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Effects of cold plasma, gamma and e-beam irradiations on reduction of fungal colony forming unit levels in medical cannabis inflorescences

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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% (ED50)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.
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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
inflorescences
Shachar Jerushalmi
1,2
, Marcel Maymon
1
, Aviv Dombrovsky
1
and Stanley Freeman
1*
Abstract
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
50
)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: freeman@volcani.agri.gov.il
1
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
s
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esea
r
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h
Jerushalmi et al. Journal of Cannabis Research (2020) 2:12
https://doi.org/10.1186/s42238-020-00020-6
Background
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
2
O
2
, 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 (
60
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
2
O
2
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.
Methods
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-typecannabis
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 8090 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
uninoculatedinflorescences 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
6
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. CFUsofthe
microbial cultures from affected floral parts, before and
after each sterilization treatment, were determined (see
below).
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 35 days, and developing CFUs of total
yeasts and mold (TYM) species were enumerated and
characterized.
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
60
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
2
O
2
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
2
O
2
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 CFUs
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 CFUs 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
2
O
2
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.
ED
50
calculation
In order to determine the effective radiation dosage for
eliminating 50% (ED
50
) CFUs, a response curve with
R
2
> 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
50
). All other ED values were cal-
culated using the same method.
Results
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.
Irradiation treatments
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-
ments, respectively.
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
not shown).
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).
Discussion
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
50
), 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
50
), 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
60
Co
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 (Sádec
2007). Likewise, in a recent report,
60
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
inflorescences
a
Irradiation dosage
(KGy) of B. cinerea
inoculated
inflorescences
b
90 8.1 12.4
70 5.5 8
50 3.6 5.2
10 0.6 1
a
Calculated using the polynomial formula in Fig. 2
b
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.
ED
10
, 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
10
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
(Sádecká 2007).
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
9
-THC, which is one of
Table 2 Effect of radiation and sterilization methods on fungal contamination of cannabis plant samples
Treatment Experiment
number
Sample type Average CFU before
treatment [log 10 CFU/g
inflorescence]
Radiation
dose
[kGy]
Plasma
exposure
time [min]
Average CFU after
treatment [log 10 CFU/g
inflorescence]
Gamma irradiation 1 Uninoculated
inflorescence
6.16 ± 0.26 7.5 0
1Botrytis cinerea-
inoculated
inflorescence
8.05 ± 0.12 7.5 1.88 ± 0.96
2 Uninoculated
inflorescence
6.04 ± 0.08 8.37 0
2Botrytis cinerea-
inoculated
inflorescence
7.7 ± 0.11 8.37 3.02 ± 0.11
E-beam 1 Uninoculated
inflorescence
6.16 ± 0.26 10.26 0
1Botrytis cinerea-
inoculated
inflorescence
8.05 ± 0.12 10.26 0
2 Uninoculated
inflorescence
6.04 ± 0.08 10.26 0
2Botrytis cinerea-
inoculated
inflorescence
7.7 ± 0.11 10.26 1.75 ± 0.5
1 Naturally infected
commercial
inflorescence
5 ± 0.25 11.99 0.211 ± 0.1
2 Naturally infected
commercial
inflorescence
2.2 ± 0.47 11.99 0
1 Naturally infected
commercial trimmed
leaves
4.3 ± 0.03 11.99 0.314 ± 0.095
2 Naturally infected
commercial trimmed
leaves
5.27 ± 0.05 11.99 0.6 ± 0.48
Cold plasma 1 Uninoculated
inflorescence
3 ± 0.08 10 0
2 Uninoculated
inflorescence
2.81 ± 0.91 10 0.25 ± 0.25
2Botrytis cinerea-
inoculated
inflorescence
7.82 ± 0.04 10 3.84 ± 0.08
Cold plasma with
changing vacuum
time
a
1 Uninoculated
inflorescence
6.6 ± 0.35 12.5 min + 10
min vacuum
0.25 ± 0.25
1Botrytis cinerea-
inoculated
inflorescence
6.88 ± 0.11 10 min + 8
min vacuum
4.05 ± 0.31
Abbreviations:CFU colony forming units, kGy kiloGray
a
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.
Conclusions
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.
Abbreviations
60
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
Acknowledgements
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
sterilization experiments.
Authorscontributions
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
manuscript.
Funding
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
published article.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Plant Pathology and Weed Research, The Volcani Center,
Agriculture Research Organization, 7505101 Rishon Lezion, Israel.
2
The Robert
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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Jerushalmi et al. Journal of Cannabis Research (2020) 2:12 Page 12 of 12
... Irradiation is an approach that is also widely used to minimize growth of molds on stored food products, including dried herbs and fruits. [89][90][91] On cannabis, irradiation includes the use of gamma rays and electrobeam radiation, 47,92 as well as cold plasma treatment. 47 The use of electrobeam irradiation on dried cannabis buds is permitted in Canada to reduce final mold levels. ...
... [89][90][91] On cannabis, irradiation includes the use of gamma rays and electrobeam radiation, 47,92 as well as cold plasma treatment. 47 The use of electrobeam irradiation on dried cannabis buds is permitted in Canada to reduce final mold levels. Populations of Penicillium spp. ...
... and Botrytis were significantly reduced following this treatment. 25,47 Organic producers have to resort to other less well studied methods to reduce overall mold counts which may include postharvest exposure to ozone as irradiation is not permitted. ...
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Cultivation of cannabis plants (Cannabis sativa L., marijuana) has taken place worldwide for centuries. In Canada, legalization of cannabis in October 2018 for the medicinal and recreational markets has spurned interest in large‐scale growing. This increased production has seen a rise in the incidence and severity of plant pathogens, causing a range of previously unreported diseases. The objective of this review is to highlight the important diseases currently affecting the cannabis and hemp industries in North America and to discuss various mitigation strategies. Progress in molecular diagnostics for pathogen identification and determining inoculum sources and methods of pathogen spread have provided useful insights. Sustainable disease management approaches include establishing clean planting stock, modifying environmental conditions to reduce pathogen development, implementing sanitation measures, and applying fungal and bacterial biological control agents. Fungicides are not currently registered for use and hence there are no published data on their efficacy. The greatest challenge remains in reducing microbial loads (colony‐forming units) on harvested inflorescences (buds). Contaminating microbes may be introduced during the cultivation and post‐harvest phases, or constitute resident endophytes. Failure to achieve a minimum threshold of microbes deemed to be safe for utilization of cannabis products can arise from organic cultivation methods or application of beneficial biocontrol agents. The current regulatory process for approval of cannabis products presents a challenge to producers utilizing biological control agents for disease management. This article is protected by copyright. All rights reserved.
... Symptoms of bud rot on cannabis and hemp plants include decay of a portion or of the entire inflorescence, followed by characteristic sporulation of the pathogen. Despite previously published reports that the causal agent of bud rot worldwide is B. cinerea (McPartland 1996;Punja et al. 2019;Garfinkel 2020;Jerushalmi et al. 2020), there have been no studies conducted to confirm whether there may be more than one species of Botrytis infecting cannabis or hemp plants in Canada or whether other pathogens can produce similar symptoms. The period of time when infection occurs or how disease symptoms progress on affected inflorescences has not been studied. ...
... The most common pathogen associated with bud rot symptoms on cannabis and hemp grown indoors and outdoors, respectively, in British Columbia was B. cinerea, representing 88% of 178 isolates recovered from 10 sampling sites over 2 years of study. This confirms previous findings associating B. cinerea with bud rot symptoms (McPartland 1996;McPartland et al. 2000;Punja et al. 2019;Garfinkel 2020;Jerushalmi et al. 2020;Thiessen et al. 2020). Diseased samples with B. cinerea were recovered most frequently during September-February, months represented by cooler night temperatures and wet or humid daytime conditions, both of which are known to favour infection by B. cinerea on other crops (Jarvis 1962;Bika et al. 2021). ...
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Bud rot pathogens cause diseases on Cannabis sativa L. (cannabis, hemp) worldwide through pre-and post-harvest infection of the inflorescence. Seven indoor or outdoor cannabis production sites and three hemp fields were sampled for bud rot and stem canker presence during 2019-2020. Among 178 isolates recovered from diseased tissues, sequences of the ITS1-5.8S-ITS2 region of rDNA, the glyceraldehde-3-phosphate dehydrogenase (G3PDH) gene and the heat shock 60 (HSP) gene identified the following: Botrytis cinerea (162 isolates), B. pseudocinerea (2), B. porri (1), Sclerotinia sclerotiorum (5), Diaporthe eres (3) and Fusarium graminearum (5). Pathogenicity studies conducted on fresh detached cannabis buds inoculated with spore suspensions or mycelial plugs showed that B. cinerea, S. sclerotiorum and F. graminearum were the most virulent, while B. pseudocinerea, B. porri and D. eres caused significantly less bud rot. Optimal growth of Botrytis species occurred at 15-25o C. In vitro antagonism tests showed that Bacillus spp., Trichoderma asperellum and Gliocladium catenulatum inhibited B. cinerea and S. sclerotiorum colony growth. When applied as a spray 48 h prior to B. cinerea inoculation, all biocontrol agents significantly (P<0.01) reduced disease development on detached inflorescences. Prolific growth and sporulation of T. asperellum and G. catenulatum were observed on bud tissues. The pathogens B. porri, S. sclerotiorum, D. eres and F. graminearum are described for the first time as cannabis bud rot pathogens. Inoculum from neighboring fields of diseased garlic, cabbage, blueberry and hay pasture, respectively, likely initiated infection of inflorescences. Several biological control agents show potential for disease reduction through competitive exclusion.
... As the demand for MC increases, many more farms have been established, and with the growing cultivation intensity, challenges and problems have arisen. These challenges are varied and can be elaborated on as follows: (i) since MC is designed for medical purposes, there are strict regulations regarding the growth, quality, and general standards pertaining to the final product, such as pesticide residues, limits on total yeast and mold (TYM) colony forming units (CFUs), and mycotoxin levels present in MC dried inflorescences [3]; (ii) a lack of theoretical knowledge of MC plant pathogens and disease reduction methods exists, as there is a lack of scientific research on this subject, and most of the information available refers to the fiber type plants (hemp) grown outdoors, since drug type plants were illegal in the past [4]; (iii) years of MC inter-crossbreeding and the use of cuttings in commercial farms have led to low plant genetic diversity and increased susceptibility to plant pathogens and pests [5]. Specifically, it has been reported that drug type MC plants tend to be less resistant when grown in high concentrations compared to fiber type plants [6]. ...
... Uncharacterized cannabis seedlings were kindly provided by Dr. Moshe Flaishman of the ARO. Growth conditions of plant material (seedlings, leaves, and mature inflorescences) were essentially as described [3]. 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 environment, and an additional week without the top cover. ...
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The use of and research on medical cannabis (MC) is becoming more common, yet there are still many challenges regarding plant diseases of this crop. For example, there is a lack of formal and professional knowledge regarding fungi that infect MC plants, and practical and effective methods for managing the casual agents of disease are limited. The purpose of this study was to identify foliar, stem, and soilborne pathogens affecting MC under commercial cultivation in Israel. The predominant major foliage pathogens were identified as Alternaria alternata and Botrytis cinerea, while the common stem and soilborne pathogens were identified as Fusarium oxysporum and F. solani. Other important fungi that were isolated from foliage were those producing various mycotoxins that can directly harm patients, such as Aspergillus spp. and Penicillium spp. The sampling and characterization of potential pathogenic fungi were conducted from infected MC plant parts that exhibited various disease symptoms. Koch postulates were conducted by inoculating healthy MC tissues and intact plants with fungi isolated from infected commercially cultivated symptomatic plants. In this study, we report on the major and most common plant pathogens of MC found in Israel, and determine the seasonal outbreak of each fungus.
... Botrytis cinerea causes inflorescence (bud) rot during growth of cannabis plants (Punja et al. 2019) which subsequently also manifests as a post-harvest problem. It is the most widely reported pathogen affecting bud quality (Jerushalmi et al. 2020). In the present study, the overall frequency of recovery of B. cinerea using the swab method was 11-20%. ...
... Treatments using gamma rays and electrobeam radiation have been shown to reduce mould contamination on a range of plant products (Aquino 2011;Calado et al. 2014;Jeong et al. 2015;Hazekamp 2016) and are an approved method for mould reduction for Canadian producers of cannabis (Health Canada 2013), except those certified for organic production. A recent study demonstrated that these forms of treatment, as well as cold plasma treatment, effectively reduced contamination of cannabis buds by B. cinerea (Jerushalmi et al. 2020) although the effects on Penicillium spp. were not studied. ...
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The objective of this study was to assess harvested dried inflorescences (buds) of cannabis (Cannabis sativa L., marijuana) for fungal presence and diversity. Samples from drying rooms of three licenced facilities in British Columbia were tested repeatedly during 2017–2019. A swab method was used, wherein sterile cotton swabs were gently swabbed over bud surfaces and directly streaked onto potato dextrose agar containing 140 mg L⁻¹ streptomycin sulphate. Petri dishes were incubated at 21–24°C for 5–6 days and the fungal colonies that developed were recorded. The testing was repeated to provide >40 cumulative sampling times over a 2-year period. Representative colonies of each unique morphological type were identified to genus and species by PCR of the ITS1-5.8-ITS2 region of rDNA and sequence analysis. Among 34 different fungal species identified, the most prevalent were Penicillium (comprising 17 different species), followed by species of Cladosporium, Botrytis, Aspergillus, Fusarium, Talaromyces and Alternaria. All samples had several fungal species present and the number and composition varied at different sampling times and within different facilities. The swab method provided a qualitative assessment of viable mould contaminants on cannabis buds and reflected the diversity of mycoflora present, many of which are previously unreported. Fungi on cannabis buds may originate from spores released from diseased or decomposing plant materials, from growing substrates used in cannabis production, or as airborne contaminants in post-harvest trimming and drying rooms. Samples of dried buds exposed to electrobeam (e-beam) radiation treatment had no detectable fungal contamination when assessed using the swab method.
... When a plasma jet was directly applied to symptomatic leaves of Philodendron erubescens infected with fungi, no further symptom development occurred, and the leaves recovered from the infected state [122]. Inflorescences of medical cannabis inoculated with Botrytis cinerea were efficiently disinfected with plasma (5-log reduction in fungal spore CFU number) [121]. ...
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In addition to being key pathogens in plants, animals, and humans, fungi are also valuable resources in agriculture, food, medicine, industry, and the environment. The elimination of pathogenic fungi and the functional enhancement of beneficial fungi have been the major topics investigated by researchers. Non-thermal plasma (NTP) is a potential tool to inactivate pathogenic and food-spoiling fungi and functionally enhance beneficial fungi. In this review, we summarize and discuss research performed over the last decade on the use of NTP to treat both harmful and beneficial yeast- and filamentous-type fungi. NTP can efficiently inactivate fungal spores and eliminate fungal contaminants from seeds, fresh agricultural produce, food, and human skin. Studies have also demonstrated that NTP can improve the production of valuable enzymes and metabolites in fungi. Further studies are still needed to establish NTP as a method that can be used as an alternative to the conventional methods of fungal inactivation and activation.
... There are three physical, chemical, and biological approaches that are used to decontaminate food and feed from mycotoxin. Physical methods include electron beam irradiation, cold atmospheric plasma, and the high hydrostatic pressure that are non-thermal approaches to eliminate mycotoxin in food products [74,75]. The usage of oxidizing and hydrolyzing agents and various types of gases are also studied as chemical methods for mycotoxin decontamination [76][77][78]. ...
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Probiotics are living microorganisms that have favorable effects on human and animal health. The most usual types of microorganisms recruited as probiotics are lactic acid bacteria (LAB) and bifidobacteria. To date, numerous utilizations of probiotics have been reported. In this paper, it is suggested that probiotic bacteria can be recruited to remove and degrade different types of toxins such as mycotoxins and algal toxins that damage host tissues and the immune system causing local and systemic infections. These microorganisms can remove toxins by disrupting, changing the permeability of the plasma membrane, producing metabolites, inhibiting the protein translation, hindering the binding to GTP binding proteins to GM1 receptors, or by preventing the interaction between toxins and adhesions. Here, we intend to review the mechanisms that probiotic bacteria use to eliminate and degrade microbial toxins.
... Values compared for each cannabinoid between treatments, with a different letter are significant, according to Tukey HSD (α = 0.05). Cannabis sativa was cultivated in the Agriculture Research Organization (ARO) Volcani Center (authorized by the Israeli Medical Cannabis Agency, IMCA, Ministry of Health, State of Israel) for this research, as described12 . Two different ARO cultivars, AO 235 and AO 325, comprising different profiles of cannabinoid and terpene compounds were used in the noncommercial experiments. ...
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Medical cannabis (MC) production is a rapidly expanding industry. Over the past ten years, many additional phytocannabinoids have been discovered and used for different purposes. MC was reported beneficial for the treatment of a variety of clinical conditions such as analgesia, multiple sclerosis, spinal cord injuries, Tourette's syndrome, epilepsy, glaucoma, Parkinson disease and more. Yet, there is still a major lack of research and knowledge related to MC plant diseases, both at the pre- and postharvest stages. Many of the fungi that infect MC, such as Aspergillus and Penicillium spp., are capable of producing mycotoxins that are carcinogenic, or otherwise harmful when consumed, and especially by those patients who suffer from a weakened immune system, causing invasive contamination in humans. Therefore, there are strict limits regarding the permitted levels of fungal colony forming units (CFU) in commercial MC inflorescences. Furthermore, the strict regulation on pesticide appliance application in MC cultivation exacerbates the problem. In order to meet the permitted CFU limit levels, there is a need for pesticide-free postharvest treatments relying on natural non-chemical methods. Thus, a decontamination approach is required that will not damage or significantly alter the chemical composition of the plant product. In this research, a new method for sterilization of MC inflorescences for reduction of fungal contaminantstes was assessed, without affecting the composition of plant secondary metabolites. Inflorescences were exposed to short pulses of steam (10, 15 and 20 s exposure) and CFU levels and plant chemical compositions, pre- and post-treatment, were evaluated. Steam treatments were very effective in reducing fungal colonization to below detection limits. The effect of these treatments on terpene profiles was minor, resulting mainly in the detection of certain terpenes that were not present in the untreated control. Steaming decreased cannabinoid concentrations as the treatment prolonged, although insignificantly. These results indicate that the steam sterilization method at the tested exposure periods was very effective in reducing CFU levels while preserving the initial molecular biochemical composition of the treated inflorescences.
... In recent years, the use of cannabis (Cannabis sativa) has gained popularity for medical and other purposes, and its cultivation worldwide has expanded rapidly (Ruchlemer et al., 2015;Jerushalmi et al., 2020). ...
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Medical cannabis products contain dozens of active pharmaceutical ingredients (APIs) derived from the cannabis plant. However, their actual compositions and relative doses significantly change according to the production methods. Product compositions are strongly dependent on processing step conditions and on components' evaporation during those steps. Review of the documentation presented to caregivers and to patients show erroneous data or misinterpretation of data related to the evaporation, for example, cannabinoids' boiling points, as well as confusions between terms, such as boiling, vaporization, and evaporation. Clarifying these aspects is essential for caregivers, for researchers, and for developers of manufacturing processes. Original and literature data were analyzed, comparing composition changes during various processing steps and correlating the extent of change to components' vapor pressures at the corresponding temperature. Evaporation-related composition changes start at temperatures as low as those of drying and curing and become extensive during decarboxylation. The relative rate of components' evaporation is determined by their relative vapor pressure and monoterpenes are lost first. On vaping, terpenes are inhaled before cannabinoids do. Commercial medical cannabis products are deficient in terpenes, mainly monoterpenes, compared with the cannabis plants used to produce them. Terms, such as "whole plant" and "full spectrum," are misleading since no product actually reflects the original cannabis plant composition. There are important implications for medical cannabis manufacturing and for the ability to make the most out of the terpene API contribution. Medical cannabis products' composition and product delivery are controlled by the relative vapor pressure of the various APIs. Quantitative data provided in this study can be used for improvement to reach better accuracy, reproducibility, and preferred medical cannabis compositions.
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Plant pathogens infecting marijuana (Cannabis sativa L.) plants reduce growth of the crop by affecting the roots, crown, and foliage. In addition, fungi (molds) that colonize the inflorescences (buds) during development or after harvest, and which colonize internal tissues as endophytes, can reduce product quality. The pathogens and molds that affect C. sativa grown hydroponically indoors (in environmentally controlled growth rooms and greenhouses) and field-grown plants were studied over multiple years of sampling. A PCR-based assay using primers for the internal transcribed spacer region (ITS) of ribosomal DNA confirmed identity of the cultures. Root-infecting pathogens included Fusarium oxysporum, Fusarium solani, Fusarium brachygibbosum, Pythium dissotocum, Pythium myriotylum, and Pythium aphanidermatum, which caused root browning, discoloration of the crown and pith tissues, stunting and yellowing of plants, and in some instances, plant death. On the foliage, powdery mildew, caused by Golovinomyces cichoracearum, was the major pathogen observed. On inflorescences, Penicillium bud rot (caused by Penicillium olsonii and Penicillium copticola), Botrytis bud rot (Botrytis cinerea), and Fusarium bud rot (F. solani, F. oxysporum) were present to varying extents. Endophytic fungi present in crown, stem, and petiole tissues included soil-colonizing and cellulolytic fungi, such as species of Chaetomium, Trametes, Trichoderma, Penicillium, and Fusarium. Analysis of air samples in indoor growing environments revealed that species of Penicillium, Cladosporium, Aspergillus, Fusarium, Beauveria, and Trichoderma were present. The latter two species were the result of the application of biocontrol products for control of insects and diseases, respectively. Fungal communities present in unpasteurized coconut (coco) fiber growing medium are potential sources of mold contamination on cannabis plants. Swabs taken from greenhouse-grown and indoor buds pre- and post-harvest revealed the presence of Cladosporium and up to five species of Penicillium, as well as low levels of Alternaria species. Mechanical trimming of buds caused an increase in the frequency of Penicillium species, presumably by providing entry points through wounds or spreading endophytes from pith tissues. Aerial distribution of pathogen inoculum and mold spores and dissemination through vegetative propagation are important methods of spread, and entry through wound sites on roots, stems, and bud tissues facilitates pathogen establishment on cannabis plants.
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Food irradiation is a process of exposing food to ionising radiation such as gamma rays emitted from the radioisotopes 60Co and 137Cs, or high energy electrons and X-rays produced by machine sources. The use of ionising radiation to destroy harmful biological organisms in food is considered a safe, well proven process that has found many applications. Depending on the absorbed dose of radiation, various effects can be achieved resulting in reduced storage losses, extended shelf life and/or improved microbiological and parasitological safety of foods. The most common irradiated commercial products are spices and vegetable seasonings. Spice irradiation is increasingly recognised as a method that reduces post-harvest losses, ensures hygienic quality, and facilitates trade with food products. This article reviews recent activities concerning food irradiation, focusing on the irradiation of spices and dried vegetable seasonings from the food safety aspect.
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Microbiological contaminants pose a potential threat to cannabis consumers. Bacteria and fungi may cause opportunistic infections in immunocompromized individuals. Even dead organisms may trigger allergies and asthma. Toxins from microbial overloads, such as Shigla toxin and aflatoxins, may pose a problem—unlikely, but possible. The Cannabis plant hosts a robust microbiome; the identification of these organisms is underway. Cannabis bioaccumulates heavy metals in its tissues, so avidly that hemp crops have been used for bioremediation. Heavy metals cause myriad human diseases, so their presence in crops destined for human consumption must be minimized. Pesticide residues in cannabis pose a unique situation among crop plants—the Environmental Protection Agency (EPA) will not propose pesticides guidelines, because Cannabis is illegal on the federal level. The use of illegal pesticides is a rising crisis, and a breakdown in ethics. Testing for pesticide residues and maximal limits are proposed.
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The efficacy of cold atmospheric pressure plasma (CAPP) with ambient air as working gas for the degradation of selected mycotoxins was studied. Deoxynivalenol, zearalenone, enniatins, fumonisin B1, and T2 toxin produced by Fusarium spp., sterigmatocystin produced by Aspergillus spp. and AAL toxin produced by Alternaria alternata were used. The kinetics of the decay of mycotoxins exposed to plasma discharge was monitored. All pure mycotoxins exposed to CAPP were degraded almost completely within 60 s. Degradation rates varied with mycotoxin structure: fumonisin B1 and structurally related AAL toxin were degraded most rapidly while sterigmatocystin exhibited the highest resistance to degradation. As compared to pure compounds, the degradation rates of mycotoxins embedded in extracts of fungal cultures on rice were reduced to a varying extent. Our results show that CAPP efficiently degrades pure mycotoxins, the degradation rates vary with mycotoxin structure, and the presence of matrix slows down yet does not prevent the degradation. CAPP appears promising for the decontamination of food commodities with mycotoxins confined to or enriched on surfaces such as cereal grains.
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Cold plasma treatment is a promising intervention in food processing to boost product safety and extend the shelf‐life. The activated chemical species of cold plasma can act rapidly against micro‐organisms at ambient temperatures without leaving any known chemical residues. This review presents an overview of the action of cold plasma against molds and mycotoxins, the underlying mechanisms, and applications for ensuring food safety and quality. The cold plasma species act on multiple sites of a fungal cell resulting in loss of function and structure, and ultimately cell death. Likewise, the species cause chemical breakdown of mycotoxins through various pathways resulting in degradation products that are known to be less toxic. We argue that the preliminary reports from cold plasma research point at good potential of plasma for shelf‐life extension and quality retention of foods. Some of the notable food sectors which could benefit from antimycotic and antimycotoxin efficacy of cold plasma include, the fresh produce, food grains, nuts, spices, herbs, dried meat and fish industries.
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BACKGROUND Due to difference in radiation sources (electron beam from electron accelerator, gamma ray from ⁶⁰Co radionuclide) and energy‐delivery time (dose rate, kGy/time), the resulting effects are expected to be different in chemical quality change and microbial decontamination in food. To better understand this impact, effects of variable dose rates of electron beam (EB, kGy/s) and gamma ray (GR, kGy/h) on microbial reduction, capsanthin content, and color parameters of red pepper (Capsicum annuum L.) powders (RPP) were determined. RPP samples were irradiated with 3 kGy absorbed dose, at variable dose rates of 1 and 5 kGy/s of EB (10 MeV/10 kW), and 1.8 and 9 kGy/h of GR (⁶⁰Co). RESULTS Aerobic plate counts (APC) as well as yeast and mold counts of non‐irradiated samples were 7.12 log CFU/g and 6.62 log CFU/g, respectively. EB and GR reduced these by 2‐3 log CFU/g. Lower dose rate (1 kGy/s) of EB was more effective for microbial reduction than higher dose rate (5 kGy/s). In contrast, higher dose rate (9 kGy/h) of GR efficiently decreased APC compared to lower dose rate (1.8 kGy/h). Higher EB and GR dose rates significantly decreased the capsanthin content and Hunter's red color (a* value). CONCLUSION Low EB (kGy/s) and high GR (kGy/h) dose rates are recommended for microbiological safety of RPP with negligible changes in color attributes visible to human eye, in contrast to the measured values. Thus, the study demonstrates that the influence of absorbed dose is dependent on the applied dose rates. This article is protected by copyright. All rights reserved.
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Despite of the constant development of novel thermal and non-thermal technologies, knowledge on the mechanisms of microbial inactivation is still very limited. Technologies such as high pressure, ultraviolet light, pulsed light, ozone, power ultrasound and cold plasma (advanced oxidation processes) have shown promising results for inactivation of microorganisms. The efficacy of inactivation is greatly enhanced by combination of conventional (thermal) with non-thermal, or non-thermal with another non-thermal technique. The key advantages offered by non-thermal processes in combination with sub lethal mild temperature (<60 °C) can inactivate microorganisms synergistically. Microbial cells, when subjected to environmental stress, can be either injured or killed. In some cases, cells are believed to be inactivated, but may only be sub lethally injured leading to their recovery or, if the injury is lethal, to cell death. It is of major concern when microorganisms adapt to stress during processing. If the cells adapt to a certain stress it is associated with enhanced protection against other subsequent stresses. One of the most striking problems during inactivation of microorganisms is spores. They are the most resistant form of microbial cells and relatively difficult to inactivate by common inactivation techniques, including heat sterilization, radiation, oxidizing agents and various chemicals. Various novel non-thermal processing technologies, alone or in combination, have shown potential for vegetative cells and spores inactivation. Predictive microbiology can be used to focus on the quantitative description of the microbial behaviour in food products, for a given set of environmental conditions.
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