Plant and fungal samples
Cannabis sativa cultivar (BB 734) was grown in the Agriculture Research Organization (ARO) Volcani Center research 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 generation mother plants. This strain is a “drug-type” cannabis with Cannabis indica characteristics.
Shoots were rooted under continuous 24 h light photoperiodic 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. The rooted shoots were replanted in 0.2 L pots and transferred 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 photoperiodic 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 inflorescences, (ii) artificially inoculated mature inflorescences with a culture of Botrytis cinerea originating from naturally infected cannabis flowers, isolated and characterized by morphological and molecular methods. It should be noted that “uninoculated” inflorescences from the ARO facility contained asymptomatic microbial infections comprised of a wide variety of different fungal species. B. cinerae was cultured 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 suspension 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 106 spores/ml. The inoculum was sprayed till run-off on healthy mature cannabis 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 (Sacramento, California, USA) at 35 °C for 12 h, then stored in a STATUS innovations vacuum pack (Metlika, Slovenia) at room temperature before experimentation. CFU’s of the 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. Thereafter, 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 characterized.
Survey of CFU levels from commercial MC farms
In order to evaluate common CFU levels under commercial conditions, samples of MC inflorescences were collected 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. Variability in sampled inflorescences existed, as certain samples exhibited disease symptoms while others remained asymptomatic, and certain inflorescences were dry.
Gamma and beta irradiation
Commercial cannabis samples were received from a number 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 radiation was based on a 60Co isotope, doubly encapsulated in stainless steel source pencils type C-188, with radiation dosages of 7.5 and 8.37 KGy (KiloGray) in two consecutive experiments, 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 H2O2 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 H2O2 liquid had no direct contact with the MC, it affects the gaseous environment and generates a highly reactive plasma with elevated concentrations 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 asymptomatic infections) and artificially inoculated Botrytis cinerea] of noncommercial plant material were obtained from the ARO Volcani Center facilities and divided 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 noncommercial floral material. The experimental design was identical to that described for the noncommercial irradiation experiments. Floral parts were placed on the electrode and H2O2 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.
ED50 calculation
In order to determine the effective radiation dosage for eliminating 50% (ED50) CFUs, a response curve with R2 > 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% (ED50). All other ED values were calculated using the same method.