DANIEL HENDRICKS, GRASS VALLEY PATIENT RESOURCE GROUP DBA HENDRX FARMS
Cannabis is an economically important crop, with a known receptibility to microbial colonization of root tissue. Rhizosphere bacteria have been known to colonize rhizoplane soil as well as plant tissue. Specifically, Plant Growth Promoting Rhizobacteria (PRGR) has been reported to support plant growth, suppress plant diseases, and increase the yield productivity of many crop producing plants.  When inoculated, cannabis/hemp seedlings have proven themselves highly receptive to PGPRs, allowing bacteria to adhere and colonize root and vascular plant tissue. In a recent study under greenhouse conditions, PGPR-inoculated cannabis seedlings favored plant growth and development of plant valuable secondary metabolites in the form of phytocannabinoids. 
In a molecular and phylogenetic analysis of cannabis cultivars Yvonne, Anka, and CRS-1, researchers cited the most abundant naturally occurring bacterial endophytes were of the genera Pseudomonas, Pantoea, and Bacillus, respectively.  The experiment provided evidence that cannabis harbors a diverse population of microbial endophytes which may be utilized in plant growth promotion and plant health promotion (PHP).
Cannabis root tissue colonization receptibility has been reported to be specific to individual cultivars.  Gelato is a popular commercially-produced cannabis cultivar from a clonally propagated mother plant. The study described herein aims to test the PGPR biological fertilization potential, on plant growth parameters, and secondary metabolites in the form of plant-flower yield, THC, THCA, CBD, CBDA, CBG, and CBGA cannabinoid content, as well as the total cannabinoid accumulation of the Cannabis sativa L. cultivar Gelato. The researchers targeted Rhodopseudomonas palustris as a biological inoculant due to the abundant occurrence of the Pseudomonas genera naturally occurring in cannabis hemp varieties. Rhodopseudomonas palustris as a PGPR has demonstrated in previous studies, an increased grain yield of 9% vs. controls. Consortiums utilizing R. paustris combined with other PGPR consortiums have proven useful to agricultural crops, increasing soil fertility and nutrient uptake. 
During the study, researchers tested R. palustris and Bacillus megaterium, in conjunction with a consortium of PGPRs, in the form of the agricultural product Sym3iotc+ (SYM3). SYM3 was applied at two concentrations along with one control, for a total of three batches of clonally-propagated cannabis plant start treatments. The inoculation was introduced to clonally reproduced cannabis plants, at a licensed cannabis nursery in Humboldt County, California. Following inoculation, clonally-propagated cannabis plant starts were then cultivated until harvest, in conjunction with the farmer’s normal fertilization method at a flower-producing licensed cannabis farm in Humboldt County. The two inoculum strengths and the control batches were randomized to ensure the test trial received no bias.
Inoculation: HENDRX, a licensed cannabis nursery, produced and inoculated trial cannabis plants starts after the roots were established and quality control screened for consistency. 72 starts received inoculum (1); 72 starts received inoculum (2); 72 starts received no treatment and served as a Control for the experiment. HENDRX then randomized the batches and labeled them X, Y, and Z to create a blind assignment so the producer did not unknowingly apply bias. Treated plants were submerged in liquid inoculum at the following dilutions:
- Inoculum (1): ¼ ounce per gallon Sym3iotic+
- Inoculum (2): 1 ounce per gallon Sym3iotic+
- Control: No treatment
It was hypothesized that either inoculum (1) or (2), in conjunction with a normal feeding program, will yield the highest average dried flower weight, as well as the highest accumulation of secondary metabolites.
Test Trials: Rambling Rose, a licensed cannabis producer, cultivated three randomized batches assigned X, Y, and Z to full maturation. Batch’s X, Y, and Z were cultivated in separate, but identical grow beds and growing media. The soil mixture in the beds contained native soil and Royal Gold Kings Mix. Soil was re-amended after prior cultivation in the soil, based on recommendations consistent with soil analysis. There were six beds total in two separate greenhouses (Lower Twin and Upper Twin). Lower Twin contained 3 beds, batch X, batch Y, and batch Z. Upper Twin contained 3 beds, batch X, batch Y, and batch Z.
Two variables were targeted to be observed. (1) Mature plant harvest weight: by regulatory standards, mature cannabis total harvest must be weighed at the time of harvest. The data was recorded at the time of harvest by Rambling Rose according to each grow bed and batch assignment. Common commercial practices require the cannabis to be dried and/or have the leaves and stems trimmed before sale. Rambling Rose chose to dry and/or remove leaves and stems from the flower of beds X, Y, and Z per the standard business practice for commercial sale. (2) Cannabinoid content: by regulatory standards, mature cannabis flowers are tested in batches for cannabinoid content. Cannabinoid content was recorded in separate batches in conjunction with batch assignments X, Y, and Z.
|Nursery Source: HENDRX||County: Humboldt|
|Farm Name: Rambling Rose||Watershed: Redwood Creek|
|Trial Size: 72 per Treatment||Total Plants: 246|
|Lat/Long: 40.8658/123.7608||Elevation: 2600 ft.|
|Plant Date: 8/13/18||Harvest Date: 11/6/19
The data was analyzed for comparison of Inoculum (1), Inoculum (2), and Control.
Sampling: Samples were collected from 3 of the 6 greenhouses. Due to the remote location of the cannabis farm, the crop was vulnerable to the environment, as wild deer were attracted to the flowering cannabis. One set of greenhouses (3 grow beds, all from Upper Twin Greenhouse) were compromised when the deer fed on the growing cannabis plants in early flower. As a result, the Upper Twin Greenhouse set of X, Y, and Z were not included in the data analysis. Only the Lower Twin Greenhouse set of X, Y, and Z were used for the data set.
Flowers from the Lower Twin Greenhouse were collected from the bottom and top of the plant from grow beds X, Y, and Z. Flower samples were collected from premature plants, and would be collected again later after full maturation. Premature flower samples were prepared, labeled, and frozen for three months prior to testing. Liquid chromatography was utilized by Humboldt Quality Assurance Laboratory for cannabinoid content analysis (Table 1).
Upon full maturation, cannabis plants were harvested. Batch’s X, Y, and Z from Lower Twin Greenhouses were kept separate. Flowers were dried and manicured per standard operating procedure for Rambling Rose Farm. Manicured flower and trim by-product were weighed separately for each batch X, Y and Z. (Table 2)
After completed harvest, dried, manicured samples of flower were tested for cannabinoid content from each batch X, Y, and Z. Samples were stored in a dry cool environment for six weeks prior to testing. Liquid chromatography was utilized by Humboldt Quality Assurance Laboratory for cannabinoid content analysis (Table 3).
|Sample||Bottom Flowers||Top Flowers|
|Lower Twin X||2.66||3.36|
|Lower Twin Y||2.78||4.13|
|Lower Twin Z||2.57||4.47|
Table 1. Immature Fresh Flower Total Cannabinoids %
|Sample||Flower by Weight||Trim by Weight||Total Harvest|
|Lower Twin X||3,405 g||2,562 g||5,967 g|
|Lower Twin Y||3,068 g||2,054 g||5,122 g|
|Lower Twin Z||3,343 g||2,032 g||5,375 g|
Table 2. Total Harvest By Gram Weight
|Lower Twin X||0.0793||0.4666||17.97|
|Lower Twin Y||0.0568||0.2985||15.27|
|Lower Twin Z||0.0827||0.2342||14.6|
Table 3. Full Maturation Individual Cannabinoids %
|Lower Twin X||Inoculate 2||1 OZ Per Gallon|
|Lower Twin Y||Inoculate 1||¼ OZ Per Gallon|
|Lower Twin Z||Control||No Treatment|
Table 4. Sample Treatment Key
To the author’s knowledge, no prior experiments have been conducted on the viability of microbial inoculation of PGPRs and the effects on the harvest quality, quantity, or the production of secondary metabolites on cannabis cultivars that have been clonally propagated. Of the three randomized batches inoculated and then cultivated in the field, the batch containing the highest overall harvest weight was the batch that received the highest concentration of bacterial inoculation (Lower Twin X). The increase in harvest data is consistent with the Pagnani 2018 study, which also provided evidence of an increase in harvest data in the hemp cultivar Finola with inoculation. 
Similarly, of the three randomized batches, the sample with the highest overall cannabinoid content at the time of harvest was also the batch that received the highest concentration of bacterial inoculation (Lower Twin X). This data is also consistent with the Pagnani 2018 study, which also resulted in an increase of overall and individual cannabinoids with inoculation.  However, a limitation to the positive effects was discovered when inoculation concentrations were excessive.  In our study, no limitation was discovered, suggesting additional research is required to discover the optimal inoculation concentration of SYM3.
Inverse to the data recorded at final harvest, the immature flowers in the data set contained the highest concentrations of cannabinoids in the flower tops in Lower Twin Z, which is the batch that received no inoculation. The reason for this data point is currently unknown. Rambling Rose reported that no Botrytis cinerea was observed in the batches containing inoculation, so perhaps the inoculation was acting as a PHPR later in the plants’ lives, increasing efficiency in late flower allowing increased conversions of cannabinoid precursors into cannabinoids. This is highly speculative, and displays further need for additional research.
Another note worth mentioning is the significant decrease in cannabinoid concentrations in the lower flowers as opposed to the top flowers in the premature plant samples. In all samples, the flowers from the bottom of the plant were drastically lower in the concentration of cannabinoids. Similar results were discovered in a study of the interplay between cannabis plant morphology and the effect on secondary metabolites where quantified THC levels in upper flowers yielded 12% versus 6% in lower flowers. 
The data in the study were consistent with previous studies of PGPR inoculation studies. Cannabis clonally-propagated cultivars receiving early inoculation of PGPR or PHPRs may create lasting positive effects on cannabis plant yields and conversion of secondary metabolites. In this study, the batch that received the highest strength inoculation demonstrated an 18.75% increase of THC and THCA combined versus the control, which received no inoculation. CBG and CBGA, which precursor both CBD and THC, demonstrated an increase of 49.8% when inoculated at the highest strength versus the control. Overall harvest yield also demonstrated a significant increase of 9.9% when comparing the highest inoculation strength versus the control.
If this data is representative of the potential results of early inoculation of cannabis plant starts, specifically clonally-propagated cannabis cultivars, then the potential economic impact for cannabis producers is significant. Additionally, in many crops PGPRs have demonstrated a decrease for the need of fertilizer, as well as reduction the crop’s water consumption.  Further studies of specific isolated bacterial inoculations could create opportunity to reduce the environmental impact of cannabis production.
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