Performing Department
Bioag Science and Pest Mgmt
Non Technical Summary
Despite the long history of hemp production in North America, industrial hemp being grown in the current era can be considered to be a new crop in most all aspects. Historical research on the arthropod pest management needs is essentially non-existent; what is known about the subject at this point in time is presented in the recently submitted review article (Cranshaw et al. 2019). This includes a summary of the present state of information derived from work done over the past three years in three states (Colorado, Virginia, Tennessee) that have made some effort, involving graduate entomology student research, to define and address pest management needs of the crop.There have been several important arthropod pest problems identified on the crop. For some of these (e.g., corn earworm) interim pest management plans on hemp can be fairly easily adapted using IPM strategies developed to manage the insect in other crops (e.g., organic sweet corn). Where the pest is unique to the crop (e.g., cannabis aphid) strategies used to manage similar insects (e.g., aphids on greenhouse vegetables) can guide development of interim pest management plans for hemp.Hemp russet mite is a species presently thought to be restricted to Cannabis spp. crops with essentially no significant associated research, even regarding basic information on life history. As a russet mite on an herbaceous crop, there is little to guide development of research-based IPM systems; presently most is inferred from information developed for tomato russet mite, the closest example of a crop pest similar to hemp russet mite. There are very significant differences between these two systems involving russet mites on North American herbaceous hosts (tomato russet mite on tomato; hemp russet mite on hemp), including the far greater geographic range of hemp russet mite as a significant pest species within North America (coast to coast) and the far greater number of producers with interest in this pest.Current pest management technologies and practices used for control of hemp russet mite are inadequate, at best. This project, over a two year period, seeks to develop the methods that can be used to eliminate hemp russet mite during the point in hemp production, clonal propagation, where it is most vulnerable to control. The developments of these methods, and their subsequent communication to hemp producers in the United States, should provide a means to produce plants that are free of this pest obviating the need for any subsequent use of pesticides for this pest on hemp crops.There are three Research-led objectives in this project:1. Determine efficacy of pest management products presently allowed for use on hemp to be used in hemp russet mite eradication strategies during early stages of hemp plant production;2. Develop the use of high CO2 atmosphere treatment to disinfest living hemp plants of hemp russet mite;3. Develop the use of quantitative-PCR (qPCR) for detection and quantification of hemp russet mite populations on hemp plants.There is one Extension-led objective in this project:1. Develop effective methods to provide research-based information regarding hemp russet mite IPM to hemp producers throughout the United States.
Animal Health Component
75%
Research Effort Categories
Basic
(N/A)
Applied
75%
Developmental
25%
Goals / Objectives
There are three Research-led objectives in this project:1. Determine efficacy of pest management products presently allowed for use on hemp to be used in hemp russet mite eradication strategies during early stages of hemp plant production;2. Develop the use of high CO2 atmosphere treatment to disinfest living hemp plants of hemp russet mite;3. Develop the use of quantitative-PCR (qPCR) for detection and quantification of hemp russet mite populations on hemp plants.There is one Extension-led objective in this project:1. Develop effective methods to provide research-based information regarding hemp russet mite IPM to hemp producers throughout the United States
Project Methods
Objective 1.Criteria for the specific products to be considered in trials for this project will be based on the criteria originally developed by the Washington State Department of Agricultureand subsequently modified and adopted by Washington and Colorado. Products on this list considered to have likelihood of some success in suppressing hemp russet mite on plants include as active ingredients horticultural oils (mineral, neem, canola, etc.), insecticidal soaps, sodium lauryl sulfate, Beauveria bassiana, Isaria fumosorosea, and, perhaps, certain essential oils and citric acid.Evaluations of these products for ability to control hemp russet mite will be conducted at indoor/greenhouse settings both at grower facilities and at the greenhouse facility described above used for plant production at Colorado State University. Standard protocols for conduct of these types of efficacy-evaluation trials will be followed including replications (minimum four/trial), arrangement in an appropriate design (e.g., randomized complete block, completely randomized) as conditions dictate, use of appropriate controls (e.g., water check, untreated check). Methods for bestmeasuring hemp russet mite populations will be an early focus of methods development.In addition to determining hemp russet mite populations on plants, observations will be made of evidence of phytotoxicity.Use rates, application methods, and treatment frequency are variables that can be used in studies to refine treatments with products that appear to have greatest promise. It will be the ultimate goal of this work to identify treatments that can completely eradicate hemp russet mite - and not produce significant plant injury - so that hemp russet mite-free plants can be produced by growers.Objective 2. The primary trials used to evaluate the use of high CO2 atmosphere treatments on live hemp plants will involve the use of specially constructed cabinetslarge enough to accommodate hemp plants of fair size and to accomodate trays of seedlings. A proposed size of these containers is 24-in (l) x 36-in (w) x 36-in (h).Four identical containers will be constructed, which can allow for concurrent studies of different treatments. CO2 monitors will be incorporated into the design to monitor levels present at three points in the container (bottom, middle, top). Other sensors will be included to record temperature, oxygen, and relative humidity.For the primary studies, bottled CO2 will be used, introduced into the lower area of the chamber and levels maintained at specified levels for different studies.With this system in place a sequence of studies can be conducted using live hemp plants infested with hemp russet mite. These include differences in CO2 concentrations, length of exposure and temperature. All trials will include a minimum of four replicated runs of the test treatments. All trials will include as plants maintained in ambient air as a control. With four treatment containers, this would allow tests of three treatments, plus the control, for each trial run. Based on preliminary results during proof of concept testing, an effective concentration of CO2 may be found within the range of 35-50%, sustained over 48 hours.Plants included in all trials will be infested with hemp russet mites before introduction into the containers and exposed to CO2 treatment. After the exposure period has been achieved, the boxes will be vented then the plants removed. Leaf samples will be taken immediately upon plant removal to examine for the presence of live stages of hemp russet mite. Treated plants will then be transferred to a greenhouse where they will be maintained under netting sufficient to exclude eriophyid mites. After one month these plants will be again examined for the presence of eriophyid mites and for effects on plant growth (e.g., stunting, yellowing, premature flowering).This system can subsequently be used in tests to examine other variables (e.g., length of exposure at a single CO2 concentration). The specific trials will all be directed to meeting the goals of this objective - developing an effective system that can consistently disinfest live plants of hemp russet mite with minimal effects on subsequent growth of hemp plants.In addition, this project would also like to pursue trials using small scale systems with self-generated CO2. The pilot tests of CO2 disinfestation on hemp plants, described in Background and Preliminary Results, involved such a system where CO2 was generated through a combination of sugar, water and yeast within an enclosed container. Such systems, if they can be shown to effectively meet the goals of this Objective, could allow CO2 disinfestation of hemp to be more broadly accessible to small scale producers.Objective 3. Two different DNA extraction protocols will be performed: one for mite samples and another for a composite of mite and plant samples. For each method, there will be varying numbers of mites (1, 10, 20, 50 and 100). DNA will be extracted usingfollowing the manufacturer's instructions and eluted inpurified water. DNA will bestored at -20 °C until qPCR can be conducted.To quantify mite levels from samples, a standard curve will be generated using a plasmid containing either species-specific Cytochrome oxidase sub unit I (COI) or Internal transcribed spacer 2 (ITS2) at a known concentration as per Keough et al. (2016) and Han et al. (2019). The level of expression of the gene will be directly correlated with the number of mites in the sample. Briefly, the COI and ITS2 gene fragments will be amplified using PCR. The cycling conditions as follows: 2 minutes incubation at 94ºC followed by 40 cycles of 30 seconds denaturation at 94ºC, 10 seconds annealing at 55ºC, and 1 minute extension at 72ºC and a final 10 minutes incubation at 72ºC. The amplicons will be cloned into pCR8TM TA vector using One Shot TOP10 chemically competent Escherichia coli (Thermo Scientific, Pittsburgh, PA). Plasmid DNA will be extracted using Qiagen Miniprep Kit following manufacturer's protocol (Qiagen, Valencia, CA) and sequenced.The sequence cannot be compared to related sequences in GenBank because there are no gene sequences for hem russet mite. This will be the first sequence of russet mite to be deposited in NCBI GenBank. The total plasmid size will be estimated by adding product size expected from the COI and ITS2 primers to 2817-bp of plasmid. The mass of the plasmid containing the COI and ITS2 genes will be calculated using the following formula (Biosystems, 2013), where m = mass and n = plasmid size (bp): m = (n) (1.096e-21g/bp). Ten-fold dilutions series, ranging from 7.54 x 108 copies/µl to 7.54 x 101 copies/µl, will be prepared in RNase-free water for obtaining a standard curve. The qPCR Mastermix for SYBR Green® (BioRad, Berkeley, CA) will be used according to manufacturer's instructions. Each reaction contained 5.0 µl of SYBR Green Taq, 0.5 µl of F primer, 0.5 µl of R primer, 2.0 µl of deionized water, and 2.0 µl of cDNA template.The qPCR will be run on a CFX Connect® (BioRad, Berkeley, CA) thermocycler, and the mean threshold cycles (Cq) values were calculated by the CFX Manager™ Software Version 3.1. The cycling conditions will likely be: 95°C for 2 minutes; followed by 40 cycles of 95°C for 10 seconds, 55°C for 30 seconds, and then the final melt curve, starting at 65°C increasing to 95°C, in increments of 0.5°C every 5 seconds. Each reaction plate contained a negative (water) and positive control and standard curve along with mite samples will be performed in duplicate. PCR efficiencies (E) of COI and ITS2 were determined using the LinRegPCR software (Ruijter et al., 2009). The mite counts in each sample will be estimated by interpolating the Cq value of COI and ITS2 against the standard curve.