(incubation)
Phlebia radiata Cel 26 Outdoor
dew-retting field
Increased fiber quality and
retting ease
Liu et al., 2017
Drug-type hemp Roots (irrigation
system)
Pseudomonas putida, Enterobacter
cloacae,Citrobacter freundii,
Comamonas testosteroni (consortium,
Mammoth PTM)
Indoor
hydroponic/soil-
less
systems
Increased inflorescence yield,
plant height, stem thickness
Conant et al.,
2017
Drug-type
marijuana
Rooted stem
cuttings
Trichoderma harzianum,Trichoderma
asperellum, Gliocladium catenulatum
Indoor hydroponic
system
Stem colonization, biocontrol
against Fusarium
Punja, 2021
Drug-type
marijuana (Afghani
Kush, White Rhino)
Inflorescences
(post-harvest spray)
Bacillus amyloliquefaciens F727
(Stargus), Gliocladium catenulatum
J1446 (Prestop), Trichoderma
asperellum T34 (Asperello)
Detached
inflorescences
Biocontrol against Botrytis Punja and Ni,
2021
Drug-type
marijuana
(Copenhagen Kush)
Leaves (foliar spray) Streptomyces lydicus WYEC108
(Actinovate), Bacillus subtilis QST713
(Rhapsody), Bacillus amyloliquefaciens
F727 (Stargus)
Indoor growing
room
Biocontrol against powdery
mildew
Scott and
Punja, 2021
Pseudomonas spp. highlighted in bold.
Interestingly, if beneficial microorganisms colonize marijuana
inflorescences, they could also potentially influence the
marketability of the finished products by directly improving
(or deteriorating) their taste and aroma (Winston et al., 2014).
Indeed, post-harvest microbiome management is suggested
as a valuable application, yet often unexplored, of food-safe
microbial inoculants (Berg et al., 2020). This prospect is
illustrated by the essential role played by regional microbial
communities in defining the unique organoleptic profile
(“terroir”) of premium wines, artisan cheeses, and craft beers
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Balthazar et al. Beneficial Pseudomonas for Cannabis Production
FIGURE 1 | Potential modes of action of beneficial Pseudomonas spp. inoculants and traits they could improve in cannabis crops.
(Cocolin and Ercolini, 2015). This daring but feasible avenue
could be explored by innovative marijuana producers who are
noteworthily concerned about the organoleptic distinctiveness of
their products (Small, 2015), pending biosafety risk assessments
as discussed below. In this context, it should be mentioned
that fermenting strains of Pseudomonas, especially P. putida,
are already exploited to synthesize natural flavoring agents
like pyrazines (roasted aroma), esters (fruity aroma), vanillin
(vanilla aroma), and benzaldehyde (cherry aroma) to alter
the organoleptic properties of several food, cosmetic and
pharmaceutical products (Bhowmik and Patil, 2018).
Biotic Stresses and Biocontrol by
Beneficial Pseudomonas spp.
Biotic stress management is possibly the greatest challenge facing
cannabis producers in North America where the recent surge
in large-scale cultivation has led to emerging disease outbreaks
and increased incidence and severity of pathogens (Punja,
2021). While most hemp and marijuana diseases are caused by
fungi and oomycetes, and occasionally by a few bacteria and
viruses, other common biotic aggressors include insects, mites,
nematodes, weeds, and parasitic plants (McPartland et al., 2000).
The prevalence of each disease and pest varies between indoor
and outdoor cropping systems (Punja, 2021), but also between
fiber, oilseed, and drug-type crops (Bakro et al., 2018;Thiessen
et al., 2020).
Common Biotic Stresses and Challenges for Their
Management
In hemp outdoor production, dense direct-seeded plant stands
tend to promote soilborne damping-off pathogens such as
Pythium,Thielaviopsis,Fusarium, and Rhizoctonia (Thiessen
et al., 2020), while various stem-infecting pathogens, including
Fusarium,Sclerotinia,Phoma, and Verticillium, further reduce
oilseed and fiber yields by wilting tissues and collapsing
mature plants (McPartland et al., 2000;Bakro et al., 2018).
Common outdoor pests include lepidopterous stem borers,
beetles, root grubs, caterpillars, leaf miners, seed-eating birds
and weeds (McPartland, 1996b). In addition to causing damages
to living plants, decaying fungi like Botrytis,Alternaria,
Trichothecium, and Cladosporium also cause post-harvest quality
issues by ruining entire lots of stored seeds (Jian et al.,
2019), by spoiling fibers during the retting process (McPartland
et al., 2000;Di Candilo et al., 2010), or by releasing mold
emissions from finished fiber-based construction materials
(Nykter, 2006). Contrastingly, in drug-type crops, inflorescence-
infecting pathogens, such as Botrytis and Fusarium, are often
the most damaging with up to 20% of direct yield losses and
over 10–15% of post-harvest losses (Punja, 2021). A plethora of
other concerning fungal pathogens also thrive in indoor and/or
outdoor production, causing leaf spots, blights, mildews, stem
cankers and inflorescence rots (McPartland et al., 2000), while
saprophytic storage molds like Aspergillus and Penicillium release
mycotoxins and may render finished products unsuitable for
human consumption (Montoya et al., 2020). The predominant
pests of marijuana plants are spider mites, aphids, whiteflies,
mealybugs, and thrips, seemingly bypassing the toxicity of surface
cannabinoids thanks to their piercing-sucking mouthparts
(McPartland, 1996b). On the other hand, weeds and parasitic
plants are not as relevant in well managed greenhouses and
cultivation rooms as they are in open fields.
In both hemp and marijuana crops, managing biotic stresses
is especially challenging because of the limited range of registered
pesticides available (Punja, 2021), the lack of formal agricultural
recommendations and mitigation strategies based on reliable
research (Sandler and Gibson, 2019), the high susceptibility
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Balthazar et al. Beneficial Pseudomonas for Cannabis Production
of modern cultivars to fungal pathogens (Clarke and Merlin,
2016), and the regional variabilities in pest and disease pressures
(Thiessen et al., 2020). Additionally, for drug-type crops,
finished products destined to human consumption must comply
with strict regulations on pesticide residues and microbial
contaminants (McPartland and McKernan, 2017). Therefore, in
addition to good management practices and crop resistance
breeding efforts reviewed recently (Punja, 2021), inoculation with
beneficial Pseudomonas spp. have been proposed to help cannabis
plants cope with biotic stresses (Lyu et al., 2019;Söderström,
2020). Promising research avenues can be loosely sorted into two
categories based on a local or systemic mode of action of the
inoculants, as detailed below.
Local Effects of Pseudomonas spp. Inoculants
By colonizing soil niches and plant tissues and producing
biofilms, beneficial Pseudomonas spp. can compete for space
and nutrients, shape microbiome communities, and locally
inhibit the growth of pathogens by antibiosis. A plethora of cell
wall-degrading enzymes (extracellular chitinases, cellulases, β-
1,3-glucanases, proteases, lipases, etc.), antibiotics (pyrrolnitrin,
pyoluteorin, phenazines, 2,4-diacetyl phloroglucinol DAPG,
etc.), siderophores (pyoverdine, pseudomonine, pyochelin,
etc.) and other antimicrobial compounds (including volatile
organic compounds like hydrogen cyanide HCN) produced by
Pseudomonas strains have been characterized (Fischer et al.,
2013;Khan et al., 2016;Backer et al., 2018). Notably, their
antagonistic activities can be used to control fungal and bacterial
diseases, but also herbivorous insects (Flury et al., 2016;Khan
et al., 2016), parasitic nematodes (Trivedi and Malhotra, 2013)
and weeds (Trognitz et al., 2016). Accordingly, antibiotic
production was suggested as the main antifungal mechanism of
beneficial P. protegens and P. synxantha strains when sprayed
on hemp leaves to control Botrytis gray mold, and/or inhibiting
the in vitro growth of Botrytis,Sclerotinia,Fusarium,Alternaria,
and Phoma (Balthazar et al., 2021). Previous studies also
demonstrated that two strains of Pseudomonas koreensis and
Pseudomonas taiwanensis found in the hemp rhizosphere could
inhibit the growth of Aspergillus and/or Fusarium in vitro by
producing siderophores, cellulases, pectinases and proteases
(Afzal et al., 2015), while hemp endophytes Pseudomonas
fulva and Pseudomonas orientalis inhibited the growth of
Botrytis, Sclerotinia and/or Rhizoctonia in vitro by producing
siderophores, cellulases, and antibiotics (Scott et al., 2018).
However, these results were not validated on plants (Afzal
et al., 2015;Scott et al., 2018). Ongoing research with other
beneficial bacteria and fungi, like Bacillus,Streptomyces,
Trichoderma, and/or Gliocladium, further supports the potential
of antagonistic biocontrol agents against Botrytis on marijuana
inflorescences (Punja and Ni, 2021) and hemp leaves (Balthazar
et al., 2021), and against Fusarium and powdery mildew on
marijuana stem cuttings and leaves, respectively (Punja, 2021;
Scott and Punja, 2021). Besides, various fungal endophytes were
also isolated from hemp and marijuana plants previously, but
their antimicrobial activities were only tested in vitro (Gautam
et al., 2013;Kusari et al., 2013;Qadri et al., 2013). Guidelines for
timing of application are also important to establish as curative
effects are usually harder to achieve than preventive protection
(Balthazar et al., 2021;Punja and Ni, 2021).
Systemic Effects of Pseudomonas spp. Inoculants
Regarding their second mode of action, many rhizospheric
Pseudomonas spp. are known to trigger a systemic state of alert
in their host plant during root colonization, which activates the
plant defenses against a broad spectrum of pathogens, viruses,
and herbivorous insects. This immune response, acting beyond
the site of inoculation and enhancing the defensive capacity of the
entire plant, is called Induced Systemic Resistance (ISR) (Pieterse
et al., 2014). In prevision of pathogen attacks, plants primed with
ISR may exhibit faster and/or stronger expression of basal defense
responses such as deposition of callose, lignin, and phenolic
compounds, increased activities of chitinase, peroxidase, and
phenylalanine ammonia lyase, production of phytoalexins and
primed expression of stress-related genes (Fischer et al.,
2013). While potential molecular processes and phytohormonal
signaling have been investigated, the exact ISR triggers and onset
mechanisms remain largely unknown and seem to depend on
the specificity of the mutual interactions between certain plants,
bacteria and pathogens (Beneduzi et al., 2012). In hemp, an early
study suggested that ISR responses were triggered by drenching
the soil with a Pseudomonas strain, thereby reducing infestations
of Orobanche ramosa (broomrape parasitic weed) by 80%.
However, this presumed mechanism was only hypothetical and
not experimentally validated (Gonsior et al., 2004). Conversely, a
recent study concluded that drenching the soil with P. synxantha,
P. simiae and/or Bacillus spp. rhizobacteria did not protect hemp
against Botrytis foliar infection and failed to trigger defense-
related gene expression in hemp leaves (Balthazar et al., 2020).
This unsuccessful attempt was tentatively attributed to a lack of
production of ISR-inducing compounds by the bacteria, or to the
inability of hemp to perceive such compounds in the rhizosphere
(Beneduzi et al., 2012). As ISR interactions are notoriously
complex, many possibilities remain to be explored regarding this
intriguing mode of action of Pseudomonas spp. inoculants.
Abiotic Stresses Tolerance With
Beneficial Pseudomonas spp.
Unfavorable environmental factors like salinity, drought, heat
or cold, soil pollutants, or nutrient deficiencies are known to
impact cannabis physiology and development, and to predispose
crops to diseases (McPartland, 1996a). In the context of global
climate change and growing human population, abiotic stresses
are expected to take an increasing toll on agricultural production
worldwide (Camaille et al., 2021). Unfortunately, as with most
modern crops, domesticated cannabis cultivars tend to have
narrower tolerances to stressful environments compared to
their wild counterparts (Small, 2015). In this context, beneficial
Pseudomonas spp. inoculants may be advantageous to promote
cannabis growth under adverse conditions and/or expand its
ecological range.
Salinity, Water, and Temperature Stresses
Cannabis plants do not tolerate excessive salt (NaCl) in soil, nor
brackish waters and salty breezes which can stunt their growth
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in coastal environments (McPartland and McKernan, 2017).
Additionally, in irrigated and indoor production settings, over-
fertilization (Caplan et al., 2017a) or the use of water softening
systems (McPartland et al., 2000) can also lead to sodium
accumulation in the root zone. Cultivar-specific adaptative
responses are being investigated in hope of breeding hemp
cultivars with broader tolerance to saline conditions (Liu et al.,
2016). Regarding water stresses, exposure to drought causes
cannabis plants to wilt and predisposes them to fungal canker
diseases (McPartland, 1996a), whereas overwatering and flooding
also cause wilting (McPartland et al., 2000) but favor root and
crown rot diseases in indoor production systems (Punja, 2021).
Air humidity further promotes the development of bud rot
pathogens within the compact moisture-retaining inflorescences
of drug-type cultivars (Clarke and Merlin, 2016), while soil water
content and salinity have significant impacts on the rhizosphere
microbiome structure (Winston et al., 2014). Field-grown hemp
crops can also be significantly damaged by excessive rainfalls
and waterlogging (Thiessen et al., 2020) or, conversely, by
water shortage and hot temperature in semi-arid environments
(Cosentino et al., 2013). However, field-grown hemp generally
withstands drought periods thanks to a long taproot allowing
access to deep water sources (Small, 2015). Unfortunately, in
contrast to fiber and oilseed crops that are direct-seeded, most
mass-produced drug-type plants are propagated vegetatively
and therefore do not develop taproots (Jin et al., 2021).
When transplanted outdoors, these clones commonly exhibit
root binding issues (poor root development) and suffer from
inadequate water supply (Thiessen et al., 2020). Additionally,
since gradual drought stress can be deliberately applied to
container-grown cannabis plants to maximize inflorescence
and cannabinoid yield (Caplan et al., 2019), drug-type crops
under controlled horticultural management may also experience
dehydration stress. Finally, regarding adaptation to temperatures,
hemp plants are usually better adapted to cool temperate climates
than marijuana cultivars originating from hot semi-tropical
regions (Small, 2017), even though cultivar-specific differences
are reported (Cosentino et al., 2013;Mayer et al., 2015). Hemp
seeds still require rather elevated temperatures to germinate,
and oilseed cultivars also require a warmer and longer season
than fiber crops to allow seed maturation (Small, 2017). While
brief exposure to freezing temperatures can be tolerated by most
hemp seedlings and mature plants (Mayer et al., 2015;Small,
2017), frosted inflorescences of drug-type crops can turn black
and develop a harsh taste compromising their market value
(McPartland et al., 2000).
Owing to their great metabolic versatility, Pseudomonas spp.
can maintain their growth in stressful environments where
they can also help plants cope with various abiotic stresses
(Nadeem et al., 2016). Successful reduction of salinity stress
has been reported in maize, rice, wheat, soybean, alfalfa, bean,
tomato, and radish inoculated with halotolerant Pseudomonas
strains, as referenced in previous reviews (Nadeem et al., 2016;
Khan et al., 2019;Bhat et al., 2020). Frequently reported
mechanisms of action include the improvement of water and
nutrient uptake by roots, upregulation of plant osmoprotectants
(proline and glycine betaine) and antioxidant activities (catalase,
peroxidase, superoxide dismutase, etc.), maintenance of ionic
homeostasis within plant tissues (sodium to potassium ratio),
modulation of stress-induced phytohormones (abscisic acid,
ethylene, auxins, etc.), and secretion of exopolysaccharide
biofilms that trap sodium cations in excess in the rhizosphere
(Piccoli and Bottini, 2013;Nadeem et al., 2016;Bhat et al.,
2020). In particular, endophytic bacteria with enzymatic 1-
aminocyclopropane-1-carboxylate (ACC) deaminase activity can
reduce the production of stress-induced ethylene in the roots,
thus suppressing its adverse effect on plant growth (Backer
et al., 2018). Interestingly, a salt-tolerant Pseudomonas geniculata
endophyte has been isolated from hemp and promoted canola
growth under salinity stress, even though this effect was not
validated toward hemp growth (Afzal et al., 2015). Similarly,
Pseudomonas-induced tolerance to drought or flood has been
reported in other plants than cannabis, including maize, rice,
wheat, sunflower, pea, mung bean, chickpea, Aleppo pine, and
Arabidopsis thaliana, as referenced in previous reviews (Liu and
Zhang, 2015;Nadeem et al., 2016;Backer et al., 2018;Kour
et al., 2019;Camaille et al., 2021). Reported mechanisms of action
included stomatal closure preventing water loss, modification
of root length and architecture increasing soil exploration and
water access, protection of membrane integrity, secretion of
exopolysaccharide biofilm improving soil aggregation and water
retention, as well as several of the mechanisms presented above
(regulation of osmoprotectants, antioxidants, phytohormones,
etc.) since osmotic and oxidative stresses are experienced by
plants in both dry and saline environments (Nadeem et al., 2016;
Kour et al., 2019;Camaille et al., 2021). Regarding exposure
to unfavorable temperatures, inoculation with psychrotolerant
Pseudomonas spp. increased cold tolerance of canola, lentil,
mung bean, wheat, and tomato (Nadeem et al., 2016;Yadav
et al., 2019), while thermotolerant strains improved tolerance
to elevated temperatures in wheat, sorghum, chickpea, and
potato (Nadeem et al., 2016;Singh et al., 2019). Reported
mechanisms included the production of antifreeze proteins
and biofilms, protection of membrane integrity, modulation of
phytohormones and antioxidants, improved nutrient acquisition,
and increased plant metabolite levels (Nadeem et al., 2016;Yadav
et al., 2019). Additionally, foliar applications of P. fluorescens
could prevent frost injuries on pear trees by competing against
ice-nucleating P. syringae pathogens that trigger ice crystals
formation at low temperatures to breach plant tissues (Lindow,
1983). Interestingly, ice-nucleating abilities have been identified
for cannabis pathogen P. cannabina (Bull et al., 2010;Xin et al.,
2018), suggesting that foliar applications of antagonistic bacteria
could also potentially protect hemp and marijuana crops against
pathogen-triggered frost injuries.
Soil Pollutants and Bioremediation
In the wake of industrial and agricultural intensification,
anthropogenic activities have led to the accumulation of
concerning pollutants in the environment, like heavy metals,
pesticides, and petroleum hydrocarbons. Soil bioremediation
is a process that uses microorganisms and plants to detoxify
these contaminants and restore soil health in an eco-friendly
manner (Kahlon, 2016). Inheriting from its weedy ancestors
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the ability to thrive in human-disturbed habitats (Small, 2015),
hemp is one of the most investigated plants in phytoremediation
and exhibits a great capacity to accumulate pollutants in its
tissues (Wu et al., 2021). Benefiting from a short life cycle, large
root system, and high biomass yield, hemp has been widely
used to decontaminate industrial wastewaters and polluted soils
from metals like cadmium, copper, nickel, zinc, lead, selenium,
cobalt; organic contaminants like benzo[a]pyrene, naphthalene,
chrysene and petrol hydrocarbons; and radioactive isotopes
of cesium and strontium (McPartland and McKernan, 2017;
Wu et al., 2021). While numerous non-food applications are
available for the disposal of harvested plant parts, like biofuels
and fiber-based construction materials (Wu et al., 2021), seeds
containing high levels of particular elements that are valuable
for human consumption, such as selenium, can also be used as
dietary supplements and biofortified nutraceuticals, thus offering
additional application prospects (Stonehouse et al., 2020). On
the other hand, contrasting with their ability to accumulate
metals and hydrocarbons, cannabis plants are very susceptible to
herbicide injuries caused by agrochemicals like clomazone and
paraquat that are carried over by air drift and water runoff from
adjacent fields (McPartland et al., 2000), or by soil residues from
previous land use (Thiessen et al., 2020).
While these compounds are translocated within hemp tissues
to be detoxified and/or sequestrated, associated phytotoxic
effects might damage plants grown on contaminated soil,
causing stunted growth, reduced seed germination (Liste
and Prutz, 2006), and chlorosis (Thiessen et al., 2020).
Such deleterious effects can be mitigated by beneficial
rhizobacteria like Pseudomonas spp. which contribute to ex
planta biodegradation and sequestration processes in the
rhizosphere, thereby reducing the contaminant load beforehand
(Wu et al., 2021). For example, hemp plants growing in soil
from a former manufactured gas plant were found to recruit
specific Pseudomonas spp. that could degrade polycyclic aromatic
hydrocarbons in the soil, thus reducing their phytotoxic effects
(Liste and Prutz, 2006). Similarly, hemp plants irrigated
by wastewaters from an oil refinery harbored endophytic
Pseudomonas spp. with phenol and benzene degradation
activities, which could be developed into bacterial inoculants to
accelerate hydrocarbon phytoremediation (Iqbal et al., 2018).
Inoculation of hemp with mycorrhizal fungi also significantly
enhanced the translocation of toxic metals from roots to
shoots, thus improving the collection of contaminated plant
parts and the phytoextraction efficiency of hemp (Citterio
et al., 2005). In other crops, beneficial Pseudomonas spp. have
been reported to degrade and/or reduce the phytotoxic effects
of herbicides 2,4-D, glyphosate, atrazine, quizalofop-p-ethyl,
and clodinafop, insecticides DDT, aldrin, lindane, fipronil
and pyriproxyfen, fungicide tebuconazole, and hazardous
chemicals trichloroethylene, anthracene, naphthalene, and
phenanthrene, among many other aromatic and chlorinated
compounds (Kahlon, 2016;Nadeem et al., 2016;Trognitz
et al., 2016). Similarly, increased plant tolerance to various
heavy metals is mediated by Pseudomonas spp. rhizobacteria
through extracellular chelation, rhizosphere acidification, redox
transformation, intracellular sequestration, and modulation of
plant oxidative stress and ethylene production (Nadeem et al.,
2016;Lata et al., 2018;Khan et al., 2019).
These promising results could play a central role in enhancing
both the range of contaminants that can be remediated with
hemp and the rate of their degradation. However, in oilseed
and drug-type crops, as harmful contaminant accumulation in
consumed seeds and inflorescences can cause human health
issues, maximum concentration levels are mandated by laws for
pesticides, carcinogenic hydrocarbons, and several heavy metals
(Mihoc et al., 2012;McPartland and McKernan, 2017;Montoya
et al., 2020). Therefore, for these crops, the use of bacterial
inoculants that would enhance the translocation of pollutants
from roots to shoots should be avoided, with a potential exception
for biofortified seed crops mentioned above. Nevertheless,
inoculants with other modes of action, like ex planta degradation
and below-ground sequestration, could still be advantageous,
especially since fertilizers and rockwool media used in marijuana
hydroponic systems are particularly vulnerable to heavy metal
contamination (McPartland and McKernan, 2017).
Nutrient Deficiencies
Fertilization is one of the most important factors in indoor
marijuana production and several recent studies have sought
to determine the optimal nutrient application rates and timing
to achieve high yields and marketable phytochemical profiles
(Caplan et al., 2017a,b;Saloner and Bernstein, 2021;Shiponi
and Bernstein, 2021). Moreover, soil nitrogen content has
a strong structuring effect on the rhizosphere microbiome
communities across distinct marijuana cultivars (Winston et al.,
2014). Hemp plants also respond with significantly increased
growth to adequate soil nutrient supply (Vera et al., 2010;Small,
2015;Deng et al., 2019), yet the lack of updated fertilization
recommendations is commonly reported as an important crop
management limitation by hemp producers (Thiessen et al.,
2020). The most common deficiencies for cannabis usually arise
from shortages of macronutrients (nitrogen N, phosphorus P,
potassium K, calcium Ca, magnesium Mg, and sulfur S), while
micronutrients (zinc Zn, manganese Mn, iron Fe, copper Cu,
boron B, chlorine Cl, and molybdenum Mo) are also essential
in minute quantities and in excess may cause phytotoxicity
(McPartland et al., 2000;Thiessen et al., 2020). Compared
to fiber hemp, oilseed and drug-type crops require about as
much of soil nitrogen, less potassium but more phosphorus
for flowering or seed production (McPartland et al., 2000).
Other elements like magnesium, iron, and manganese are also
involved in cannabinoid biosynthesis regulation (McPartland
et al., 2000) and oilseed production (Mihoc et al., 2012).
Accordingly, optimal fertilization during marijuana vegetative
stage allows for larger plants resulting in increased inflorescence
weight, higher THC content, and potentially more frequent crop
turnover due to reduced maturation time (Caplan et al., 2017b).
However, fertilization during marijuana flowering stage usually
leads to a diluting effect on phytochemical yield, meaning that
increasing the biomass of inflorescences lowers their cannabinoid
concentration, even though the total cannabinoid production
may still increase (Caplan et al., 2017a;Saloner and Bernstein,
2021;Shiponi and Bernstein, 2021).
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As introduced above, soil microbial communities can
promote plant growth through a variety of mechanisms,
including improving the availability of nutrients and their
acquisition by plants (Backer et al., 2018). Plant growth-
promoting Pseudomonas spp. thus hold great potential to
help cannabis crops cope with nutrient deficiencies (Lyu
et al., 2019;Söderström, 2020). For example, diazotrophic
Pseudomonas stutzeri strains can fix atmospheric nitrogen
gas into biologically usable forms like ammonia, therefore
increasing nitrogen available to plants like Indian grass and
rice (Khan et al., 2016). Many rhizospheric Pseudomonas strains
also synthesize low amounts of phytohormonal indole-3-acetic
acid (IAA, auxin) and/or decrease growth-inhibiting ethylene
production in plant tissues via ACC deaminase activity. These
phytohormonal modulations stimulate the development of lateral
and adventitious roots and improve soil exploration and nutrient
uptake by wheat, maize, canola, tomato (Patten and Glick, 2002;
Nadeem et al., 2016), and many medicinal and aromatic plants
(Çakmakçı et al., 2020). Treating seeds with root-promoting
Pseudomonas spp. also improves germination and seedling
survival in canola (Patten and Glick, 2002), maize, oilseed rape,
sorghum, and sugar beet (Zachow et al., 2013), which could
be beneficial for seeded crops of fiber and oilseed hemp. Root-
promoting Pseudomonas spp. also improve the rooting success
of vegetative cuttings in mint (Kaymak et al., 2008), mung
bean (Mayak et al., 1999;Patten and Glick, 2002), ficus (Sezen
et al., 2014), eucalyptus (Teixeira et al., 2007), and blackcurrant
(Dubeikovsky et al., 1993). As mentioned above, rooting success
is very important for drug-type cannabis clones that are mass-
produced by vegetative cuttings (Thiessen et al., 2020). However,
preliminary assays with PGPR inoculants in commercial settings
did not substantially improve root emergence on marijuana
cuttings (Conant et al., 2017). Finally, Pseudomonas spp.
rhizobacteria secrete organic acids, HCN, phosphatases, phytases
and siderophores that help release important nutrients from
their non-labile forms in soil (Backer et al., 2018). Such
mineralization and/or solubilization processes are mediated
by enzymatic activities, chelating substances, and rhizosphere
acidification, and can increase the bioavailability of phosphorus
(Rathinasabapathi et al., 2018;Kalayu, 2019), iron (Lemanceau
et al., 2009), potassium (Kour et al., 2020), and zinc (Kumar
et al., 2019). Interestingly, in hemp, the endophytes P. geniculata
and P. koreensis were found to produce siderophores, IAA,
and phosphorus-solubilizing compounds, and to promote canola
root development in vitro. However, these beneficial traits
were not further validated toward hemp growth (Afzal et al.,
2015). Other studies reported that a consortium of phosphorus-
solubilizing bacteria—including P. putida (Baas et al., 2016)—
significantly increased marijuana inflorescence yield by 16.5%
and promoted faster plant maturation under commercial
production (Conant et al., 2017), while a consortium of four
diazotrophic bacteria—other than Pseudomonas spp.—promoted
hemp growth comparably to a conventional nitrogen fertilizer
(Pagnani et al., 2018). Nutrient-solubilizing or mycorrhizal fungi
like Trichoderma,Glomus, and Rhizophagus also influenced hemp
growth, phosphorus uptake and/or seedling quality (Citterio
et al., 2005;Kakabouki et al., 2021a,b). Additional studies
reported positive effects of Pseudomonas spp. inoculants on hemp
growth but without investigating the underlying mechanisms
(Balthazar et al., 2020;Comeau et al., 2021).
STRATEGIES TO IDENTIFY PROMISING
Pseudomonas spp.
The many promising avenues presented above suggest that
Pseudomonas spp. inoculants with multiple modes of action
could be developed for use in integrated management of cannabis
crops to promote yield and harvest quality under various biotic
and abiotic stresses. The following sections explore where to find
Pseudomonas strains with such beneficial attributes, and suggest
screening strategies to assess their abilities in vitro,in planta, and
in consortia (Figure 2).
Where to Look for Beneficial
Pseudomonas spp.
Two main strategies are usually distinguished when selecting
beneficial microorganisms for plant inoculation and microbiome
engineering: the bottom-up or synthetic approach which
pieces together candidates exhibiting desired traits from an
extensive pool gathered from various sources, and the top-
down or stepwise approach, which starts from a complex
existing microbial community and identifies its keystone players
(Tabacchioni et al., 2021). In cannabis, bottom-up studies seem
undeniably promising given the recent results obtained with
existing PGPR strains (Citterio et al., 2005;Jin et al., 2014;
Pagnani et al., 2018;Comeau et al., 2021), biocontrol agents
(Balthazar et al., 2020, 2021), or commercialized bioproducts
(Gonsior et al., 2004;Conant et al., 2017;Kakabouki et al.,
2021a,b;Punja, 2021;Punja and Ni, 2021;Scott and Punja, 2021),
thus paving the way for future work with existing Pseudomonas-
based bioproducts that are already registered for cereals, fruit
trees, and greenhouse vegetables (Fischer et al., 2013;Khan
et al., 2016). On the other hand, several top-down studies have
also started to examine the potential of harnessing cannabis
microbiome residents to improve pathogen control (Gautam
et al., 2013;Kusari et al., 2013;Qadri et al., 2013;Scott et al.,
2018;Ahmed et al., 2021), salinity tolerance (Afzal et al., 2015),
soil phytoremediation (Liste and Prutz, 2006;Iqbal et al., 2018),
cannabinoid production (Ahmed and Hijri, 2021), and fiber
retting process (Di Candilo et al., 2010;Ribeiro et al., 2015;
Liu et al., 2017;Law et al., 2020). In this regard, exploring
the microbiome of wild cannabis plants and ancestral heirloom
cultivars within their native Eurasian habitats could be of crucial
importance to identify beneficial microorganisms that have
associated with cannabis over long evolutionary periods. Indeed,
such microorganisms were likely lost during the domestication
process, which often reduces the variety of plant-associated
microbes compared to geographic areas with higher biodiversity
on the plant side (Ludwig-Müller, 2015;Berg and Raaijmakers,
2018). This unintended process may even be exacerbated in
cannabis grown indoors because of the strict sanitation measures
and repeated establishment of microbe-free planting materials
(Punja, 2021), inadvertently filtering out beneficial organisms in
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FIGURE 2 | Strategies and bottlenecks to identify beneficial Pseudomonas strains and develop bioproducts for cannabis crops.
highly domesticated cultivars. Alternatively, beneficial bacteria
could also be sourced from environments where a specific
benefit is observed, such as biocontrol strains typically found
in disease suppressive soils (Pieterse et al., 2014), plant growth-
promoting bacteria isolated from rich undisturbed forest soils
(Ahmed and Hijri, 2021;Pirttilä et al., 2021), microorganisms
altering organoleptic properties of food products (Cocolin and
Ercolini, 2015;Bhowmik and Patil, 2018), and abiotic stress-
ameliorating microbes retrieved from extreme environments like
dry, hypersaline, cold, or geothermal sites (Zachow et al., 2013;
Backer et al., 2018;Pirttilä et al., 2021).
In vitro Screening for Promising
Pseudomonas spp.
Specific in vitro assays are commonly used to quickly screen
promising microorganisms for their plant growth-promoting
characteristics. For examples, phosphate solubilization abilities
can be assessed by growing bacteria on Pikovskaya’s (PVK)
agar, nitrogen fixation by using N-free medium, siderophore
production with chrome azurol S (CAS) agar, IAA and
other phytohormones production with Salkowski reagent and
colorimetric methods, ACC deaminase activity with Dworkin
and Foster’s (DF) salts medium (Afzal et al., 2015;Ngalimat
et al., 2021), biofilm formation with crystal violet staining (Selin
et al., 2009), and metabolic capabilities with Biolog microarrays
(Gómez-Lama Cabanás et al., 2018;Zboralski et al., 2020). For
screening of biocontrol determinants, in vitro confrontational
assays are commonly used to assess the growth inhibition
of culturable pathogens (Afzal et al., 2015;Kusari et al.,
2017;Balthazar et al., 2021), Mueller Hinton (MH) media
are used for antibiotic diffusion assays, Cyantesmo paper for
HCN production, UV-visible spectroscopy for phenazines and
pyoverdine detection, and gas and/or liquid chromatography
methods for quantification of volatile compounds (VOCs) and
soluble antibiotics (Selin et al., 2009;Gómez-Lama Cabanás
et al., 2018;Ngalimat et al., 2021). Additionally, lytic enzyme
activities like proteases, pectinases and chitinases, can be detected
on minimal media amended, respectively, with skim milk,
pectin, and colloidal chitin (Afzal et al., 2015;Gómez-Lama
Cabanás et al., 2018). Finally, tolerance to abiotic stresses
such as salinity, drought and soil pollutants can be assessed
by amending growing media, respectively, with NaCl (Afzal
et al., 2015), polyethylene glycol (PEG) (Kour et al., 2019),
and the pollutant targeted for bioremediation (Iqbal et al.,
2018), while plates may be incubated between 4 and 50◦C to
isolate psychrotolerant and thermotolerant bacteria, respectively
(Kour et al., 2019). Screening conditions based on requirements
for future commercialization steps can also advantageously
pre-select bacteria that grow well on inexpensive media and
in industrial fermenters used for mass production, that can
withstand formulation processes such as freeze-drying, and
that can thrive in the targeted cropping system in terms
of temperature range, light intensity and compatibility with
agrochemicals used (Köhl et al., 2011). Ultimately, the whole
genome of selected strains can be sequenced and analyzed
by in silico gene mining tools to provide a comprehensive
overview of their potential beneficial traits, unveiling prospective
bioactive compounds that would otherwise be overlooked
by classical laboratory experimental methods and artificial
conditions (Paterson et al., 2017).
In planta Screening for Promising
Pseudomonas spp.
Even though in vitro methods are appealing for high-throughput
screening of microorganisms harboring beneficial traits, their
shortcomings and biases are frequently pointed out, cautioning
for example that nutrient-rich media and favorable growing
conditions often do not reflect the complex natural environments
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in which the microorganisms should be active (Köhl et al., 2019).
Accordingly, selecting the best Pseudomonas spp. performers
during in vitro screening assays does not always guarantee success
when subsequently inoculated on hemp plants (Balthazar et al.,
2021). Inspiring innovations like leaf-based custom culture media
(Elsawey et al., 2020) could help mimick cannabis intricate
phyllosphere environment for rapid screening, while using
cannabis hosts as “bait plants” or “recruiters” should also capture
more host-adapted isolates from the soil (Zachow et al., 2013;
Afzal et al., 2015;Ahmed and Hijri, 2021).
Additionally, many important bacterial modes of action
cannot be properly assessed in bioassays without host plants,
like ISR elicitation, rhizosphere colonization, microbiome
interactions, phytohormonal and phytochemical modulations,
plant growth promotion, and biocontrol activity against obligate
biotrophic pathogens. In this regard, since processes like ISR
elicitation (Balthazar et al., 2020), endosphere colonization
(Winston et al., 2014;Comeau et al., 2020) and plant growth
promotion (Afzal et al., 2015) seem to be genetically determined
by host-microbe combinations in cannabis, as is generally the
case in other plants (Bulgarelli et al., 2013;Pieterse et al., 2014),
it would be important to start the screening process with the
same cannabis cultivar as that intended for the end-use; as well
as to use either seeds or clonal cuttings as intended later since
this impacts the crop genetic uniformity. In planta bioassays
can also reveal deleterious effects of the plant on the bacteria,
such as antibacterial activity of cannabis phytochemicals (Ali
et al., 2012), or, conversely, undesirable effects of the bacteria
on the plant, such as HCN phytotoxicity (Trognitz et al.,
2016), inhibition of root elongation at high IAA concentrations
(Patten and Glick, 2002;Çakmakçı et al., 2020), reduced seed
germination due to ACC deaminase activity (Nadeem et al.,
2016), or altered sex determination of inflorescences due to
phytohormonal disturbance (e.g., masculinization of female
flowers or hermaphroditism, which is problematic in marijuana
production where male flowers and pollination are undesirable)
(Ganger et al., 2019;Punja and Holmes, 2020;Adal et al.,
2021). These effects also illustrate that unilateral pre-screening
of bacteria for higher secondary metabolite production and
activity is not always a good strategy. Similarly, Pseudomonas
strains selected for weed biocontrol should be tested for their
selectivity against the targeted weeds and lack of damage to
cannabis plants. If weed seeds are not readily available to screen
for bioherbicidal effects, lettuce seeds are commonly used instead
(Trognitz et al., 2016).
For all these reasons, it is advisable to introduce the
intended cannabis host plant early in the screening process,
under controlled conditions that would best represent the
epidemiological and environmental realities of the crop, even
if these large-scale experiments would require more time and
resources (Köhl et al., 2011, 2019). Hopefully, high-throughput
plant-based bioassays could benefit from recent innovations
in cannabis biotechnological research, allowing for example
the live visualization of root development and responses to
elicitors (Suwanchaikasem et al., 2021), the automated estimation
of hemp fiber yield and quality from scanner image analysis
(Müssig and Amaducci, 2018), the high-resolution profiling of
cannabinoids and plant extracts by chromatography coupled
to mass spectrometry (Delgado-Povedano et al., 2020), the
field-scale detection of diseases by drone remote sensing
and machine learning (Bates, 2021), and the marker-assisted
monitoring of cannabis pathways linked to pathogen defenses
(Balthazar et al., 2020;McKernan et al., 2020;Pépin et al., 2021),
abiotic stress responses (Mayer et al., 2015;Liu et al., 2016),
phytochemical biosynthesis (Booth et al., 2017;Grassa et al., 2018;
Jalali et al., 2019;Hesami et al., 2020), seed protein accumulation
(Ponzoni et al., 2018), and fiber quality (Guerriero et al., 2017;
Hesami et al., 2020).
Consortia Versus Single-Strain
Inoculants
Many studies in different crops have reported positive effects
when combining several Pseudomonas strains together and/or
with other microorganisms, as compared to single-strain
inoculants. Beneficial microorganisms commonly used in
consortia inoculants with Pseudomonas spp. include the bacterial
genera Bacillus,Rhizobium,Acinetobacter,Azospirillum, and
Burkholderia, and fungi Glomus and Trichoderma (Nadeem
et al., 2016). Synergistic effects of such consortia are likely
due to complementary modes of action and/or ecological
requirements, resulting in more reliable functional outcomes
than single organisms (Kaminsky et al., 2019). In this context,
innovative holistic approaches have also been proposed, such as
the design of synthetic microbial communities (SynComs, e.g.,
complex consortia mimicking microbiome functions, interaction
networks and/or phylogenetic profiles) (Paredes et al., 2018;de
Souza et al., 2020), core-microbiome therapy (e.g., transfer of
an artificially cultivated microbiome from a healthy plant to a
diseased one, like performed in clinical gastroenterology) (Gopal
et al., 2013), prebiotic approaches (e.g., molecules stimulating
the bioactivity of the resident microbiome and the growth of
beneficial organisms) (Massart et al., 2015;Tabacchioni et al.,
2021), or combination with helper strains (e.g., bacteria without
beneficial properties by themselves but enhancing the efficacy
of co-inoculated partners) (Massart et al., 2015;Berninger et al.,
2018). In cannabis, several Pseudomonas strains in consortia with
Bacillus strains promoted hemp growth significantly more than
the corresponding single-strain inoculants (Comeau et al., 2021),
but did not improve ISR elicitation against Botrytis gray mold
disease (Balthazar et al., 2020). Other bacterial consortia also
significantly promoted hemp growth and/or retting process, but
comparisons with single-strain inoculants were not investigated
(Di Candilo et al., 2010;Conant et al., 2017;Pagnani et al.,
2018). Finally, two hemp endophytic Pseudomonas strains were
found to be compatible for growth alongside fungal biocontrol
agents Trichoderma and Stachybotrys in vitro (Scott et al., 2018),
suggesting that they could be combined effectively in a joint
bioproduct formulation. In silico screening (Tabacchioni et al.,
2021), computer-assisted combination optimization (de Souza
et al., 2020), and predictive association modeling strategies
(Paredes et al., 2018) could also be implemented as preliminary
steps to help with the rational design of custom microbial
consortia for cannabis.
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COMMERCIALIZATION BOTTLENECKS
AND CHALLENGES
Many traits contribute to the success of Pseudomonas spp.
developed as commercial bioproducts, like their rapidity of
growth and ease of mass production, their ability to readily use
a wide array of nutrients and compete in the rhizosphere, their
multiple modes of action and broad-spectrum activities, and their
adaptability to a variety of plant-soil environments and stresses
(Fischer et al., 2013).
In cannabis, the potential benefits of Pseudomonas spp.
inoculants have already been demonstrated in vitro (Afzal et al.,
2015;Iqbal et al., 2018;Scott et al., 2018;Balthazar et al.,
2021) and in different cropping systems including greenhouses
(Gonsior et al., 2004), hydroponic and soil-less systems (Conant
et al., 2017), and indoor growth chambers (Balthazar et al., 2020,
2021;Comeau et al., 2021), while experiments in outdoor fields
are still lacking. Notably, two of the aforementioned studies
used commercially available Pseudomonas-based bioproducts
(Gonsior et al., 2004;Conant et al., 2017).
A plethora of good reviews can be consulted for a
general overview of commercialization steps, challenges, and
bottlenecks commonly encountered when developing microbe-
based bioproducts, including efficacy testing, risk assessments,
formulation design, mass production, commercial registration,
and marketing (Köhl et al., 2011;Backer et al., 2018;
Kaminsky et al., 2019;Pirttilä et al., 2021). The following
sections will therefore mainly focus on specific considerations
when developing Pseudomonas spp. inoculants for cannabis
crops (Figure 2).
Risks for Crops and Human Health
As introduced above, Pseudomonas strains related to the species
P. syringae and P. aeruginosa are common pathogens of plants
and animals, including cannabis and humans, whereas plant-
beneficial strains from the P. fluorescens group are generally
considered safe for humans and crops (Holmes et al., 2015).
Even though some P. syringae strains have been described
as biocontrol agents and exhibit plant-beneficial attributes,
the possibility of a switch to phytopathogenic behavior by
horizontal gene transfer would make their introduction into
crops extremely risky (Passera et al., 2019). Similarly, in some
jurisdictions, P. aeruginosa is part of the blacklisted organisms
and bile-tolerant Gram-negative (BTGN) bacteria that must be
absent in marijuana products destined to human consumption
(McPartland and McKernan, 2017;McKernan et al., 2018;
Boyar, 2021), even though the need to test cured products has
been questioned (Holmes et al., 2015). Therefore, to prevent
any biosafety risk, bioproducts containing bacteria related to
P. syringae or P. aeruginosa should not be used on cannabis
crops. Consequently, proper taxonomic identification when
screening candidate strains should be carried out with methods
able to differentiate unambiguously between closely related
Pseudomonas species, such as genome-wide comparisons or
multilocus sequence analyzes (MLSA), rather than 16S ribosomal
gene sequencing which mostly allows genera discrimination
(Lalucat et al., 2020). The selected candidates can be further
scrutinized to exclude potential producers of virulence factors
and harmful toxins, based on whole-genome mining strategies
and/or production detection assays (Balthazar et al., 2021). This
step can help support their classification as safe organisms,
thus facilitating the commercial registration process (Paterson
et al., 2017). In this regard, beneficial Pseudomonas strains
might have an advantage over mycotoxin-producing biocontrol
agents like Trichoderma and Stachybotrys (Vujanovic et al.,
2020), although the relevance of in-depth toxicological risk
assessments of microbial metabolite residues on plants has
been questioned, due to the low amounts produced in planta
(Köhl et al., 2019) and subsequent heat degradation during
marijuana product processing (Holmes et al., 2015). Besides,
the common fear of introducing harmless microorganisms into
edible plant parts has no real basis since plants tissues are already
naturally colonized by such microorganisms and many healthy
food products also contain microorganisms safe for human
consumption (Pirttilä et al., 2021).
Unfortunately, even with carefully selected beneficial
microorganisms, concerns about potential carry-over of
contaminants from treated plants into medical or recreational
products could still limit the use of bioproducts on drug-type
crops. Indeed, commercial quality control procedures and
regulatory requirements usually do not distinguish between
beneficial and harmful microorganisms, which could lead to
unwarranted rejection of the product (McKernan et al., 2016;
Boyar, 2021;Punja and Ni, 2021). Restricting treatments to
plant roots and vegetative parts, and/or long before harvest
time, has been suggested in order to spare the valuable
inflorescences from residues while still offering beneficial
effects like plant growth promotion, abiotic stress tolerance
and below-ground microbiome wealth (Conant et al., 2017;
Pagnani et al., 2018;Comeau et al., 2021;Kakabouki et al.,
2021a,b), as well as disease prevention by direct antibiosis
against soilborne pathogens (Punja, 2021), and ISR elicitation
(Balthazar et al., 2020) or sporulation suppression (Balthazar
et al., 2021;Scott and Punja, 2021) against aerial pathogens.
Additionally, more specific bacterial contaminant testing
methods are urgently needed to resolve this important issue
in cannabis cultivation, for example testing methods based on
specific nucleic acid detection (McKernan et al., 2016, 2018,
2021;Boyar, 2021).
Surprisingly, the detection of fungal contaminants can also
be compromised in the presence of chloramphenicol-resistant
P. fluorescens, when culture-based methods that fail to prevent
their proliferation are used (McKernan et al., 2016). Similarly,
salicylic acid-producing Pseudomonas spp. can falsely elevate the
total yeast and mold (TYM) count in marijuana products by
interfering with pH-based detection methods (McKernan et al.,
2018). These false-positive results may lead to undue product
rejection and unnecessary fungicide applications. The necessary
implementation of more accurate fungal detection methods,
such as multiplex molecular assays, could therefore also play an
important role in enabling the use of beneficial Pseudomonas spp.
in marijuana cultivation (McKernan et al., 2016, 2021;Boyar,
2021).
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Risks for the Environment
Before microbial inoculants can be applied to cannabis crops,
potential risks associated with non-target environmental effects
should be examined (Mawarda et al., 2020), especially for
outdoor crops. Indeed, in many countries, registration of
microbial inoculants often require that they do not persist after
a target time period, do not migrate off-site, nor have long-
term impacts on natural communities. Such requirements can
be in direct opposition to traits that were favored earlier in
the screening process, like aggressive growth, dispersal, and
competitiveness of microbial candidates, thus aggravating their
invasive potential after large-scale release (Kaminsky et al., 2019).
Approaches aiming to modify already established microbial
communities would thus be ecologically sounder than methods
aiming to replace them (Agoussar and Yergeau, 2021). Besides,
plants under stress tend to mostly promote microorganisms
that are already present in their environment, rather than
recruiting new ones from applied inoculants (Giard-Laliberté
et al., 2019). Survival and dispersal of Pseudomonas spp. are
also expected to differ between indoor and outdoor cannabis
cropping systems, with factors like weather, intercropping, crop
debris, and natural competitors impacting open fields but not
protected environments which are sanitized between growing
cycles. In this context, non-sporulating Pseudomonas spp. might
have an advantage over bacteria like Bacillus spp. whose stress-
tolerant spores are particularly amenable to dry formulation and
extended shelf-life but also promote undesired persistence in
soil (Kaminsky et al., 2019). Environmental risks also depend
on the mode of action exerted by the microorganisms (Köhl
et al., 2019), with lower risks associated with mechanisms
that do not require establishment and long-term survival of
the bacteria, like ISR elicitation, early modulation of root
architecture, or transient effects at a critical crop stage. However,
disappearance of the inoculated strain does not necessarily
imply a lack of lasting legacy on the native communities. In
fact, the vast majority of studies report persistent changes in
community composition as long-term consequences of microbial
inoculation (Mawarda et al., 2020). To address these issues,
it has been recommended to use native strains isolated from
local ecosystems, and to appraise potential lasting impacts
on microbiome composition, functioning and resilience with
sensitive high throughput methods, for at least several months
and/or growing seasons after application at multiple location sites
(Köhl et al., 2011;Mawarda et al., 2020).
Finally, the possibility of an intensified spread of antibiotic
resistance genes because of the introduction of beneficial bacteria
into soils has been raised. Indeed, most PGPR strains, including
common Pseudomonas spp. crop inoculants, have been found to
be resistant to multiple antibiotics, likely because of inadvertent
screening and/or co-selection with competitiveness abilities
(Kang et al., 2017). While bacterial biofilms and plant-associated
micro-environments are known hotspots for horizontal gene
transfers (HGT) (Van Elsas et al., 2003), the occurrence of
plasmid exchange from inoculated P. fluorescens to indigenous
Gram-negative rhizobacteria in soil has been demonstrated
under field conditions (van Elsas et al., 1998). Moreover,
antibiotic-resistant microorganisms associated with edible crops
may exacerbate the risk of dissemination and human exposure
through the food chain and worldwide trade exchanges (Chen
et al., 2019). To mitigate these issues, crop inoculants should
neither contain bacteria resistant to multiple antibiotics or to
important human drugs, nor contain genetically engineered
strains marked with antibiotic resistance genes. Applications on
crop leaves or seeds rather than by soil drench can also help
mitigate the problem in soil, as would applications of bioactive
metabolites instead of the living organisms that produced them
(Kang et al., 2017).
Formulations and Practical Applications
Developing bioproduct formulations that maintain high cell
viability is an important bottleneck when working with non-
sporulating Gram-negative bacteria like Pseudomonas spp.
Attention should thus be given to inoculum concentration
and product shelf-life. For commercial purposes, Pseudomonas
spp. are usually mass-produced using liquid fermentation
technologies and formulated as liquids, slurries, or solid forms
(powders and granules) (Nakkeeran et al., 2005;Berninger et al.,
2018). Since desiccation is often mainly responsible for the
loss of viability of non-sporulating bacteria during bioproduct
processing, storage, and field application, mitigation strategies
are important to consider and include the validation of suitable
drying methods, the addition of proper carriers and protectants
(peat, talc, skimmed milk, etc.), and optional storage at cold
temperature (Berninger et al., 2018). While most Pseudomonas-
based bioproducts are commercialized at concentration superior
to 106CFU/g with an average shelf-life of 6 months to a year
(Nakkeeran et al., 2005;Berninger et al., 2018), cannabis plant
growth can already be appreciably promoted at low inoculum
density (Pagnani et al., 2018) and excessive concentrations
could even lead to deleterious effects, as reported with other
PGPR tested on cannabis (Pagnani et al., 2018;Comeau et al.,
2021). However, considering the wide variety of cannabis
production systems, formulating a range of bioproducts for
reliable and consistent results under contrasting environments
could be an exciting challenge. For example, the efficacy
of inoculated bacteria will likely vary between outdoor and
indoor crops, because of differential abiotic conditions (naturally
fluctuating or controlled environments), exposure to adverse
weather and rain (open fields or sheltered greenhouses and
cultivation rooms), edaphic factors and resident microbiomes
(natural soils or standardized growing substrates and soil-less
hydroponic systems). The growing substrate alone was shown
to substantially influence the outcome of synergistic interactions
between Pseudomonas spp. and Bacillus spp. inoculants to
promote cannabis growth and modulate resident microbiome in
indoor cultivation (Comeau et al., 2021).
Compatibility with existing production practices and
agrochemicals is another important hurdle when developing
bioproducts for a broad market (Köhl et al., 2011). Agrochemicals
applied simultaneously should not reduce Pseudomonas spp.
viability, and the abilities of certain Pseudomonas spp. to degrade
and/or reduce the effects of several herbicides, insecticides
and fungicides—as detailed above—should also be considered
(Kahlon, 2016;Nadeem et al., 2016;Trognitz et al., 2016).
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Furthermore, to ensure practical utilization by cannabis growers,
formulations should be easy to apply with standard machinery
and equipment and should be compatible with traditional
cultivation techniques and organic certifications (Nakkeeran
et al., 2005;Berninger et al., 2018). Thanks to their remarkable
versatility, Pseudomonas spp. inoculants have been adapted to
a variety of delivery methods commonly used in agriculture
and horticulture, including seed coatings, soil amendments, root
dips, foliar sprays, or multiple combination of these treatments
(Nakkeeran et al., 2005;Berninger et al., 2018). Notably, in
intensive cannabis cultivation systems, microbial inoculants
can be circulated in irrigation water and hydroponic nutritive
solution (Conant et al., 2017;Kakabouki et al., 2021a,b;Punja,
2021), while bacterial volatiles might be dispensed through
atmosphere conditioning and ventilation infrastructures in
greenhouses and indoor facilities (Garbeva and Weisskopf, 2020).
Moreover, for drug-type crops reproduced by vegetative cuttings,
shoot endophytic organisms inoculated on mother plants could
be vertically transmitted to clonal cuttings (Comeau et al., 2020;
Pirttilä et al., 2021). Similarly, for seeded oilseed and fiber crops,
endophytic bacteria introduced into the inflorescences of parent
plants could be transmitted inside the seeds to the offspring
generation (Mitter et al., 2017;Berg and Raaijmakers, 2018).
However, regulatory requirements for the marijuana market
could substantially increase some production costs, such as the
need to manufacture the bioproducts in sterile conditions and
to use only food-safe additives to prevent marijuana product
contaminations (Berninger et al., 2018;Pirttilä et al., 2021). In
this regard, consortia-based products are also usually associated
with additional research and production expenses compared to
single-strain inoculants (Köhl et al., 2019), even though direct
co-cultivation approaches exist for fully compatible Pseudomonas
strains to spare the costly process of mass-producing each strain
separately (Berninger et al., 2018).
Finally, guidelines for timing of application will need to
be adapted according to crop-type, production goals, and
bioproduct intended purpose. As discussed above, increased
risk of microbial contaminants on marijuana products should
be carefully examined when treating close to harvest (Punja,
2021). Application timing for PGPR strains that increase plant
biomass should be aligned with phytochemical production
goals (either maximizing inflorescence weight, or maximizing
phytochemical concentration), as indicated by the distinctive
effects of fertilizers when used at different cultivation stages—as
explained above—(Caplan et al., 2017a,b;Saloner and Bernstein,
2021;Shiponi and Bernstein, 2021). Additionally, the use of
biocontrol agents for preventive disease management should
be based on forecasted risks for pathogen infestations and
environmental factors (Balthazar et al., 2021;Punja and Ni,
2021). Multi-application schedules can also be advantageous for
inoculants with transient effects which are preferable for their low
environmental impacts (Kaminsky et al., 2019).
CONCLUSION
The many different modes of action exerted by beneficial
Pseudomonas spp. hold great potential to promote the yield
and harvest quality of marijuana and hemp crops and
could be exploited to target specific biotic and abiotic issues
encountered by each crop type (grown for fibers, oilseeds,
and/or phytochemicals). Traditional and innovative strategies to
identify promising bacteria and formulate suitable bioproducts
should provide useful avenues toward the development of
effective inoculants.
AUTHOR CONTRIBUTIONS
CB: conceptualization, writing-original draft, and review
and editing. DJ and MF: writing-review and editing and
funding acquisition. All authors have read and approved the
submitted version.
FUNDING
TRICHUM (Translating Research into Innovation for Cannabis
Health at Université de Moncton) was supported by grants
from Genome Canada (Genome Atlantic NB-RP3), the Atlantic
Canada Opportunities Agency (project 212090), and the New
Brunswick Innovation Foundation (RIF2018-036).
REFERENCES
Abdulla, D. O., and Karademir, C. (2019). The effect of biofertilizers on cotton
(Gossypium hirsutum L.) development yield and fiber technological properties.
Glob. J. Bot. Sci. 7, 1–9. doi: 10.12974/2311-858x.2019.07.1
Adal, A. M., Doshi, K., Holbrook, L., and Mahmoud, S. S. (2021). Comparative
RNA-Seq analysis reveals genes associated with masculinization in
female Cannabis sativa.Planta 253, 1–17. doi: 10.1007/s00425-020-03
522-y
Afzal, I., Shinwari, Z. K., and Iqrar, I. (2015). Selective isolation and
characterization of agriculturally beneficial endophytic bacteria from wild hemp
using canola. Pakistan J. Bot. 47, 1999–2008.
Agoussar, A., and Yergeau, E. (2021). Engineering the plant microbiota in the
context of the theory of ecological communities. Curr. Opin. Biotechnol. 70,
220–225. doi: 10.1016/j.copbio.2021.06.009
Ahmed, B., and Hijri, M. (2021). Potential impacts of soil microbiota manipulation
on secondary metabolites production in cannabis. J. Cannabis Res. 3, 1–9.
doi: 10.1186/s42238-021- 00082-0
Ahmed, B., Smart, L., and Hijri, M. (2021). Microbiome of field grown hemp
reveals potential microbial interactions with root and rhizosphere soil. Front.
Microbiol. 12:741597. doi: 10.3389/fmicb.2021.741597
Ali, E. M., Almagboul, A. Z., Khogali, S. M., and Gergeir, U. M. (2012).
Antimicrobial activity of Cannabis sativa L. Chinese Med. 3:18123. doi: 10.4236/
cm.2012.31010
Andre, C. M., Hausman, J.-F., and Guerriero, G. (2016). Cannabis sativa: the plant
of the thousand and one molecules. Front. Plant Sci. 7:19. doi: 10.3389/fpls.2016.
00019
Apicella, P., Sands, L., Ma, Y., and Berkowitz, G. (2021). Delineating the genetic
regulation of cannabinoid biosynthesis during female flower development in
Cannabis sativa.BioRxiv [Preprint]. doi: 10.1101/2021.11.05.464876
Frontiers in Microbiology | www.frontiersin.org 16 January 2022 | Volume 12 | Article 833172

fmicb-12-833172 January 10, 2022 Time: 14:26 # 17
Balthazar et al. Beneficial Pseudomonas for Cannabis Production
Baas, P., Bell, C., Mancini, L., Lee, M., Conant, R., and Wallenstein, M. (2016).
Phosphorus mobilizing consortium Mammoth PTM enhances plant growth.
PeerJ 4:e2121. doi: 10.7717/peerj.2121
Backer, R., Rokem, J. S., Ilangumaran, G., Lamont, J., Praslickova, D., Ricci, E., et al.
(2018). Plant growth-promoting rhizobacteria: context, mechanisms of action,
and roadmap to commercialization of biostimulants for sustainable agriculture.
Front. Plant Sci. 9:1473. doi: 10.3389/fpls.2018.01473
Backer, R. G., Rosenbaum, P., McCarty, V., Eichhorn Bilodeau, S., Lyu, D., Ahmed,
M. B., et al. (2019). Closing the yield gap for cannabis: a meta-analysis of factors
determining cannabis yield. Front. Plant Sci. 10:495. doi: 10.3389/fpls.2019.
00495
Bakro, F., Wielgusz, K., Bunalski, M., and Jedryczka, M. (2018). An overview of
pathogen and insect threats to fibre and oilseed hemp (Cannabis sativa L.) and
methods for their biocontrol. Integr. Control Oilseed Crops IOBC WPRS Bull.
136, 9–20.
Balestrini, R., Brunetti, C., Cammareri, M., Caretto, S., Cavallaro, V., Cominelli, E.,
et al. (2021). Strategies to modulate specialized metabolism in Mediterranean
crops: from molecular aspects to field. Int. J. Mol. Sci. 22:2887. doi: 10.3390/
ijms22062887
Balthazar, C., Cantin, G., Novinscak, A., Joly, D. L., and Filion, M. (2020).
Expression of putative defense responses in cannabis primed by Pseudomonas
and/or Bacillus strains and infected by Botrytis cinerea.Front. Plant Sci. 11:1873.
doi: 10.3389/fpls.2020.572112
Balthazar, C., Novinscak, A., Cantin, G., Joly, D. L., and Filion, M. (2021).
Biocontrol activity of Bacillus spp. and Pseudomonas spp. against Botrytis
cinerea and other cannabis fungal pathogens. Phytopathology [Epub ahead of
print]. doi: 10.1094/phyto-03- 21-0128-r
Barnett, S. E., Cala, A. R., Hansen, J. L., Crawford, J., Viands, D. R., Smart, L. B.,
et al. (2020). Evaluating the microbiome of hemp. Phytobiomes J. 4, 351–363.
doi: 10.1094/pbiomes-06- 20-0046-r
Bates, T. A. (2021). Science TakesFlight: Detection of Black Leg on Turnip Gray Mold
on Hemp. Master Thesis. Corvallis, OR: Oregon State University.
Beneduzi, A., Ambrosini, A., and Passaglia, L. M. (2012). Plant growth-promoting
rhizobacteria (PGPR): their potential as antagonists and biocontrol agents.
Genet. Mol. Biol. 35, 1044–1051. doi: 10.1590/s1415-47572012000600020
Berg, G., and Raaijmakers, J. M. (2018). Saving seed microbiomes. ISME J. 12,
1167–1170. doi: 10.1038/s41396-017- 0028-2
Berg, G., Rybakova, D., Fischer, D., Cernava, T., Vergˇ
cs, M.-C. C., Charles, T.,
et al. (2020). Microbiome definition re-visited: old concepts and new challenges.
Microbiome 8, 1–22. doi: 10.1186/s40168-020-00875- 0
Berninger, T., González López, Ó, Bejarano, A., Preininger, C., and Sessitsch, A.
(2018). Maintenance and assessment of cell viability in formulation of non-
sporulating bacterial inoculants. Microb. Biotechnol. 11, 277–301. doi: 10.1111/
1751-7915.12880
Bhat, M. A., Kumar, V., Bhat, M. A., Wani, I. A., Dar, F. L., Farooq, I., et al. (2020).
Mechanistic insights of the interaction of plant growth-promoting rhizobacteria
(PGPR) with plant roots toward enhancing plant productivity by alleviating
salinity stress. Front. Microbiol. 11:1952. doi: 10.3389/fmicb.2020.01952
Bhowmik, S. N., and Patil, R. T. (2018). “Application of microbial biotechnology in
food processing,” in New and Future Developments in Microbial Biotechnology
and Bioengineering: Crop Improvement Through Microbial Biotechnology, eds R.
Prasad, S. S. Gill, and N. Tuteja (Amsterdam: Elsevier), 73–106.
Bona, E., Lingua, G., and Todeschini, V. (2016). “Effect of bioinoculants on the
quality of crops,” in Bioformulations: for Sustainable Agriculture, eds N. K.
Arora, S. Mehnaz, and R. Balestrini (New Delhi: Springer), 93–124.
Booth, J. K., Page, J. E., and Bohlmann, J. (2017). Terpene synthases from Cannabis
sativa.PLoS One 12:e0173911. doi: 10.1371/journal.pone.0173911
Boyar, K. (2021). “Cannabis microbial testing: methodologies and considerations,”
in Recent Advances in the Science of Cannabis, eds R. M. Strongin, J. Meehan-
Atrash, and M. Vialpando (Boca Raton: CRC Press), 131–160.
Bulgarelli, D., Schlaeppi, K., Spaepen, S., Van Themaat, E. V. L., and Schulze-
Lefert, P. (2013). Structure and functions of the bacterial microbiota of plants.
Annu. Rev. Plant Biol. 64, 807–838. doi: 10.1146/annurev-arplant-050312-12
0106
Bull, C. T., Manceau, C., Lydon, J., Kong, H., Vinatzer, B. A., and Fischer-
Le Saux, M. (2010). Pseudomonas cannabina pv. cannabina pv. nov., and
Pseudomonas cannabina pv. alisalensis (Cintas Koike and Bull, 2000) comb.
nov., are members of the emended species Pseudomonas cannabina (ex Šutiè
& Dowson 1959) Gardan, Shafik, Belouin, Brosch, Grimont & Grimont 1999.
Syst. Appl. Microbiol. 33, 105–115. doi: 10.1016/j.syapm.2010.02.001
Çakmakçı, R., Mosber, G., Milton, A. H., Alatürk, F., and Ali, B. (2020). The effect
of auxin and auxin-producing bacteria on the growth, essential oil yield, and
composition in medicinal and aromatic plants. Curr. Microbiol. 77, 564–577.
doi: 10.1007/s00284-020- 01917-4
Camaille, M., Fabre, N., Clément, C., and Ait Barka, E. (2021). Advances in wheat
physiology in response to drought and the role of plant growth promoting
rhizobacteria to trigger drought tolerance. Microorganisms 9:687. doi: 10.3390/
microorganisms9040687
Caplan, D., Dixon, M., and Zheng, Y. (2017b). Optimal rate of organic fertilizer
during the vegetative-stage for cannabis grown in two coir-based substrates.
HortScience 52, 1307–1312. doi: 10.21273/HORTSCI11903-17
Caplan, D., Dixon, M., and Zheng, Y. (2017a). Optimal rate of organic fertilizer
during the flowering stage for cannabis grown in two coir-based substrates.
HortScience 52, 1796–1803. doi: 10.21273/HORTSCI12401-17
Caplan, D., Dixon, M., and Zheng, Y. (2019). Increasing inflorescence dry weight
and cannabinoid content in medical cannabis using controlled drought stress.
HortScience 54, 964–969. doi: 10.21273/HORTSCI13510-18
Chen, Q.-L., Cui, H.-L., Su, J.-Q., Penuelas, J., and Zhu, Y.-G. (2019). Antibiotic
resistomes in plant microbiomes. Trends Plant Sci. 24, 530–541. doi: 10.1016/j.
tplants.2019.02.010
Citterio, S., Prato, N., Fumagalli, P., Aina, R., Massa, N., Santagostino, A., et al.
(2005). The arbuscular mycorrhizal fungus Glomus mosseae induces growth
and metal accumulation changes in Cannabis sativa L. Chemosphere 59, 21–29.
doi: 10.1016/j.chemosphere.2004.10.009
Clarke, R. C., and Merlin, M. D. (2016). Cannabis domestication, breeding history,
present-day genetic diversity, and future prospects. Crit. Rev. Plant Sci. 35,
293–327. doi: 10.1080/07352689.2016.1267498
Cocolin, L., and Ercolini, D. (2015). Zooming into food-associated microbial
consortia: a ‘cultural’ evolution. Curr. Opin. Food Sci. 2, 43–50. doi: 10.1016/
j.cofs.2015.01.003
Comeau, D., Balthazar, C., Novinscak, A., Bouhamdani, N., Joly, D. L., and Filion,
M. (2021). Interactions between Bacillus spp., Pseudomonas spp. and Cannabis
sativa promote plant growth. Front. Microbiol. 12:715758. doi: 10.3389/fmicb.
2021.715758
Comeau, D., Novinscak, A., Joly, D. L., and Filion, M. (2020). Spatio-temporal and
cultivar-dependent variations in the cannabis microbiome. Front. Microbiol.
11:491. doi: 10.3389/fmicb.2020.00491
Conant, R., Walsh, R., Walsh, M., Bell, C., and Wallenstein, M. (2017). Effects
of a microbial biostimulant, Mammoth PTM, on Cannabis sativa bud yield.
J. Hortic. 4:191. doi: 10.4172/2376-0354.1000191
Cosentino, S. L., Riggi, E., Testa, G., Scordia, D., and Copani, V. (2013). Evaluation
of European developed fibre hemp genotypes (Cannabis sativa L.) in semi-arid
Mediterranean environment. Ind. Crops Product. 50, 312–324. doi: 10.1016/j.
indcrop.2013.07.059
de Souza, R. S. C., Armanhi, J. S. L., and Arruda, P. (2020). From microbiome to
traits: designing synthetic microbial communities for improved crop resiliency.
Front. Plant Sci. 11:1179. doi: 10.3389/fpls.2020.01179
Delgado-Povedano, M., Callado, C. S.-C., Priego-Capote, F., and Ferreiro-Vera,
C. (2020). Untargeted characterization of extracts from Cannabis sativa L.
cultivars by gas and liquid chromatography coupled to mass spectrometry in
high resolution mode. Talanta 208:120384. doi: 10.1016/j.talanta.2019.120384
Deng, G., Du, G., Yang, Y., Bao, Y., and Liu, F. (2019). Planting density and
fertilization evidently influence the fiber yield of hemp (Cannabis sativa L.).
Agronomy 9:368. doi: 10.3390/agronomy9070368
Di Candilo, M., Bonatti, P., Guidetti, C., Focher, B., Grippo, C., Tamburini, E., et al.
(2010). Effects of selected pectinolytic bacterial strains on water-retting of hemp
and fibre properties. J. Appl. Microbiol. 108, 194–203. doi: 10.1111/j.1365-2672.
2009.04409.x
Dubeikovsky, A., Mordukhova, E., Kochetkov, V. T., Polikarpova, F., and
Boronin, A. (1993). Growth promotion of blackcurrant softwood cuttings by
recombinant strain Pseudomonas fluorescens BSP53a synthesizing an increased
amount of indole-3-acetic acid. Soil Biol. Biochem. 25, 1277–1281. doi: 10.1016/
0038-0717(93)90225-z
Eichhorn Bilodeau, S., Wu, B.-S., Rufyikiri, A.-S., MacPherson, S., and Lefsrud,
M. (2019). An update on plant photobiology and implications for cannabis
production. Front. Plant Sci. 10:296. doi: 10.3389/fpls.2019.00296
Frontiers in Microbiology | www.frontiersin.org 17 January 2022 | Volume 12 | Article 833172

fmicb-12-833172 January 10, 2022 Time: 14:26 # 18
Balthazar et al. Beneficial Pseudomonas for Cannabis Production
Elsawey, H., Patz, S., Nemr, R. A., Sarhan, M. S., Hamza, M. A., Youssef, H. H.,
et al. (2020). Plant broth-(Not bovine-) based culture media provide the most
compatible vegan nutrition for in vitro culturing and in situ probing of plant
microbiota. Diversity 12:418. doi: 10.3390/d12110418
Fischer, S., Príncipe, A., Alvarez, F., Cordero, P., Castro, M., Godino, A., et al.
(2013). “Fighting plant diseases through the application of Bacillus and
Pseudomonas strains,” in Symbiotic Endophytes, ed. R. Aroca (Berlin: Springer),
165–193.
Flury, P., Aellen, N., Ruffner, B., Péchy-Tarr, M., Fataar, S., Metla, Z., et al.
(2016). Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic
distribution and comparative genomics. ISME J. 10, 2527–2542. doi: 10.1038/
ismej.2016.5
Ganger, M. T., Hiles, R., Hallowell, H., Cooper, L., McAllister, N., Youngdahl, D.,
et al. (2019). A soil bacterium alters sex determination and rhizoid development
in gametophytes of the fern Ceratopteris richardii.AoB Plants 11:lz012. doi:
10.1093/aobpla/plz012
Garbeva, P., and Weisskopf, L. (2020). Airborne medicine: bacterial volatiles and
their influence on plant health. New Phytol. 226, 32–43. doi: 10.1111/nph.16282
Gautam, A. K., Kant, M., and Thakur, Y. (2013). Isolation of endophytic fungi from
Cannabis sativa and study their antifungal potential. Arch. Phytopathol. Plant
Protect. 46, 627–635. doi: 10.1080/03235408.2012.749696
Giard-Laliberté, C., Azarbad, H., Tremblay, J., Bainard, L., and Yergeau, É (2019).
A water stress-adapted inoculum affects rhizosphere fungi, but not bacteria nor
wheat. FEMS Microbiol. Ecol. 95:fiz080. doi: 10.1093/femsec/fiz080
Gómez-Lama Cabanás, C., Legarda, G., Ruano-Rosa, D., Pizarro-Tobías, P.,
Valverde-Corredor, A., Niqui, J. L., et al. (2018). Indigenous Pseudomonas spp.
strains from the olive (Olea europaea L.) rhizosphere as effective biocontrol
agents against Verticillium dahliae: from the host roots to the bacterial
genomes. Front. Microbiol. 9:277. doi: 10.3389/fmicb.2018.00277
Gonsior, G., Buschmann, H., Szinicz, G., Spring, O., and Sauerborn, J. (2004).
Induced resistance—an innovative approach to manage branched broomrape
(Orobanche ramosa) in hemp and tobacco. Weed Sci. 52, 1050–1053. doi: 10.
1614/ws-04-088r1
Gopal, M., Gupta, A., and Thomas, G. V. (2013). Bespoke microbiome therapy to
manage plant diseases. Front. Microbiol. 4:355. doi: 10.3389/fmicb.2013.00355
Gorelick, J., and Bernstein, N. (2017). “Chemical and physical elicitation for
enhanced cannabinoid production in Cannabis,” in Cannabis sativa L.-Botany
and Biotechnology, eds S. Chandra, H. Lata, and M. A. ElSohly (Cham:
Springer), 439–456.
Grassa, C. J., Wenger, J. P., Dabney, C., Poplawski, S. G., Motley, S. T., Michael,
T. P., et al. (2018). A complete Cannabis chromosome assembly and adaptive
admixture for elevated cannabidiol (CBD) content. BioRxiv [Preprint]. doi:
10.1101/458083v4
Guerriero, G., Behr, M., Legay, S., Mangeot-Peter, L., Zorzan, S., Ghoniem, M., et al.
(2017). Transcriptomic profiling of hemp bast fibres at different developmental
stages. Sci. Rep. 7:4961. doi: 10.1038/s41598-017- 05200-8
Hesami, M., Pepe, M., Alizadeh, M., Rakei, A., Baiton, A., and Jones, A. M. P.
(2020). Recent advances in cannabis biotechnology. Ind. Crops Product.
158:113026. doi: 10.1016/j.indcrop.2020.113026
Holmes, M., Vyas, J., Steinbach, W., and McPartland, J. (2015). Microbiological
Safety Testing of Cannabis. London: Cannabis Safety Institute.
Iqbal, A., Arshad, M., Hashmi, I., Karthikeyan, R., Gentry, T. J., and Schwab, A. P.
(2018). Biodegradation of phenol and benzene by endophytic bacterial strains
isolated from refinery wastewater-fed Cannabis sativa.Environ. Technol. 39,
1705–1714. doi: 10.1080/09593330.2017.1337232
Jalali, S., Salami, S. A., Sharifi, M., and Sohrabi, S. (2019). Signaling compounds
elicit expression of key genes in cannabinoid pathway and related metabolites in
cannabis. Ind. Crops Product. 133, 105–110. doi: 10.1016/j.indcrop.2019.03.004
Jian, F., Al Mamun, M. A., White, N. D., Jayas, D. S., Fields, P. G., and McCombe,
J. (2019). Safe storage times of FINOLA R
hemp (Cannabis sativa) seeds with
dockage. J. Stored Product. Res. 83, 34–43. doi: 10.1016/j.jspr.2019.05.013
Jiménez, J., Novinscak, A., and Filion, M. (2020). Pseudomonas fluorescens
LBUM677 differentially increases plant biomass, total oil content and lipid
composition in three oilseed crops. J. Appl. Microbiol. 128, 1119–1127. doi:
10.1111/jam.14536
Jin, D., Henry, P., Shan, J., and Chen, J. (2021). Identification of phenotypic
characteristics in three chemotype categories in the genus Cannabis.
HortScience 56, 481–490. doi: 10.21273/hortsci15607-20
Jin, R., Yang, M., and Liu, F. (2014). Effects of endophytic fungi re-inoculation
on physiological and agronomic characters of hemp (Cannabis sativa). Plant
Divers. Resour. 36, 65–69.
Kahlon, R. S. (2016). “Biodegradation and bioremediation of organic chemical
pollutants by Pseudomonas,” in Pseudomonas: Molecular and Applied Biology,
ed. R. S. Kahlon (Cham: Springer), 343–417.
Kakabouki, I., Mavroeidis, A., Tataridas, A., Kousta, A., Efthimiadou, A.,
Karydogianni, S., et al. (2021a). Effect of Rhizophagus irregularis on growth
and quality of Cannabis sativa seedlings. Plants 10:1333. doi: 10.3390/
plants10071333
Kakabouki, I., Tataridas, A., Mavroeidis, A., Kousta, A., Karydogianni, S., Zisi,
C., et al. (2021b). Effect of colonization of Trichoderma harzianum on growth
development and CBD content of hemp (Cannabis sativa L.). Microorganisms
9:518. doi: 10.3390/microorganisms9030518
Kalayu, G. (2019). Phosphate solubilizing microorganisms: promising approach as
biofertilizers. Int. J. Agron. 2019, 1–7. doi: 10.1155/2019/4917256
Kaminsky, L. M., Trexler, R. V., Malik, R. J., Hockett, K. L., and Bell, T. H.
(2019). The inherent conflicts in developing soil microbial inoculants. Trends
Biotechnol. 37, 140–151. doi: 10.1016/j.tibtech.2018.11.011
Kang, Y., Shen, M., Xia, D., Ye, K., Zhao, Q., and Hu, J. (2017). Caution of
intensified spread of antibiotic resistance genes by inadvertent introduction of
beneficial bacteria into soil. Acta Agric. Scand. B Soil Plant Sci. 67, 576–582.
doi: 10.1080/09064710.2017.1314548
Kaymak, H., Yarali, F., Guvenc, I., and Donmez, M. F. (2008). The effect of
inoculation with plant growth rhizobacteria (PGPR) on root formation of mint
(Mentha piperita L.) cuttings. Afr. J. Biotechnol. 7, 4479–4483.
Khan, H., Parmar, N., and Kahlon, R. S. (2016). “Pseudomonas-plant interactions I:
plant growth promotion and defense-mediated mechanisms,” in Pseudomonas:
Molecular and Applied Biology, ed. R. S. Kahlon (Cham: Springer), 419–468.
Khan, N., Asadullah, and Bano, A. (2019). “Rhizobacteria and abiotic stress
management,” in Plant Growth Promoting Rhizobacteria for Sustainable Stress
Management - Volume 1: Rhizobacteria in Abiotic Stress Management, eds R. Z.
Sayyed, N. K. Arora, and M. S. Reddy (Singapore: Springer), 65–80.
Köhl, J., Kolnaar, R., and Ravensberg, W. J. (2019). Mode of action of microbial
biological control agents against plant diseases: relevance beyond efficacy.
Front. Plant Sci. 10:845. doi: 10.3389/fpls.2019.00845
Köhl, J., Postma, J., Nicot, P., Ruocco, M., and Blum, B. (2011). Stepwise screening
of microorganisms for commercial use in biological control of plant-pathogenic
fungi and bacteria. Biol. Control 57, 1–12. doi: 10.1016/j.biocontrol.2010.12.004
Kour, D., Rana, K. L., Kaur, T., Yadav, N., Halder, S. K., Yadav, A. N., et al. (2020).
“Potassium solubilizing and mobilizing microbes: biodiversity, mechanisms
of solubilization, and biotechnological implication for alleviations of abiotic
stress,” in New and Future Developments in Microbial Biotechnology and
Bioengineering: Trends of Microbial Biotechnology for Sustainable Agriculture
and Biomedicine Systems: Diversity and Functional Perspectives, eds A.
Rastegari, A. Yadav, and N. Yadav (Amsterdam: Elsevier), 177–202.
Kour, D., Rana, K. L., Yadav, A. N., Yadav, N., Kumar, V., Kumar, A., et al.
(2019). “Drought-tolerant phosphorus-solubilizing microbes: biodiversity and
biotechnological applications for alleviation of drought stress in plants,” in Plant
Growth Promoting Rhizobacteria for Sustainable Stress Management - Volume 1:
Rhizobacteria in Abiotic Stress Management, eds R. Z. Sayyed, N. K. Arora, and
M. S. Reddy (Singapore: Springer), 255–308.
Kumar, A., Dewangan, S., Lawate, P., Bahadur, I., and Prajapati, S. (2019). “Zinc-
solubilizing bacteria: a boon for sustainable agriculture,” in Plant Growth
Promoting Rhizobacteria for Sustainable Stress Management - Volume 1:
Rhizobacteria in Abiotic Stress Management, eds R. Z. Sayyed, N. K. Arora, and
M. S. Reddy (Singapore: Springer), 139–155.
Kumar, A. N., Soo, C. I., Ng, B. H., Hassan, T., Ban, A. Y. L., and Manap,
R. A. (2018). Marijuana “bong” pseudomonas lung infection: a detrimental
recreational experience. Respirol. Case Rep. 6:e00293. doi: 10.1002/rcr2.293
Kumar, S., Pandey, P., and Maheshwari, D. (2009). Reduction in dose of chemical
fertilizers and growth enhancement of sesame (Sesamum indicum L.) with
application of rhizospheric competent Pseudomonas aeruginosa LES4. Eur. J.
Soil Biol. 45, 334–340. doi: 10.1016/j.ejsobi.2009.04.002
Kusari, P., Kusari, S., Spiteller, M., and Kayser, O. (2013). Endophytic fungi
harbored in Cannabis sativa L.: diversity and potential as biocontrol agents
against host plant-specific phytopathogens. Fungal Divers. 60, 137–151. doi:
10.1007/s13225-012-0216-3
Frontiers in Microbiology | www.frontiersin.org 18 January 2022 | Volume 12 | Article 833172

fmicb-12-833172 January 10, 2022 Time: 14:26 # 19
Balthazar et al. Beneficial Pseudomonas for Cannabis Production
Kusari, P., Kusari, S., Spiteller, M., and Kayser, O. (2017). “Cannabis endophytes
and their application in breeding and physiological fitness,” in Cannabis sativa
L.-Botany and Biotechnology, eds S. Chandra, H. Lata, and M. A. ElSohly (Cham:
Springer), 419–437.
Lalucat, J., Mulet, M., Gomila, M., and García-Valdés, E. (2020). Genomics in
bacterial taxonomy: impact on the genus Pseudomonas.Genes 11:139. doi:
10.3390/genes11020139
Lata, R., Chowdhury, S., Gond, S. K., and White, J. F. Jr. (2018). Induction of
abiotic stress tolerance in plants by endophytic microbes. Lett. Appl. Microbiol.
66, 268–276. doi: 10.1111/lam.12855
Law, A. D., McNees, C. R., and Moe, L. A. (2020). The microbiology of
hemp retting in a controlled environment: steering the hemp microbiome
towards more consistent fiber production. Agronomy 10:492. doi: 10.3390/
agronomy10040492
Lemanceau, P., Bauer, P., Kraemer, S., and Briat, J.-F. (2009). Iron dynamics in the
rhizosphere as a case study for analyzing interactions between soils, plants and
microbes. Plant Soil 321, 513–535. doi: 10.1007/s11104-009- 0039-5
Lindow, S. (1983). Methods of preventing frost injury caused by epiphytic ice-
nucleation-active bacteria. Plant Dis. 67:327. doi: 10.1094/pd-67- 327
Liste, H.-H., and Prutz, I. (2006). Plant performance, dioxygenase-expressing
rhizosphere bacteria, and biodegradation of weathered hydrocarbons in
contaminated soil. Chemosphere 62, 1411–1420. doi: 10.1016/j.chemosphere.
2005.05.018
Liu, J., Qiao, Q., Cheng, X., Du, G., Deng, G., Zhao, M., et al. (2016). Transcriptome
differences between fiber-type and seed-type Cannabis sativa variety exposed to
salinity. Physiol. Mol. Biol. Plants 22, 429–443. doi: 10.1007/s12298-016- 0381-z
Liu, M., Ale, M. T., Kołaczkowski, B., Fernando, D., Daniel, G., Meyer, A. S.,
et al. (2017). Comparison of traditional field retting and Phlebia radiata Cel
26 retting of hemp fibres for fibre-reinforced composites. AMB Express 7, 1–15.
doi: 10.1186/s13568-017- 0355-8
Liu, X.-M., and Zhang, H. (2015). The effects of bacterial volatile emissions on plant
abiotic stress tolerance. Front. Plant Sci. 6:774. doi: 10.3389/fpls.2015.00774
Ludwig-Müller, J. (2015). Plants and endophytes: equal partners in secondary
metabolite production? Biotechnol. Lett. 37, 1325–1334. doi: 10.1007/s10529-
015-1814-4
Lyu, D., Backer, R. G., Robinson, W. G., and Smith, D. L. (2019). Plant-growth
promoting rhizobacteria for cannabis production: yield, cannabinoid profile
and disease resistance. Front. Microbiol. 10:1761. doi: 10.3389/fmicb.2019.
01761
Majeed, A., Abbasi, M. K., Hameed, S., Yasmin, S., Hanif, M. K., Naqqash, T., et al.
(2018). Pseudomonas sp. AF-54 containing multiple plant beneficial traits acts
as growth enhancer of Helianthus annuus L. under reduced fertilizer input.
Microbiol. Res. 216, 56–69. doi: 10.1016/j.micres.2018.08.006
Mansouri, H., Asrar, Z., and Szopa, J. (2009). Effects of ABA on primary terpenoids
and 19-tetrahydrocannabinol in Cannabis sativa L. at flowering stage. Plant
Growth Regul. 58, 269–277. doi: 10.1007/s10725-009-9375-y
Massart, S., Martinez-Medina, M., and Jijakli, M. H. (2015). Biological control in
the microbiome era: challenges and opportunities. Biol. Control 89, 98–108.
doi: 10.1016/j.biocontrol.2015.06.003
Mawarda, P. C., Le Roux, X., Van Elsas, J. D., and Salles, J. F. (2020). Deliberate
introduction of invisible invaders: a critical appraisal of the impact of microbial
inoculants on soil microbial communities. Soil Biol. Biochem. 148:107874. doi:
10.1016/j.soilbio.2020.107874
Mayak, S., Tirosh, T., and Glick, B. (1999). Effect of wild-type and mutant plant
growth-promoting rhizobacteria on the rooting of mung bean cuttings. J. Plant
Growth Regul. 18, 49–53. doi: 10.1007/PL00007047
Mayer, B. F., Ali-Benali, M. A., Demone, J., Bertrand, A., and Charron, J. B.
(2015). Cold acclimation induces distinctive changes in the chromatin state and
transcript levels of COR genes in Cannabis sativa varieties with contrasting cold
acclimation capacities. Physiol. Plant. 155, 281–295. doi: 10.1111/ppl.12318
McKernan, K., Helbert, Y., Ebling, H., Cox, A., Kane, L., and Zhang, L. (2018).
Microbiological examination of nonsterile cannabis products: molecular
microbial enumeration tests and the limitation of colony forming units. OSF
[Preprints]. doi: 10.31219/osf.io/vpxe5
McKernan, K., Helbert, Y., Kane, L., Houde, N., Zhang, L., and McLaughlin, S.
(2021). Whole genome sequencing of colonies derived from cannabis flowers
and the impact of media selection on benchmarking total yeast and mold
detection tools. F1000Research 10:264. doi: 10.12688/f1000research.53467.1
McKernan, K., Spangler, J., Helbert, Y., Lynch, R. C., Devitt-Lee, A., Zhang, L.,
et al. (2016). Metagenomic analysis of medicinal Cannabis samples; pathogenic
bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast
and mold tests. F1000Research 5:2471. doi: 10.12688/f1000research.9662.1
McKernan, K. J., Helbert, Y., Kane, L. T., Ebling, H., Zhang, L., Liu, B., et al.
(2020). Sequence and annotation of 42 cannabis genomes reveals extensive
copy number variation in cannabinoid synthesis and pathogen resistance genes.
BioRxiv [Preprint]. doi: 10.37473//10.1101/2020.01.03.894428
McPartland, J. M. (1996b). Cannabis pests. J. Int. Hemp Assoc. 3, 52–55.
McPartland, J. M. (1996a). A review of Cannabis diseases. J. Int. Hemp Assoc. 3,
19–23.
McPartland, J. M., Clarke, R. C., and Watson, D. P. (2000). Hemp Diseases and
Pests: Management and Biological Control: An Advanced Treatise. Wallingford:
CABI.
McPartland, J. M., and Hillig, K. W. (2004). Striatura ulcerosa. J. Ind. Hemp 9,
89–96. doi: 10.1300/j237v09n01_10
McPartland, J. M., and McKernan, K. J. (2017). “Contaminants of concern in
cannabis: microbes, heavy metals and pesticides,” in Cannabis sativa L.-Botany
and Biotechnology, eds S. Chandra, H. Lata, and M. A. ElSohly (Cham:
Springer), 457–474.
Mihoc, M., Pop, G., Alexa, E., and Radulov, I. (2012). Nutritive quality of romanian
hemp varieties (Cannabis sativa L.) with special focus on oil and metal contents
of seeds. Chem. Central J. 6, 1–12. doi: 10.1186/1752-153X-6-122
Mitter, B., Pfaffenbichler, N., Flavell, R., Compant, S., Antonielli, L., Petric, A., et al.
(2017). A new approach to modify plant microbiomes and traits by introducing
beneficial bacteria at flowering into progeny seeds. Front. Microbiol. 8:11. doi:
10.3389/fmicb.2017.00011
Montoya, Z., Conroy, M., Vanden Heuvel, B. D., Pauli, C. S., and Park, S.-
H. (2020). Cannabis contaminants limit pharmacological use of cannabidiol.
Front. Pharmacol. 11:1439. doi: 10.3389/fphar.2020.571832
Müssig, J., and Amaducci, S. (2018). Scanner based image analysis to characterise
the influence of agronomic factors on hemp (Cannabis sativa L.) fibre width.
Ind. Crops Product. 113, 28–37. doi: 10.1016/j.indcrop.2017.12.059
Nadeem, S. M., Naveed, M., Ayyub, M., Khan, M. Y., Ahmad, M., and Zahir, Z. A.
(2016). Potential, limitations and future prospects of Pseudomonas spp. for
sustainable agriculture and environment: a review. Soil Environ. 35, 106–145.
Nakkeeran, S., Fernando, W. D., and Siddiqui, Z. A. (2005). “Plant growth
promoting rhizobacteria formulations and its scope in commercialization for
the management of pests and diseases,” in PGPR: Biocontrol and Biofertilization,
ed. Z. A. Siddiqui (The Netherlands: Springer), 257–296.
Ngalimat, M. S., Mohd Hata, E., Zulperi, D., Ismail, S. I., Ismail, M. R., Mohd
Zainudin, N. A. I., et al. (2021). Plant growth-promoting bacteria as an
emerging tool to manage bacterial rice pathogens. Microorganisms 9:682. doi:
10.3390/microorganisms9040682
Nykter, M. (2006). Microbial Quality of Hemp (Cannabis sativa L.) and flax (Linum
usitatissimum L.) from Plants to Thermal Insulation. Doctoral thesis. Helsinki:
University of Helsinki.
Pagnani, G., Pellegrini, M., Galieni, A., D’Egidio, S., Matteucci, F., Ricci, A.,
et al. (2018). Plant growth-promoting rhizobacteria (PGPR) in Cannabis sativa
‘Finola’cultivation: an alternative fertilization strategy to improve plant growth
and quality characteristics. Ind. Crops Products 123, 75–83. doi: 10.1016/j.
indcrop.2018.06.033
Paredes, S. H., Gao, T., Law, T. F., Finkel, O. M., Mucyn, T., Teixeira, P. J. P. L.,
et al. (2018). Design of synthetic bacterial communities for predictable
plant phenotypes. PLoS Biol. 16:e2003962. doi: 10.1371/journal.pbio.200
3962
Passera, A., Compant, S., Casati, P., Maturo, M. G., Battelli, G., Quaglino, F., et al.
(2019). Not just a pathogen? Description of a plant-beneficial Pseudomonas
syringae strain. Front. Microbiol. 10:1409. doi: 10.3389/fmicb.2019.01409
Paterson, J., Jahanshah, G., Li, Y., Wang, Q., Mehnaz, S., and Gross, H. (2017).
The contribution of genome mining strategies to the understanding of active
principles of PGPR strains. FEMS Microbiol. Ecol. 93:fiw249. doi: 10.1093/
femsec/fiw249
Patten, C. L., and Glick, B. R. (2002). Role of Pseudomonas putida indoleacetic
acid in development of the host plant root system. Appl. Environ. Microbiol.
68, 3795–3801. doi: 10.1128/AEM.68.8.3795-3801.2002
Pépin, N., Hebert, F. O., and Joly, D. L. (2021). Genome-wide characterization of
the MLO gene family in Cannabis sativa reveals two genes as strong candidates
Frontiers in Microbiology | www.frontiersin.org 19 January 2022 | Volume 12 | Article 833172

fmicb-12-833172 January 10, 2022 Time: 14:26 # 20
Balthazar et al. Beneficial Pseudomonas for Cannabis Production
for powdery mildew susceptibility. Front. Plant Sci. 12:729261. doi: 10.3389/fpls.
2021.729261
Perea, K. R. (2019). Determining the Influence of Cultivar and Proximity to
Plant Roots on the Resulting Cannabis Soil Microbiome using Microbial
Community Analysis. Master thesis. Socorro: New Mexico Institute of Mining
and Technology.
Petit, J., Salentijn, E. M., Paulo, M.-J., Thouminot, C., van Dinter, B. J., Magagnini,
G., et al. (2020). Genetic variability of morphological, flowering, and biomass
quality traits in hemp (Cannabis sativa L.). Front. Plant Sci. 11:102. doi: 10.3389/
fpls.2020.00102
Piccoli, P., and Bottini, R. (2013). “Abiotic stress tolerance induced by endophytic
PGPR,” in Symbiotic Endophytes, ed. R. Aroca (Berlin: Springer-Verlag), 151–
163.
Pieterse, C. M., Zamioudis, C., Berendsen, R. L., Weller, D. M., Van Wees, S. C., and
Bakker, P. A. (2014). Induced systemic resistance by beneficial microbes. Annu.
Rev. Phytopathol. 52, 347–375. doi: 10.1146/annurev-phyto-082712-102340
Pirttilä, A. M., Mohammad Parast Tabas, H., Baruah, N., and Koskimäki, J. J.
(2021). Biofertilizers and biocontrol agents for agriculture: how to identify
and develop new potent microbial strains and traits. Microorganisms 9:817.
doi: 10.3390/microorganisms9040817
Ponzoni, E., Brambilla, I., and Galasso, I. (2018). Genome-wide identification and
organization of seed storage protein genes of Cannabis sativa. Biol. Plant. 62,
693–702. doi: 10.1007/s10535-018- 0810-7
Punja, Z. K. (2021). Emerging diseases of Cannabis sativa and sustainable
management. Pest. Manag. Sci. 77, 3857–3870. doi: 10.1002/ps.6307
Punja, Z. K., and Holmes, J. E. (2020). Hermaphroditism in marijuana (Cannabis
sativa L.) inflorescences – impact on floral morphology, seed formation,
progeny sex ratios, and genetic variation. Front. Plant Sci. 11:718. doi: 10.3389/
fpls.2020.00718
Punja, Z. K., and Ni, L. (2021). The bud rot pathogens infecting cannabis (Cannabis
sativa L., marijuana) inflorescences: symptomology, species identification,
pathogenicity and biological control. Can. J. Plant Pathol. 43, 827–854. doi:
10.1080/07060661.2021.1936650
Qadri, M., Johri, S., Shah, B. A., Khajuria, A., Sidiq, T., Lattoo, S. K., et al. (2013).
Identification and bioactive potential of endophytic fungi isolated from selected
plants of the Western Himalayas. SpringerPlus 2:8. doi: 10.1186/2193-18
01-2-8
Rajabi-Khamseh, S., Danesh Shahraki, A., Rafieiolhossaini, M., and Saeidi, K.
(2020). Bacterial inoculation positively affects the quality and quantity of flax
under deficit irrigation regimes. J. Appl. Microbiol. 131, 321–338. doi: 10.1111/
jam.14934
Rathinasabapathi, B., Liu, X., Cao, Y., and Ma, L. Q. (2018). “Phosphate-
solubilizing Pseudomonads for improving crop plant nutrition and agricultural
productivity,” in New and Future Developments in Microbial Biotechnology and
Bioengineering: Crop Improvement Through Microbial Biotechnology, eds R.
Prasad, S. S. Gill, and N. Tuteja (Amsterdam: Elsevier B.V.), 363–372.
Ribeiro, A., Pochart, P., Day, A., Mennuni, S., Bono, P., Baret, J.-L., et al. (2015).
Microbial diversity observed during hemp retting. Appl. Microbiol. Biotechnol.
99, 4471–4484. doi: 10.1007/s00253-014- 6356-5
Russo, E. B. (2019). The case for the entourage effect and conventional breeding of
clinical cannabis: no “strain,” no gain. Front. Plant Sci. 9:1969. doi: 10.3389/fpls.
2018.01969
Salentijn, E. M., Zhang, Q., Amaducci, S., Yang, M., and Trindade, L. M. (2015).
New developments in fiber hemp (Cannabis sativa L.) breeding. Ind. Crops
Product. 68, 32–41. doi: 10.1016/j.indcrop.2014.08.011
Saloner, A., and Bernstein, N. (2021). Nitrogen supply affects cannabinoid and
terpenoid profile in medical cannabis (Cannabis sativa L.). Ind. Crops Product.
167:113516. doi: 10.1016/j.indcrop.2021.113516
Sandler, L., and Gibson, K. (2019). A call for weed research in industrial hemp
(Cannabis sativa L). Weed Res. 59, 255–259. doi: 10.1111/wre.12368
Sasse, J., Martinoia, E., and Northen, T. (2018). Feed your friends: do plant exudates
shape the root microbiome? Trends Plant Sci. 23, 25–41. doi: 10.1016/j.tplants.
2017.09.003
Schluttenhofer, C., and Yuan, L. (2017). Challenges towards revitalizing hemp: a
multifaceted crop. Trends Plant Sci. 22, 917–929. doi: 10.1016/j.tplants.2017.08.
004
Scott, C., and Punja, Z. K. (2021). Evaluation of disease management approaches
for powdery mildew on Cannabis sativa L.(marijuana) plants. Can. J. Plant
Pathol. 43, 394–412. doi: 10.1080/07060661.2020.1836026
Scott, M., Rani, M., Samsatly, J., Charron, J.-B., and Jabaji, S. (2018). Endophytes
of industrial hemp (Cannabis sativa L.) cultivars: identification of culturable
bacteria and fungi in leaves, petioles, and seeds. Can. J. Microbiol. 64, 664–680.
doi: 10.1139/cjm-2018- 0108
Selin, C., Habibian, R., Poritsanos, N., Athukorala, S. N., Fernando, D., and De
Kievit, T. R. (2009). Phenazines are not essential for Pseudomonas chlororaphis
PA23 biocontrol of Sclerotinia sclerotiorum, but do play a role in biofilm
formation. FEMS Microbiol. Ecol. 71, 73–83. doi: 10.1111/j.1574-6941.2009.
00792.x
Sezen, I., Kaymak, H. Ç, Aytatlı, B., Dönmez, M. F., and Erci¸sli, S. (2014).
Inoculations with plant growth promoting rhizobacteria (PGPR) stimulate
adventitious root formation on semi-hardwood stem cuttings of Ficus
benjamina L. Propagation Ornamental Plants 14, 152–157.
Shiponi, S., and Bernstein, N. (2021). The highs and lows of p supply in medical
cannabis: effects on cannabinoids, the ionome, and morpho-physiology. Front.
Plant Sci. 12:657323. doi: 10.3389/fpls.2021.657323
Singh, R., Masurkar, P., Pandey, S. K., and Kumar, S. (2019). “Rhizobacteria–
plant interaction, alleviation of abiotic stresses,” in Plant Growth Promoting
Rhizobacteria for Sustainable Stress Management - Volume 1: Rhizobacteria in
Abiotic Stress Management, eds R. Z. Sayyed, N. K. Arora, and M. S. Reddy
(Singapore: Springer), 345–353.
Sitaraman, R. (2015). Pseudomonas spp. as models for plant-microbe interactions.
Front. Plant Sci. 6:787. doi: 10.3389/fpls.2015.00787
Small, E. (2015). Evolution and classification of Cannabis sativa (marijuana, hemp)
in relation to human utilization. Bot. Rev. 81, 189–294. doi: 10.1007/s12229-
015-9157-3
Small, E. (2017). “"Classification of Cannabis sativa L in relation to agricultural,
biotechnological, medical and recreational utilization,” in Cannabis sativa L.-
Botany and Biotechnology, eds S. Chandra, H. Lata, and M. A. ElSohly (Cham:
Springer), 1–62.
Söderström, L. (2020). Plant-Growth Promoting Rhizobacteria in Soilless Cannabis
Cropping Systems: Implications for Growth Promotion and Disease Suppression.
Alnarp: Swedish University of Agricultural Sciences.
Stonehouse, G. C., McCarron, B. J., Guignardi, Z. S., El Mehdawi, A. F., Lima, L. W.,
Fakra, S. C., et al. (2020). Selenium metabolism in hemp (Cannabis sativa L.)—
Potential for phytoremediation and biofortification. Environ. Sci. Technol. 54,
4221–4230. doi: 10.1021/acs.est.9b07747
Suwanchaikasem, P., Walker, R., Idnurm, A., Selby-Pham, J., and Boughton,
B. A. (2021). Root-TRAPR: a modular plant growth device to visualize root
development and monitor growth parameters, as applied to an elicitor response
of Cannabis sativa.Res. Square [Epub ahead of print]. doi: 10.21203/rs.3.rs-
764290/v1
Tabacchioni, S., Passato, S., Ambrosino, P., Huang, L., Caldara, M., Cantale, C.,
et al. (2021). Identification of beneficial microbial consortia and bioactive
compounds with potential as plant biostimulants for a sustainable agriculture.
Microorganisms 9:426. doi: 10.3390/microorganisms9020426
Taghinasab, M., and Jabaji, S. (2020). Cannabis microbiome and the role of
endophytes in modulating the production of secondary metabolites: an
overview. Microorganisms 8:355. doi: 10.3390/microorganisms8030355
Teixeira, D. A., Alfenas, A. C., Mafia, R. G., Ferreira, E. M., Siqueira, L. D., Maffia,
L. A., et al. (2007). Rhizobacterial promotion of eucalypt rooting and growth.
Brazil. J. Microbiol. 38, 118–123. doi: 10.1590/s1517-83822007000100025
Thakur, M., Bhattacharya, S., Khosla, P. K., and Puri, S. (2019). Improving
production of plant secondary metabolites through biotic and abiotic
elicitation. J. Appl. Res. Med. Aromatic Plants 12, 1–12. doi: 10.1016/j.jarmap.
2018.11.004
Thiessen, L. D., Schappe, T., Cochran, S., Hicks, K., and Post, A. R. (2020).
Surveying for potential diseases and abiotic disorders of industrial hemp
(Cannabis sativa) production. Plant Health Prog. 21, 321–332. doi: 10.1094/
php-03-20-0017- rs
Thompson, G. R., Tuscano, J., Dennis, M., Singapuri, A., Libertini, S., Gaudino, R.,
et al. (2017). A microbiome assessment of medical marijuana. Clin. Microbiol.
Infect. 23, 269–270. doi: 10.1016/j.cmi.2016.12.001
Frontiers in Microbiology | www.frontiersin.org 20 January 2022 | Volume 12 | Article 833172

fmicb-12-833172 January 10, 2022 Time: 14:26 # 21
Balthazar et al. Beneficial Pseudomonas for Cannabis Production
Trivedi, P. C., and Malhotra, A. (2013). “Bacteria in the management of plant-
parasitic nematodes,” in Bacteria in Agrobiology: Disease Management, ed. D. K.
Maheshwari (Berlin: Springer-Verlag), 349–377.
Trognitz, F., Hackl, E., Widhalm, S., and Sessitsch, A. (2016). The role of plant–
microbiome interactions in weed establishment and control. FEMS Microbiol.
Ecol. 92:fiw138. doi: 10.1093/femsec/fiw138
van Elsas, J. D., McSpadden Gardener, B. B., Wolters, A. C., and Smit, E. (1998).
Isolation, characterization, and transfer of cryptic gene-mobilizing plasmids in
the wheat rhizosphere. Appl. Environ. Microbiol. 64, 880–889. doi: 10.1128/aem.
64.3.880-889.1998
Van Elsas, J. D., Turner, S., and Bailey, M. J. (2003). Horizontal gene transfer in the
phytosphere. New Phytol. 157, 525–537. doi: 10.1046/j.1469-8137.2003.00697.x
Vera, C., Malhi, S., Phelps, S., May, W., and Johnson, E. (2010). N, P, and S
fertilization effects on industrial hemp in Saskatchewan. Can. J. Plant Sci. 90,
179–184. doi: 10.4141/cjps09101
Vujanovic, V., Korber, D. R., Vujanovic, S., Vujanovic, J., and Jabaji,
S. (2020). Scientific prospects for cannabis-microbiome research
to ensure quality and safety of products. Microorganisms 8:290.
doi: 10.3390/microorganisms8020290
Wei, G., Ning, K., Zhang, G., Yu, H., Yang, S., Dai, F., et al. (2021).
Compartment niche shapes the assembly and network of Cannabis sativa-
associated microbiome. Front. Microbiol. 12:714993. doi: 10.3389/fmicb.2021.
714993
Willman, M., Keener, H. M., and Benitez, M.-S. (2021). Sequence resource of
bacterial communities associated with hemp in Ohio. Phytobiomes J. 5, 244–
247. doi: 10.1094/pbiomes-09- 20-0062-a
Winston, M. E., Hampton-Marcell, J., Zarraonaindia, I., Owens, S. M., Moreau,
C. S., Gilbert, J. A., et al. (2014). Understanding cultivar-specificity and soil
determinants of the Cannabis microbiome. PLoS One 9:e99641. doi: 10.1371/
journal.pone.0099641
Wu, Y., Trejo, H. X., Chen, G., and Li, S. (2021). Phytoremediation of contaminants
of emerging concern from soil with industrial hemp (Cannabis sativa L.):
a review. Environ. Dev. Sustain. 23, 14405–14435. doi: 10.1007/s10668-021-
01289-0
Xin, X.-F., Kvitko, B., and He, S. Y. (2018). Pseudomonas syringae: what it takes to
be a pathogen. Nat. Rev. Microbiol. 16, 316–328. doi: 10.1038/nrmicro.2018.17
Yadav, A. N., Kour, D., Sharma, S., Sachan, S. G., Singh, B., Chauhan, V. S., et al.
(2019). “Psychrotrophic microbes: biodiversity, mechanisms of adaptation, and
biotechnological implications in alleviation of cold stress in plants,” in Plant
Growth Promoting Rhizobacteria for Sustainable Stress Management - Volume 1:
Rhizobacteria in Abiotic Stress Management, eds R. Z. Sayyed, N. K. Arora, and
M. S. Reddy (Singapore: Springer), 219–253.
Zachow, C., Müller, H., Tilcher, R., Donat, C., and Berg, G. (2013). Catch
the best: novel screening strategy to select stress protecting agents
for crop plants. Agronomy 3, 794–815. doi: 10.3390/agronomy304
0794
Zboralski, A., Biessy, A., Savoie, M.-C., Novinscak, A., and Filion, M.
(2020). Metabolic and genomic traits of phytobeneficial phenazine-producing
Pseudomonas spp. are linked to rhizosphere colonization in Arabidopsis
thaliana and Solanum tuberosum.Appl. Environ. Microbiol. 86:e02443-19. doi:
10.1128/aem.02443-19
Zhou, J. Y., Sun, K., Chen, F., Yuan, J., Li, X., and Dai, C. C. (2018).
Endophytic Pseudomonas induces metabolic flux changes that enhance
medicinal sesquiterpenoid accumulation in Atractylodes lancea.Plant Physiol.
Biochem. 130, 473–481. doi: 10.1016/j.plaphy.2018.07.016
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Frontiers in Microbiology | www.frontiersin.org 21 January 2022 | Volume 12 | Article 833172