Evaluation of the Efficacy, Agronomic Fit, and Environmental Fate of Biopesticides for Management of Economically Important Diseases and Arthropod Pests of Hemp

FFAR-BioWorks-Cornell-UKY Co-PI: Gauthier and Villanueva, UKY Evaluation of the efficacy, agronomic fit, and environmental fate of biopesticides for management of economically important diseases and arthropod pests of hemp : Goals 3 and 4 Goal 3. Microbial persistence. GAUTHIER Quantifying microbes on plant foliage is common practice in industry and academic research (BioWorks does this regularly for both bacteria and fungi). Drawing on previous expertise of Consortium scientists suggests that minimal time and effort will be spent developing test methods. Ergo, we anticipate that successfully delivering on the proposal in eighteen months is a highly achievable goal. Methods i. Test products Given the nature of the trials, i.e., to monitor persistence on flowers, we will use the highest label rate for each listed product to simplify the experiments. Treatments Test Rate PlantShield HC 10 g / L 1 4 lbs / 100 gals 4 qts / 100 gals BotryStop WP 2 qts / 100 gals 2 N/A RD00AS-1 (B. subtilis) 3 NI00OD-1 (B. bassiana) 4 Untreated control 5 ii. Application protocols. Each product will be sprayed onto plants two to three weeks into flowering a total of 4 times at 7-day intervals, with the final spray made ca. 3-weeks prior to harvest per current recommendations. Flowers will be sampled 24 hours prior to and after each spray. After the final spray, flowers will be sampled weekly to harvest (allowing repeated measures analyses). In addition to evaluating persistence, we will evaluate the efficacy of a mitigation strategy (indoors), whereby a sub-set of flowers will be sprayed with PERpose Plus (hydrogen peroxide) twice prior to harvest at 1 oz/gal. Microbes will be enumerated microbes and compared to untreated flowers. Tests will be done on flowers grown indoors and outdoors. UK: ''White'' (v. PM susceptible), ''Oregon CBG'', X-59; Cornell: Kayagene cultivars will be grown for the greenhouse experiments. Two other cultivars (Maverick and Mr. Chunk) will be grown from seed and used as mother plants for cuttings. Experiments will be replicated twice, over two field/growing seasons to accommodate variation in climatic conditions and their influence on spore survival. iii. Enumeration methods. Two methods that have previously been utilized will be tested: a. One to three flowers will be collected from at least five plants selected at random from within the treated group. Each sample will be bagged separately to provide replicate sub-samples. Flowers will be washed in sterile Tween-80 or Triton X-100 on a shaker or by vortexing with glass beads. A dilution series will be prepared from the resulting suspension; 100µL aliquots from the 0X, 10X and 100X and 1000X dilutions will be plated onto duplicate plates of selective media and CFUs counted 3- to 5-days later. Note: For plants sprayed with Bacillus, a 5 mL sample of the stock suspension will first be heat shocked at 70 °C for 30 seconds to eliminate non-spore forming bacteria, yeasts and fungi. b. Flower samples will be collected as outlined above (a). Ten grams of will be blended with 500 mL water (may include Triton/Tween to aid wetting and release of spores from flowers). The resulting mix will be filtered through 4 layers of sterile muslin gauze to remove plant debris and a dilution series prepared from the stock. Dilutions will be plated onto duplicate plates of selective media and CFUs counted 3- to 5-days later. Note: For plants sprayed with Bacillus, a 5 mL sample of the stock suspension will first be heat shocked at 70 °C for 30 seconds to eliminate non-spore forming bacteria, yeasts and fungi. iv. Statistical analysis Data for each product will be assessed using a one-way ANOVA in combination with a one-way repeated measures ANOVA where appropriate. Goal 4. Biopesticide efficacy against key target pests of hemp. VILLANUEVA Trials will be initiated in Yr 2 of the project and will focus on two primary pests of field- and greenhouse-grown hemp: cannabis aphid, Phorodon cannabis; and hemp russet mite, Aculops cannabicola. The relative efficacy of horticultural and essential oils, at different rates, will be evaluated against both pests. Methods i. Test products Product(s) Rate Frequency Method Name N/A Untreated Control N/A N/A N/A Foliar Standard (TBD) Foliar SuffOil-X-1% TBD TBD TBD Foliar SuffOil-X-2% Foliar NI02ES-1-07% SuffOil-X 1% v/v 3 Apps; 7-day interval Foliar NI02ES-1-1% SuffOil-X 2% v/v 3 Apps; 7-day interval NI02ES-1 0.7% v/v 3 Apps; 7-day interval NI02ES-1 1% v/v 3 Apps; 7-day interval All products will be applied as foliar sprays, targeting upper and lower leaf surfaces and stems. ii. Trial protocols Cannabis aphid. Trials will be conducted on confirmed cannabis aphid populations on hemp plants in greenhouse or field settings, containerized or in-ground plants. To ensure test populations are at experimentally-appropriate levels within the timeframe of the project, hemp plants will be inoculated with at least 10 adult aphids 1- to 2-weeks prior to the initial treatment application. Each treatment will have six to eight replicates arranged in a Complete Randomized Design (CRD) or Randomized Complete Block Design (RCBD). Aphid populations will be assessed prior to spraying (0 DAIT), and subsequently at 3, 7, 14, and 28 Days After Initial Treatment (DAIT). Plants will also be examined for evidence of phytotoxicity at 0, 3 and 28 DAIT. Hemp russet mite. Trials will be conducted on confirmed hemp russet mite populations on hemp plants in greenhouse or field settings, containerized or in-ground plants. Test populations will be established by placing heavily infected leaves from a colony plant onto the experimental plants. Mites will be allowed to establish for approximately 7-days or when mite density reaches ca. 50 mites per leaf. Each treatment will have 6 – 8 replicates arranged in a Complete Randomized Design (CRD) or Randomized Complete Block Design (RCBD). Hemp russet mite populations will be assessed prior to initial sprays (0 DAIT), and subsequently 3, 7, 14, and 28 Days After Initial Treatment (DAIT). iii. Statistical analysis Raw data were subject to Abbot’s transformation to allow comparison of treatments to the untreated check. The normality of the data will be confirmed and means separated using Fisher’s Protected Least Significant Difference (LSD, p<0.05).