O R I G I N A L R E S E A R C H Open Access
Effects of cold plasma, gamma and e-beam
irradiations on reduction of fungal colony
forming unit levels in medical cannabis
, Marcel Maymon
, Aviv Dombrovsky
and Stanley Freeman
Background: The use of medical cannabis (MC) in the medical field has been expanding over the last decade, as
more therapeutic beneficial properties of MC are discovered, ranging from general analgesics to anti-inflammatory
and anti-bacterial treatments. Together with the intensified utilization of MC, concerns regarding the safety of
usage, especially in immunocompromised patients, have arisen. Similar to other plants, MC may be infected by
fungal plant pathogens (molds) that sporulate in the tissues while other fungal spores (nonpathogenic) may be
present at high concentrations in MC inflorescences, causing a health hazard when inhaled. Since MC is not grown
under sterile conditions, it is crucial to evaluate current available methods for reduction of molds in inflorescences
that will not damage the active compounds. Three different sterilization methods of inflorescences were examined
in this research; gamma irradiation, beta irradiation (e-beam) and cold plasma to determine their efficacy in
reduction of fungal colony forming units (CFUs) in vivo.
Methods: The examined methods were evaluated for decontamination of both uninoculated and artificially
inoculated Botrytis cinerea MC inflorescences, by assessing total yeast and mold (TYM) CFU levels per g plant tissue.
In addition, e-beam treatment was also tested on naturally infected commercial MC inflorescences.
Results: All tested methods significantly reduced TYM CFUs at the tested dosages. Gamma irradiation reduced CFU
levels by approximately 6- and 4.5-log fold, in uninoculated and artificially inoculated B. cinerea MC inflorescences,
respectively. The effective dosage for elimination of 50% (ED
)TYM CFU of uninoculated MC inflorescence treated
with e-beam was calculated as 3.6 KGy. In naturally infected commercial MC inflorescences, e-beam treatments
reduced TYM CFU levels by approximately 5-log-fold. A 10 min exposure to cold plasma treatment resulted in 5-
log-fold reduction in TYM CFU levels in both uninoculated and artificially inoculated B. cinerea MC inflorescences.
Conclusions: Although gamma irradiation was very effective in reducing TYM CFU levels, it is the most expensive
and complicated method for MC sterilization. Both e-beam and cold plasma treatments have greater potential since
they are cheaper and simpler to apply, and are equally effective for MC sterilization.
Keywords: Botrytis cinerea, CFU, Cold plasma, E-beam, Gamma irradiation, Medical Cannabis, Sterilization
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* Correspondence: email@example.com
Department of Plant Pathology and Weed Research, The Volcani Center,
Agriculture Research Organization, 7505101 Rishon Lezion, Israel
Full list of author information is available at the end of the article
Journal of Cannabi
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12
In recent years there is a growing trend of research re-
garding the beneficial effects of medical cannabis (MC)
for treating various diseases and ailments (Ben Amar
2006; Ruchlemer et al. 2015). The use of MC is growing
exponentially, especially in patients suffering from differ-
ent types of cancer and HIV, following FDA approval
(Ruchlemer et al. 2015), MC is also used widely as a gen-
eral analgesic. Since MC is used by patients with a weak-
ened immune system, there are potential risks to their
health when exposed to microbial-infected cannabis
(fungal spores, bacteria, etc.), as shown by a growing
number of reports (Cescon et al. 2008; Gargani et al.
2011; Hazekamp 2016; Ruchlemer et al. 2015). There-
fore, it is critical to supply MC-treated patients with a
“clean”, mold-free and healthy product.
In order to achieve a high level of quality control, many
countries, including Israel, the Netherlands, and the Euro-
pean pharmacopoeia have imposed strict regulations dictat-
ing the permitted number of microbial contaminations
present in commercial MC supplied to patients as, 2000,
100 and 50,000 colony forming units (CFUs) of total yeasts
and molds (TYM) per g inflorescence, respectively (https://
www.health.gov.il/hozer/mmk151_2016.pdf, EP 8.0, 5.1.8.C)
(Hazekamp 2016). These CFU limitations are very low and
to date, effective cultivation of MC under sterile conditions
does not exist. Therefore, the need for post-harvest decon-
tamination of inhaled MC products is essential. Even though
sterilization methods such as autoclaving or ultraviolet
(U.V.) irradiation may be the first to come to mind, the
most important therapeutic compounds in MC (cannabi-
noids and terpenes) are heat and light sensitive and undergo
decarboxylation, causing their early decay when exposed to
the above decontamination methods (Hazekamp 2016;
Russo 2011;Small2016). This highlights the necessity for
novel techniques to disinfect MC without exposing the
product to high temperatures or U.V. irradiation.
Both gamma and beta radiation (e-beam) fall under
the category of ionizing radiation, containing an amount
of energy that causes excitation or ionization of atoms
and molecules, leading to the creation of free radicals
(Jeong et al. 2015). These free radicals in turn sever cer-
tain chemical bonds that lead to damage of molecules
and especially cell DNA. Damage in these cases can be
either direct, caused by Reactive Oxygen Species (ROS)
created from the radiolysis of H
, or indirect, caused
by other free radicals (Sádecká 2007). In living organ-
isms, these damaged molecules cause a disruption in the
chemical and metabolic functions of living cells thus
leading to cell death (Hazekamp 2016; Jeong et al. 2015;
Sádecká 2007). The advantages of using gamma and beta
irradiation for MC decontamination are numerous. Ion-
izing radiation leaves no residues after application (un-
like fungicides for example) and does not involve
extreme heat or U.V. irradiation which may damage the
active compounds in MC (Hazekamp 2016). Studies re-
lated to the effects of gamma and beta irradiation on de-
contamination of the final product of MC is limited,
however, these treatments do not appear to have a detri-
mental effect on the quality of food and spice products
(Arvanitoyannis et al. 2009; Guerreiro et al. 2016; Jeong
et al. 2015; Sádecká 2007).
Gamma irradiation is commonly based on the use of
cobalt 60 isotope (
Co) which is reported as safe for de-
contamination of both MC and various food products
(Arvanitoyannis et al. 2009; Jeong et al. 2015; Sádecká
2007). Moreover, long term mammalian studies have
shown that irradiated foods are both safe and nutritious
for human consumption (Thayer et al. 1996). While
gamma irradiation is more commonly used, e-beam is a
newer method showing greater promise. This technique
does not require a radioactive source as the radiation is
created using an electron accelerator making it environ-
mentally friendly. Moreover, it was reported that a simi-
lar efficacy of decontamination was observed when
Botrytis cinerea (a major MC inflorescence fungal patho-
gen) was exposed to either gamma or beta irradiation
(McPartland et al. 2017). Furthermore, another fungal
pathogen Penicillium expansum, was more sensitive to
e-beam than gamma irradiation (Jeong et al. 2015).
While there was no direct mention of P. expansum as a
specific phytopathogen of MC, Penicillium spp. spores
are ubiquitous and common in dry MC products, sug-
gesting that this fungus may be a potential pathogen of
concern (McPartland et al. 2017; Punja et al. 2019).
While gamma and e-beam irradiation possess a similar
mode of action, cold plasma treatment is a different
method for sanitation and sterilization. The general defin-
ition of plasma is a state of ionized gas, with limited net
charge. Natural examples of plasma are the sun and the
aurora (Misra et al. 2019;Turner2016). Cold plasma is
usually achieved by deploying electrical discharges in gases
at atmospheric or subatmospheric pressure. When a high
enough voltage is reached a breakdown of the gas occurs,
leading to the formation of a mix of antimicrobial ele-
ments. The mechanisms that take place during this phase
of cold plasma reaction are numerous and include vibra-
tion and excitation of gas atoms, ion-ion neutralization,
quenching and many more (Misra et al. 2019;Sahuetal.
2017). Addition of H
to the plasma, augments the
sterilization mechanism; e.g. it was shown that at a high
concentration, ROS inhibit cell proliferation and cause
apoptosis (Thannickal and Fanburg 2000). Many reports
have reported the efficacy of cold plasma treatment in in-
activating a wide spectrum of bacteria (gram positive and
negative) and in many of these studies the method was
shown to be even more effective in the reduction of fungal
viability and spore CFU counts (Hertwig et al. 2015a,
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 2 of 12
2015b; Kim et al. 2014; Misra et al. 2019;Zahoranová
et al. 2016). In recent years cold plasma sterilization has
become more popular in various medical applications
such as surface sterilization and sterilization of damaged
tissues (Heinlin et al. 2010;Kolbetal.2008; Xinpei et al.
2009). The direct mechanism of inactivation of fungi using
cold plasma is still not entirely clear. Scanning electron
microscopy examination of plasma post-treated Cordyceps
bassiana spores revealed dried, cracked and flattened
propagules, indicating that cold plasma treatment may
cause cell wall leakage and destruction, resulting in re-
duced cell viability (Lee et al. 2015). Similar results were
achieved with cold plasma treatments of Aspergillus spp.
(Dasan et al. 2017), which is of paramount importance
since Aspergillus spp. are very common in MC floral parts,
and can cause serious health complications in immuno-
compromised patients when mycotoxin-contaminated
products are inhaled in large quantities (Gargani et al.
2011; Hamadeh et al. 1988; Ruchlemer et al. 2015). Even
more intriguing is the reported ability of cold plasma
treatment to reduce the presence of these toxins as well as
pesticide residues (Misra et al. 2015; Sarangapani et al.
2016). While gamma, e-beam irradiation and cold plasma
treatments appear promising for MC sterilization, there is
a lack of evidence and knowledge regarding the efficacy of
each of these methods, specifically in the treatment of har-
vested MC inflorescences, and their effect on the desired
active chemical compounds.
In this research, we examined the efficacy of three
sterilization methods: (i) gamma irradiation, (ii) beta ir-
radiation (e-beam), and (iii) cold plasma sterilization, for
reduction and elimination of fungal colony forming units
(CFUs) in uninoculated and artificially inoculated B.
cinerea inflorescences, naturally infected commercial
trimmed floral parts and inflorescences.
Plant and fungal samples
Cannabis sativa cultivar (BB 734) was grown in the Agri-
culture Research Organization (ARO) Volcani Center re-
search facility (authorized by the Israeli Medical Cannabis
Agency, IMCA, Ministry of Health, State of Israel) for this
research. Uncharacterized cannabis seedlings were kindly
provided by Dr. Moshe Flaishman, ARO that established
the genetic source. Plants were propagated and five male
flowers were used for pollination of 30 female flower
plants. Seeds were collected from the harvested female
flowers and sown for continuous breeding and selection
for a range of parameters. The strain that was used in this
study (BB 734) was derived from shoots of third gener-
ation mother plants. This strain is a “drug-type”cannabis
with Cannabis indica characteristics.
Shoots were rooted under continuous 24 h light photo-
periodic conditions of 880 LUX, for 1 week in a closed
plastic planting container (80 × 40 × 50 cm) in a humid en-
vironment, and an additional week without the top cover.
The rooted shoots were replanted in 0.2 L pots and trans-
ferred for vegetative propagation under photoperiodic
conditions of 18 h light and 6 h dark of 3000 LUX, for 2
months. Plants were retransferred into 0.5 to 2 L pots and
placed in a flowering induction chamber 4 × 3 m, for 80–
90 days. The flowering chamber contained six 600 W high
pressure sodium lamps (SunMaster, Twinsburg, Ohio,
USA) with dual red and blue spectrum light, under photo-
periodic conditions of 11 h light (50,000 LUX) and 13 h
dark, until flowers were produced. Mature inflorescences,
that were produced 80–90 days after floral induction, were
used for sterilization experiments.
Two types of plant parts were used: (i) uninoculated (that
included asymptomatic natural infections) mature inflores-
cences, (ii) artificially inoculated mature inflorescences with
acultureofBotrytis cinerea originating from naturally in-
fected cannabis flowers, isolated and characterized by mor-
phological and molecular methods. It should be noted that
“uninoculated”inflorescences from the ARO facility con-
tained asymptomatic microbial infections comprised of a
wide variety of different fungal species. B. cinerae was cul-
tured for 2 weeks at 22 °C on 9 cm Petri plates containing
potato dextrose agar (Difco, Franklin Lakes, New Jersey,
USA) supplemented with 0.25 g/l chloramphenicol (PDAC)
(Acros Organics, Geel, Belgium). After 14 days, spores were
harvested from the plates with a sterile rod by adding a sus-
pension of 10 ml sterile saline solution (NaCl 0.85 g/l,
Tween 20, 100 μl/l). The conidia were filtered through four
layers of sterile gauze and centrifuged (Heraeus, Franklin
Lakes, New Jersey, USA) at 9000 RPM for 10 min at 4 °C.
The pellet was resuspended in 20 ml fresh saline solution
and adjusted to a concentration of 10
spores/ml. The in-
oculum was sprayed till run-offonhealthymaturecannabis
plants that were subsequently covered by a plastic bag.
After 5 days, the bags were removed and harvested flowers
were dried in an Excalibur 3548/3948 digital oven (Sacra-
mento, California, USA) at 35 °C for 12 h, then stored in a
STATUS innovations vacuum pack (Metlika, Slovenia) at
room temperature before experimentation. CFU’softhe
microbial cultures from affected floral parts, before and
after each sterilization treatment, were determined (see
Quantification of fungal colonies
One g of each floral sample was inserted into a 10 ml
sterile saline solution in 50 ml Falcon tubes, vortexed for
30 s and kept at room temperature for 10 min. There-
after, serial dilutions were conducted and spread on
PDAC plates that were maintained at room temperature
(22°-25 °C) for 3–5 days, and developing CFU’s of total
yeasts and mold (TYM) species were enumerated and
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 3 of 12
Survey of CFU levels from commercial MC farms
In order to evaluate common CFU levels under commer-
cial conditions, samples of MC inflorescences were col-
lected from 4 different farms located in Israel. CFU levels
were evaluated as described. Plating of each sample was
conducted 3 times to achieve higher reproducibility. Vari-
ability in sampled inflorescences existed, as certain sam-
ples exhibited disease symptoms while others remained
asymptomatic, and certain inflorescences were dry.
Gamma and beta irradiation
Commercial cannabis samples were received from a num-
ber of commercial farms in Israel for irradiation treatments.
Treatments comprising of e-beam (beta irradiation) and
gamma irradiation were conducted at Sorvan Radiation
Ltd., Soreq Nuclear Research Center, Israel. Gamma radi-
ation was based on a
Co isotope, doubly encapsulated in
stainless steel source pencils type C-188, with radiation dos-
ages of 7.5 and 8.37 KGy (KiloGray) in two consecutive ex-
periments, respectively. E-beam radiation was created using
an electron accelerator, with 15 kW (KWs) and an energy
capacity of 5.25 megaelectronvolt (MeV). The radiation
dosages were 4.18, 8.2 and 10.26 KGy, and 4.06, 8.5, and
10.26 KGy in two consecutive experiments, respectively.
Cold plasma irradiation
Cold plasma treatment was conducted using a prototype
created by NovaGreen company, (Kibbutz Megiddo, Israel).
The gas in this treatment was low pressure air with the
addition of H
liquid at a concentration of 35% (Chen
Shmuel Chemicals Ltd., Haifa, Israel). A vacuum chamber
was generated using an Edwards i10 dry pump to eliminate
possible oil contamination that may have occurred during
wet pump usage. Although the H
liquid had no direct
contact with the MC, it affects the gaseous environment
and generates a highly reactive plasma with elevated con-
centrations of oxygen species. An RF generator at a voltage
of 6 kV generated the plasma and exposure periods lasted
for 2.5, 5.0 and 10 min for each experiment.
Sampling procedures and experimental design
Two sample types [uninoculated (that included asymp-
tomatic infections) and artificially inoculated Botrytis
cinerea] of noncommercial plant material were ob-
tained from the ARO Volcani Center facilities and di-
vided into bags containing 5 g MC floral parts each
(total of 20 g per sample type). Artificially inoculated
Botrytis cinerea and uninoculated samples were treated
with beta and gamma radiation at Sorvan facility. A 5 g
non-irradiated sample of each floral MC type served as
a control. After irradiation treatments, four and three
biological repeats (from two consecutive experiments,
respectively) were removed from each bag and CFU’s
were determined, as described.
Irradiation treatments of naturally infected commercial
plant material including (i) dried and packed floral parts,
and (ii) dried and packed trimmed leaves were assessed
for efficacy of treatments by determining CFU counts.
Inflorescences were naturally infected indicating that
CFUs from these inflorescences were comprised of a
wide variety of fungal species. Each product contained
two 500 g vacuum-sealed bags that were treated with e-
beam irradiation at Sorvan nuclear facility. A 5 g sample
that was removed from each bag before the irradiation
treatments served as a control. To determine efficacy of
e-beam irradiation treatments at different locations in
the bag, six samples of 5 g each were removed after
treatments from different locations of each bag: from the
upper right corner, upper left corner, lower right corner,
lower left corner, upper middle area and lower middle
area, and CFU’s were determined as described.
Cold plasma treatment was conducted on noncom-
mercial floral material. The experimental design was
identical to that described for the noncommercial irradi-
ation experiments. Floral parts were placed on the elec-
trode and H
was injected around the perimeter. Each
treatment comprised of 8 min of vacuum and different
plasma exposure periods described. An untreated sample
served as control.
In order to determine the effective radiation dosage for
eliminating 50% (ED
) CFUs, a response curve with
> 0.95 was produced for each treatment. CFU levels
in the controls of each treatment were calculated as the
100%. This value divided by two was used as the Y value
in the response curve formula of each treatment, and
served as the radiation dosage required for reducing
CFU levels by 50% (ED
). All other ED values were cal-
culated using the same method.
CFU survey of inflorescences from commercial farms
The initial CFU survey that was conducted on 21 sam-
ples indicated that in all four tested commercial farms
levels of TYM fungal contamination exceeded the max-
imum CFU values of 2000 yeasts and molds per g inflor-
escence permitted by the IMCA (Fig. 1); some samples
exceeded this level by as much as 3.08 log-fold CFU/g
inflorescences. Morphological identification of the CFUs
indicated an abundance of the following fungal species;
Alternaria spp., Aspergillus spp., Botrytis cinerea,Fusar-
ium spp., and Penicillium spp.
Gamma irradiation of noncommercial MC inflorescences
Gamma irradiation treatments caused a considerable re-
duction in TYM CFU levels in noncommercial MC
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 4 of 12
inflorescences. CFU levels were reduced in the uninocu-
lated MC inflorescence samples from 6.16± 0.2 and from
6.04 ± 0.08 to 0 log CFU/g inflorescence in the first (7.5
KGy irradiation dosage) and second (8.37 KGy) experi-
In artificially inoculated Botrytis cinerea MC inflores-
cences, gamma irradiation treatments resulted in a re-
duction of CFU levels from 8.05 ± 0.12 and 7.7 ± 0.11 to
1.88 ± 0.96 and 3.02 ± 0.11 log CFU/g inflorescence, in
consequent experiments respectively, a respective reduc-
tion of 6- and 4.5 log-fold. Peak temperatures measured
during irradiation treatments reached 32.5 and 30.0 °C
in the first and second experiments, respectively.
E-beam (beta irradiation)
E-beam treatments of noncommercial material E-
beam treatments at 10.26 KGy of noncommercial MC
floral parts were very effective in completely eliminating
contamination of uninoculated inflorescences from
6.16 ± 0.26 and 6.04 ± 0.08 to 0 log CFU/g inflorescence
in two consequent experiments, respectively (Fig. 2).
A similar pattern was found when noncommercial, arti-
ficially inoculated Botrytis cinerea MC inflorescences were
treated under the same conditions, (Figs. 3and 4a). E-
beam irradiation reduced CFU levels in two consecutive
experiments to 0 and 1.75 ± 0.5 log CFU/g inflorescence,
respectively (Fig. 3). In both experiments, peak tempera-
tures during radiation were less than 27 °C.
The effective dosages calculated for reduction of per-
cent population of CFUs for e-beam treatments in artifi-
cially inoculated Botrytis cinerea and uninoculated MC
inflorescences are shown in Table 1.
While the ED values in the uninoculated MC inflores-
cences were considerably lower than those for the artifi-
cially inoculated Botrytis cinerea inflorescences, it is
worth mentioning that in the inoculated inflorescences
(Fig. 3) initial CFU levels were 100-fold higher than
those of the uninoculated inflorescences (Fig. 2).
E-beam treatments of commercial material Beta-ir-
radiation of commercial material significantly reduced
and eliminated CFUs in MC inflorescences at all loca-
tions in the vacuum-sealed packages. In the first experi-
ment, e-beam reduced CFU levels from 4.9±0.25 to 0 log
CFU/g inflorescence, compared to the untreated control.
(Fig. 5). Likewise, in the second experiment, CFUs were
reduced to undetected levels at all sampled locations in
the package (Fig. 5).
The results of irradiation of commercial MC trimmed
leaves were more varied (Fig. 6). In the first experiment,
no viable CFUs were detected from three of the sam-
pled locations, although CFU levels were significantly
reduced in three of the other locations, compared to
the untreated controls (Fig. 6). In the second experi-
ment, even though initial CFU levels were higher, no
CFUs were detected from four sampled locations after
the treatment (Fig. 6).
Fig. 1 CFU levels [log10(CFU/g inflorescence)] detected from four farms designated A, B, C and D. Consecutive numbers after each individual
farm indicate different samples of inflorescences taken from that farm. Samples D1 and D2 represent dried inflorescences. Asterisks indicate MC
inflorescence that were asymptomatic, all other samples exhibited varying degrees of disease symptoms. Bars represent SE of the mean of 3
replicates per sample. The gray line represents maximum levels of total yeasts and molds permitted according to protocols of the IMCA for
commercial MC inflorescences (2000 CFU/g)
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 5 of 12
Cold plasma treatments of noncommercial material
Cold plasma treatments resulted in a reduction in CFU
levels of uninoculated inflorescences, according to ex-
posure times (Fig. 7). After 2.5 min of plasma exposure,
CFU levels were reduced from 3.01 ± 0.08 and 2.81 ±
0.99 log CFU/g inflorescence to 0 and 0.79 ± 0.39 log
CFU/g inflorescence, in the first and second experi-
ments, respectively (Fig. 7). However, after 5 min expos-
ure the detected CFU levels were 1.83 ± 0.47 and 0.92 ±
0.46 log CFU/g inflorescence, and after 10 min of expos-
ure, CFU levels were reduced to 0 and 0.25 ± 0.25 log
CFU/g inflorescence, in the respective consecutive ex-
periments, a reduction of approximately 3-log-fold in
detected CFU levels. In an experiment performed with
heavily infected uninoculated inflorescences, a reduction
of approximately 6 logs was recorded, to below 10 CFU/
g inflorescence after 12 min of plasma exposure (data
A similar pattern was observed in artificially inoculated
Botrytis cinerea MC inflorescences treated with cold
plasma (Figs. 4b and 8). After 2.5 min of plasma expos-
ure, CFU levels were reduced by approximately 3-log-
fold, by 2-log-fold after 5 min exposure, and 4-log-fold
after 10 min exposure (Fig. 8).
The use of medical cannabis (MC) has increased tre-
mendously in the last decade (Ruchlemer et al. 2015).
Fig. 2 CFU levels [log10(CFU/g inflorescence)] of uninoculated MC inflorescences, exposed to different e-beam irradiation dosages in two
experiments. Bars represent SE of the mean of 9 replicates per sample. A value of 3.55 KGy was calculated, according to the polynominal formula
(dotted line), to reduce CFUs by 50% (ED
), represented by the dashed line
Fig. 3 CFU levels, [log10(CFU/g inflorescence)] of artificially inoculated Botrytis cinerea MC inflorecences, exposed to different e-beam irradiation
dosages in two experiments. Bars represent SE of the mean of 9 replicates per sample. A value of 5.18 KGy was calculated, according to the
polynominal formula (dotted line) to reduce CFUs by 50% (ED
), represented by the dashed line
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 6 of 12
One of the main reasons for the rising popularity and
interest in MC are the therapeutic qualities of this plant
(Ben Amar 2006; Cascio et al. 2017; Russo 2011; Sirikan-
taramas and Taura 2017). With the increase in use, both
for recreational and therapeutic means, reports are start-
ing to accumulate concerning the threat of microbial
presence in MC inflorescences and the harmful potential
to cannabis consumers, especially in immunocomprom-
ised patients (Gargani et al. 2011; Hamadeh et al. 1988;
McPartland and McKernan 2017; Ruchlemer et al.
2015). The extent of MC inflorescence infections by fun-
gal CFUs was determined in our initial survey of com-
mercial farms, indicating that without sterilization
treatments, levels of CFUs were extremely high, above
that permitted by the IMCA in all tested sites, disregard-
ing the sample condition; dried or un-dried, and with or
without visible disease symptoms (Fig. 1). Since it is not
feasible to cultivate commercial MC under a sterile en-
vironment there is an acute need for postharvest MC in-
florescence sterilization before usage (Hazekamp 2016).
There have been many reports regarding the efficacy of
different non-thermal treatments for food and herb
sterilization, especially the utilization of gamma irradiation
(Guerreiro et al. 2016; Jeong et al. 2015; Sádecká 2007). Al-
ternatively, e-beam (beta irradiation) and cold plasma, are
effective for food sterilization and are safe for human con-
sumption (Jeong et al. 2015; Misra et al. 2019;Moreno
et al. 2007; Van Impe et al. 2018). Even though these
methods have been applied or suggested for decontaminat-
ing MC inflorescences to safeguard its use by immunocom-
promised patients, there is a lack of knowledge in regards
to their efficacy in eliminating deleterious microorganisms.
In this research, we examined the effect of gamma ir-
radiation, e-beam and cold plasma treatments, on the re-
duction of CFU contamination in artificially inoculated
and naturally infected MC inflorescences and trimmed
leaves (Table 2). Gamma irradiation was very effective in
reducing the CFU levels by approximately 6-log-fold
CFU/g inflorescences, at a minimal dosage of 7.5 KGy.
Similarly, in the Netherlands, MC is sterilized using
gamma irradiation at a dosage of 10 KGy, which is well
below the authorized dosage of 30 KGy, permitted by the
FDA for irradiation of aromatic herbs and spices (Sádecká
2007). Likewise, in a recent report,
Co gamma irradi-
ation was used to sterilize cherry tomatoes with a radi-
ation dosage of 5.7 KGy that reduced CFU levels from 2.2
log CFU/g to nearly zero, an inactivation efficacy of 99.8%
(Guerreiro et al. 2016). In spite of its effectiveness, gamma
irradiation remains an expensive sterilization method re-
quiring the usage of radioactive isotopes, specialized
equipment and facilities. In contrast, e-beam does not
require the use of radioactive isotopes and as such, is con-
siderably more environmentally friendly (Leonhardt 1990).
Moreover, in this research we found that e-beam treat-
ments were very effective in eliminating CFUs from
infected MC inflorescences applied at low temperatures,
below 27 °C, that do not detrimentally affect the active in-
gredients of MC. In fact, this method is so effective that at
a radiation dosage of 10.26 KGy, CFU levels were reduced
from 6 log CFU/g inflorescence to 0 (Fig. 2). A similar
Fig. 4 Fungal CFUs from artificially inoculated Botrytis cinerea inflorescences after: ae-beam irradiation (4, 8, 10 KGy and untreated control), and b
cold plasma (2.5, 5, 10 min exposure and untreated control) treatments
Table 1 Effective dosage of e-beam treatments for reducing
percent CFU populations in both infected and artificially
inoculated Botrytis cinerea MC inflorescences
Effective dosage (ED)(%) Irradiation dosage
(KGy) of uninoculated
(KGy) of B. cinerea
90 8.1 12.4
70 5.5 8
50 3.6 5.2
10 0.6 1
Calculated using the polynomial formula in Fig. 2
Calculated using the polynomial formula in Fig. 3
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 7 of 12
reduction in CFUs was observed with artificially inocu-
lated inflorescences (Fig. 3).
ED values are an important and useful tool as they in-
dicate the efficacy of treatments and also allow for com-
parison between different sterilization systems; in
general lower ED values represent a higher acute toxicity
(Lorke 1983). ED values are extremely relevant in this
study since they indicate a measurable value for the effi-
cacy of the treatments. The ED values calculated for e-
beam irradiation (Table 1) were also very promising; e.g.
, the radiation dosage required to reduce 10% CFU
populations of uninoculated and artificially inoculated
inflorescences were calculated as 0.6 KGy and 1 KGy, re-
spectively. In comparison, the ED
value for inactivation
of enteric viruses e.g. poliovirus Type 1 on cantaloupe
surfaces using e-beam was 4.76 KGy (Shurong et al.
2006). Similarly, e-beam treatment of red pepper powder
at a dosage of 3 KGy, reduced CFU levels of total yeasts
Fig. 5 CFU levels [log10(CFU/g inflorescence)] in naturally infected commercial MC inflorescence before (control) and after e-beam treatments in
two experiments. Post-treatment samples were taken from different locations of vacuum-sealed packages. Bars represent SE of 3 replicates per
sample per location
Fig. 6 CFU levels, [log10(CFU/g inflorescence)] in naturally infected commercial MC trimmed leaves before (control) and after e-beam treatments
in two experiments. Post-treatment samples were taken from different locations of vacuum-sealed packages. Bars represent SE of 3 replicates per
sample per location
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 8 of 12
and molds from 6.62 to 3.71 log CFU/g (Kyung et al.
2019). In our research, we found that a dosage of 12.4
KGy resulted in 90% reduction in CFU levels in artifi-
cially inoculated B. cinerea MC inflorescence (Table 1).
Although 12.4 KGy is considered a rather high radiation
dosage, it is important to note that in this experiment
the initial CFU levels were very high (more than 7.7 ±
0.11 log CFU/g). Accordingly, initial CFU levels in com-
mercial MC were constantly much lower, measuring
below 5 ± 0.25 CFU/g. This indicates that irradiation
values of 8.1 KGy, resulting in a reduction of 90% of un-
inoculated MC inflorescences are very reasonable con-
sidering that the irradiation dosages are much lower
than the maximum limit of 30 KGy, according to that
authorized by the FDA for irradiation of dry herbs
Cold plasma treatment of MC inflorescences was also
found to be effective for elimination of fungal propagules
in this study. After 10 min of plasma exposure, CFU
levels in uninoculated MC inflorescences were reduced
by approximately 3 log-fold CFU/g. It is important to
note that initial recorded infection levels in these experi-
ments were approximately 3 log CFU/g indicating that
given a higher infection level, an exposure to 10 min
plasma treatment may have resulted in an even greater
CFU reduction (Fig. 7). In an additional experiment
Fig. 7 CFU levels [log10(CFU/g inflorescence)] of uninoculated MC inflorescences exposed to different cold plasma treatments. Bars represent SE
of the mean of 9 replicates per sample
Fig. 8 CFU levels, log10(CFU/g inflorescence), of Botrytis cinerea-inoculated MC inflorescences exposed to different cold plasma tretments. Bars
represent SE of the mean of 9 replicates per sample
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 9 of 12
(data not shown) a cold plasma treatment of 8 min vac-
uum time followed by 12 min of plasma exposure re-
sulted in CFU reduction of approximately 6 log CFU/g
of uninoculated MC inflorescences. Accordingly, in arti-
ficially inoculated B. cinerea MC inflorescences the re-
sults were improved with a reduction of approximately 4
log CFU/g, after 10 min of plasma exposure (Fig. 8).
Similarly, cold plasma treatments conducted in Shaare
Zedek Medical Center Israel, resulted in complete
sterilization of MC inflorescences (Ruchlemer et al.
2015). In that research, autoclaving and ethylene gas
sterilization were found to be as effective for MC inflor-
escence sterilization, although the two latter methods re-
sulted in a greater reduction of Δ
-THC, which is one of
Table 2 Effect of radiation and sterilization methods on fungal contamination of cannabis plant samples
Sample type Average CFU before
treatment [log 10 CFU/g
Average CFU after
treatment [log 10 CFU/g
Gamma irradiation 1 Uninoculated
6.16 ± 0.26 7.5 –0
8.05 ± 0.12 7.5 –1.88 ± 0.96
6.04 ± 0.08 8.37 –0
7.7 ± 0.11 8.37 –3.02 ± 0.11
E-beam 1 Uninoculated
6.16 ± 0.26 10.26 –0
8.05 ± 0.12 10.26 –0
6.04 ± 0.08 10.26 –0
7.7 ± 0.11 10.26 –1.75 ± 0.5
1 Naturally infected
5 ± 0.25 11.99 –0.211 ± 0.1
2 Naturally infected
2.2 ± 0.47 11.99 –0
1 Naturally infected
4.3 ± 0.03 11.99 –0.314 ± 0.095
2 Naturally infected
5.27 ± 0.05 11.99 –0.6 ± 0.48
Cold plasma 1 Uninoculated
3 ± 0.08 –10 0
2.81 ± 0.91 –10 0.25 ± 0.25
7.82 ± 0.04 –10 3.84 ± 0.08
Cold plasma with
6.6 ± 0.35 –12.5 min + 10
0.25 ± 0.25
6.88 ± 0.11 –10 min + 8
4.05 ± 0.31
Abbreviations:CFU colony forming units, kGy kiloGray
in the cold plasma treatment with changing vacuum time, only one experiment was conducted
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 10 of 12
the major phytocannabinoids in MC. Cold plasma was
also found useful in decontamination of wheat seeds,
resulting in complete inactivation of Fusarium nivale-ar-
tificially inoculated seeds after as little as 90 s treatment
(Zahoranová et al. 2016). An increase in CFU counts
after 5 min of plasma exposure compared to that after
2.5 min was recorded in our experiments, which is still
unclear (Figs. 7and 8) and will require further research.
In various studies, cold plasma was reported to degrade
both mycotoxins and pesticides in in vitro experiments
(Ten Bosch et al. 2017; Misra et al. 2011; Sarangapani
et al. 2016). Mycotoxins such as aflatoxins, zearalenone
and fumonisins are secondary metabolites produced by
certain fungi such as Aspergillus and Fusarium spp. that
are ubiquitous in MC inflorescences and are known to
cause health risks to humans and mammals (McPartland
et al. 2017; Misra et al. 2019). Thus, cold plasma treat-
ments may be even more beneficial by not only reducing
CFU counts but also by reducing levels of mycotoxins
(Ten Bosch et al. 2017; Misra et al. 2019).
Another important aspect when dealing with MC are
the active compounds, comprised mainly of different
phytocannabinoids and terpenoids. These compounds,
especially phytocannabinoids, are responsible for MC
therapeutic effects and currently more than 100 different
phytocannabinoids have been identified (Cascio et al.
2017; Russo 2011). In recent years, there is a growing
understanding that some of the MC therapeutic affects
are a result of synergism among the bioactive com-
pounds in certain MC lines and cultivars (Russo 2011).
Thus, it is crucial to decontaminate MC inflorescences
using a method that causes the least damage to the pro-
files of these active compounds.
In this research, we tested 3 different methods for MC
inflorescence sterilization, all three proving to be effect-
ive (Table 2). Gamma irradiation was very effective in re-
ducing total yeast and mold (TYM) CFU levels but is
not environmentally friendly and requires a nuclear facil-
ity. On the other hand, e-beam (beta) irradiation does
not require the use of radioactive isotopes and is much
faster and easier to apply, possessing high efficacy in re-
ducing TYM CFUs, achieving maximum CFU reduction
of approximately 8-log-fold (Table 2). Cold plasma was
also effective in reducing TYM CFU levels, reaching
maximum CFU reduction of approximately 6-log-fold
(Table 2). Assessing fungicide and mycotoxin degrad-
ation effects of cold plasma and e-beam in MC inflores-
cences in vivo, and also the effect of both e-beam and
cold plasma treatments on active compounds in MC,
will require further, extensive research. However, both
of these methods appear to possess the potential in pro-
ducing clean, safe and healthy MC products.
Co: 60 cobalt isotope; ARO: Agriculture Research Organization; CFU: Colony
forming unit; E-BEAM: Electron beam; ED: Effective dosage; IMCA: Israeli
Medical Cannabis Agency; KGy: KiloGray; KWs: Kilowatts; MC: Medical
Cannabis; MeV: Megaelectronvolt; PDAC: Potato dextrose agar and
chloramphenicol; ROS: Reactive oxygen species; TYM: Total yeast and mold
The authors thank M. Borenstein for technical and greenhouse assistance.
The authors are indebted to Dr. Moshe Flaishman from ARO for kindly
providing us with cannabis seedlings and to the various cannabis farms for
providing commercial plant material for the experiments. We thank Sorvan
Radiation Ltd., Soreq Nuclear Research Center, Yavne, Israel, and Novagreen
company, Kibbutz Megiddo, Israel, for cooperation in performing the
SJ conducted the research, MM assisted with technical and lab experiments,
AD raised partial funding and SF raised partial funding, conceived and
supervised the project. The author(s) read and approved the final
The authors thank the Chief Scientist of the Israeli Ministry of Agriculture,
grant numbers 20-02-0070 and 20-02-0099, for funding this research.
Availability of data and materials
All data generated or analyzed during this study are included in this
The authors declare that they have no competing interests.
Department of Plant Pathology and Weed Research, The Volcani Center,
Agriculture Research Organization, 7505101 Rishon Lezion, Israel.
H. Smith Faculty of Agriculture, Food and Environment, The Hebrew
University of Jerusalem, 7610001 Rehovot, Israel.
Received: 9 September 2019 Accepted: 18 February 2020
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