Copper Stimulation of Tetrahydrocannabinol and Cannabidiol Production in Hemp (Cannabis sativa L.) Is Copper-Type, Dose, and Cultivar Dependent.

Copper (Cu) is an element widely used as a pesticide for the control of plant diseases. Cu is also known to influence a range of plant secondary metabolisms. However, it is not known whether Cu influences the levels of the major metabolites in hemp (Cannabis sativa L.), tetrahydrocannabinol (THC) and cannabidiol (CBD). This study investigated the impact of Cu on the levels of these cannabinoids in two hemp cultivars, Wife and Merlot, under field conditions, as a function of harvest time (August-September), Cu type (nano, bulk, or ionic), and dose (50, 100, and 500 ppm). In Wife, Cu caused significant temporal increases in THC and CBD production during plant growth, reaching increases of 33% and 31% for THC and 51% and 16.5% for CBD by harvests 3 and 4, respectively. CuO nanoparticles at 50 and 100 ppm significantly increased THC and CBD levels, compared to the control, respectively, by 18% and 27% for THC and 19.9% and 33.6% for CBD. These nanospecific increases coincided with significantly more Cu in the inflorescences (buds) than in the control and bulk CuO treatments. Contrarily, no temporal induction of the cannabinoids by Cu was noticed in Merlot, suggesting a cultivar-specific response to Cu. However, overall, in Merlot, Cu ions, but not particulate Cu, induced THC and CBD levels by 27% and 36%, respectively, compared to the control. Collectively, our findings provide information with contrasting implications in the production of these cannabinoids, where, dependent on the cultivar, metabolite levels may rise above the 0.3% regulatory threshold for THC but to a more profitable level for CBD. Further investigations with a wider range of hemp cultivars, CuO nanoparticle (NP) doses, and harvest times would clarify the significance and broader implications of the findings.

Biologically synthesized zinc and copper oxide nanoparticles using Cannabis sativa L. enhance soybean (Glycine max) defense against fusarium virguliforme.

In this study, zinc and copper oxide nanoparticles (NPs) were synthesized using hemp (Cannabis sativa L.) leaves (ZnONP-HL and CuONP-HL), and their antifungal potential was assessed against Fusarium virguliforme in soybean (Glycine max L.). Hemp was selected because it is known to contain large quantities of secondary metabolites that can potentially enhance the reactivity of NPs through surface property modification. Synthesizing NPs with biologically derived materials allows to avoid the use of harsh and expensive synthetic reducing and capping agents. The ZnONP-HL and CuONP-HL showed average grain/crystallite size of 13.51 nm and 7.36 nm, respectively. The biologically synthesized NPs compared well with their chemically synthesized counterparts (ZnONP chem, and CuONP chem; 18.75 nm and 10.05 nm, respectively), confirming the stabilizing role of hemp-derived biomolecules. Analysis of the hemp leaf extract and functional groups that were associated with ZnONP-HL and CuONP-HL confirmed the presence of terpenes, flavonoids, and phenolic compounds. Biosynthesized NPs were applied on soybeans as bio-nano-fungicides against F. virguliforme via foliar treatments. ZnONP-HL and CuONP-HL at 200 µg/mL significantly (p < 0.05) increased (∼ 50%) soybean growth, compared to diseased controls. The NPs improved the nutrient (e.g., K, Ca, P) content and enhanced photosynthetic indicators of the plants by 100-200%. A 300% increase in the expression of soybean pathogenesis related GmPR genes encoding antifungal and defense proteins confirmed that the biosynthesized NPs enhanced disease resistance against the fungal phytopathogen. The findings from this study provide novel evidence of systemic suppression of fungal disease by nanobiopesticides, via promoting plant defense mechanisms.

Effectiveness of Garlic for the Control of Ixodes scapularis (Acari: Ixodidae) on Residential Properties in Western Connecticut.

We conducted field trials to evaluate the ability of a garlic juice-based product to control or suppress nymphal activity of the blacklegged tick, Ixodes scapularis Say, at residential properties in Connecticut in 2009, 2010, and 2011. The product was applied at a rate of 0.2 g AI/m2. Percent control of nymphal densities achieved by the spray treatment at 6, 11, and 18 d postspray for the 3 yr was 37.0, 59.0, and 47.4%, respectively. Differences between nymphal densities were greatest during the first post-spray sampling period. While garlic may require multiple applications for the suppression of tick activity, this product could provide a minimal-risk option for the short-term control of nymphal I. scapularis in the residential landscape.

Expansion of zoonotic babesiosis and reported human cases, Connecticut, 2001-2010.

To document the expansion of human babesiosis in Connecticut, we analyzed reservoir host sera for seroreactivity to Babesia microti Franca and reviewed Connecticut human surveillance case data collected during 2001-2010. Sera from white-footed mice, Peromyscus leucopus Rafinesque, from 10 towns in 5 counties, collected at 4-7-yr periods between 2001 and 2010, were tested for total immunoglobulins. The prevalence of B. microti-positive mice was compared with confirmed and probable human case reports tabulated by the Connecticut Department of Public Health. The highest babesiosis and rodent seroprevalence rates were in New London County, where this protozoan disease was first documented in the state. However, human cases and reservoir host infection increased significantly from 2001-2005 to 2005-2010 and in other parts of the state. Clinicians should be aware that the disease is not confined to long-established endemic areas of the state.

Efficacy and environmental persistence of nootkatone for the control of the blacklegged tick (Acari: Ixodidae) in residential landscapes.

The ability of the plant-derived compound nootkatone to control nymphs of the blacklegged tick, Ixodes scapularis Say, was evaluated at lawn perimeter plots at homes in Lyme disease endemic areas of Connecticut. Three formulations of nootkatone ranging from 0.05 to 0.84% (0.06 - 1.03 g AI/m2) were applied by a hydraulic sprayer from 2008 to 2010. In 2008, the 0.84% emulsifiable nootkatone formulation provided 100% control of I. scapularis through week 1, but declined to 49 and 0% by 2 and 3 wk posttreatment, respectively. A combination of 0.05% nootkatone and entomopathogenic fungus, Metarhizium brunneum Petch F52, resulted in 50% control for the first week posttreatment and no control in subsequent weeks. The 0.84% emulsifiable nootkatone formulation was phytotoxic, although no damage was observed with the 0.05% formulation with Metarhizium. Residual analysis of nootkatone collected on filter paper disks showed that > or = 95% of the emulsified nootkatone for both formulations was lost within 7 d after application. A lignin-encapsulated nootkatone formulation (0.56 and 0.46% in 2009 and 2010, respectively) provided 100% control of I. scapularis for 8 wk in 2009 and, in 2010, 67% control at approximately 1 wk posttreatment with respect to the pretreatment counts, although there was no difference in tick abundance posttreatment. A 0.60% Maillard-reaction encapsulated nootkatone formulation in 2010 provided a similar level of control (62%). Nootkatone in the lignin and Maillard formulations were more persistent than the emulsifiable formulation. Little or no phytotoxicity was observed with the encapsulated formulations. Encapsulating nootkatone reduced phytotoxicity and appeared to reduce environmental loss. While nootkatone can provide effective tick control, further work is needed to refine formulations to address phytotoxicity, yet provide sufficient material to control ticks.

Susceptibility of Ixodes scapularis (Acari: Ixodidae) to Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) using three exposure assays in the laboratory.

An emulsifiable concentrate (EC) and granular (G) formulation of the entomopathogenic fungus, Metarhizium brunneum strain F52 (formerly Metarhizium anisopliae strain F52) were tested against unfed adults and nymphs of Ixodes scapularis Say in the laboratory. Three exposure methods; dip, surface contact, and direct spray application, and three exposure time intervals (3, 30, and 300 min) were used to evaluate the EC formulation. Application rates ranged from 2.6 x 10(2) to 2.6 x 10(8) conidia/cm2. The surface treatment was used for granular formulation with concentrations ranging from 2.3 x 10(5) to 2.3 x 10(7) conidia/cm2 for same three exposure times. Both the EC and G formulations of this fungus were highly pathogenic against I. scapularis adults and nymphs. Logistic regression analysis found formulation, spore concentration, time of exposure, and observation period were significant or highly significant factors influencing tick mortality. For adult I. scapularis, the spray application with the EC formulation of M. brunneum F52 resulted in a lower LC50 (5.9 x 10(4) conidia/cm2) at 30 min than surface exposure to the EC (LC50 = 1.3 x 10(6) conidia/cm2) or G formulation (LC50 = 8.1 x 10(5) conidia/cm2). At higher concentrations, fungal activity was evident in adult I. scapularis held at 5 degrees C suggesting the fungus may provide control in the cooler fall season. While the observed pathogenicity of a fungus against ticks can be dependent upon the bioassay assessment, we found nymphs and adults of I. scapularis to be highly susceptible to M. brunneum F52, regardless of the exposure method used.

Potential of Tenebrio molitor (Coleoptera: Tenebrionidae) as a bioassay probe for Metarhizium brunneum (Hypocreales: Clavicipitaceae) activity against Ixodes scapularis (Acari: Ixodidae).

The yellow mealworm, Tenebrio molitor L., has been used to indicate qualitatively the presence of entomopathogenic fungi in the soil or as a model for evaluating stress and other factors on fungal activity. Although this beetle appears highly susceptible to many of these fungi, little quantitative information is available on the sensitivity of T. molitor to a specific fungus and, therefore, fungal presence or as an indicator for pathogenicity to other species. The purpose of this study was to establish the suitability of T. molitor larvae as a bioassay probe for Metarhizium brunneum for comparison against the blacklegged tick, Ixodes scapularis. Nine concentrations of M. brunneum strain F52 ranging from 1.0 x 10(1) to 8.4 x 10(8) conidial/ml were simultaneously tested against T. molitor larvae and I. scapularis adults. Larvae of yellow mealworm were less sensitive to M. brunneum than I. scapularis adults (LC50's 4.4 x 10(7) and 1.7 x 10(5) conidia/ml, respectively, 4-wk post-treatment). The greater sensitivity of I. scapularis to the fungus suggests that the detection of fungal mycosis in mealworms would indicate sufficient inoculum to be pathogenic to I. scapularis and make this insect a suitable probe for evaluation of the presence and activity of M. brunneum against the blacklegged tick in field applications.

A formulation to encapsulate nootkatone for tick control.

Nootkatone is a component of grapefruit oil that is toxic to the disease-vectoring tick, Ixodes scapularis Say, but unfortunately causes phytotoxicity to treated plants and has a short residual activity due to volatility. We prepared a lignin-encapsulated nootkatone formulation to compare with a previously used emulsifiable formulation for volatility, plant phytotoxicity, and toxicity to unfed nymphs of I. scapularis. Volatility of nootkatone was measured directly by trapping nootkatone vapor in a closed system and indirectly by measuring nootkatone residue on treated filter paper after exposure to simulated sunlight (Xenon). After 24 h in the closed system, traps collected only 15% of the nootkatone applied as the encapsulated formulation compared with 40% applied as the emulsifiable formulation. After a 1-h light exposure, the encapsulated formulation retained 92% of the nootkatone concentration compared with only 26% retained by the emulsifiable formulation. For plant phytotoxicity, cabbage, Brassica oleracea L., leaves treated with the encapsulated formulation expressed less necrosis, retaining greater leaf weight compared with leaves treated with the emusifiable formulation. The nootkatone in the emulsifiable formulation was absorbed by cabbage and oat, Avena sativa L., plants (41 and 60% recovered 2 h after application, respectively), as opposed to 100% recovery from the plants treated with encapsulated nootkatone. Using a treated vial technique, encapsulated nootkatone was significantly more toxic to I. scapularis nymphs (LC50 = 20 ng/cm2) compared with toxicity of the emulsifiable formulation (LC50 = 35 ng/cm2). Thus, the encapsulation of nootkatone improved toxicity for tick control, reduced nootkatone volatility, and reduced plant phytotoxicity.

Evaluation of Metarhizium anisopliae strain F52 (Hypocreales: Clavicipitaceae) for control of Ixodes scapularis (Acari: Ixodidae).

Field efficacy of an emulsifiable concentrate formulation of the entomopathogenic fungus Metarhizium anisopliae strain F52 for the control of Ixodes scapularis nymphs was evaluated at residential sites in northwestern Connecticut in 2007. Two spray applications with two rates, 3.2 x 10(5) and 1.3 x 10(6) spores/cm2, were made: the first on 8-9 May, 2-3 wk before nymphal activity, and the second on 29 June or 2 July when ticks were active. There was no significant difference in nymphal abundance between the three treatment groups (P = 0.490) after the first application, indicating that preseason or early applications are not effective, despite a bioasaay with yellow mealworms that showed spores in the treated areas was infective for at least 1 mo postapplication. By contrast, there was a significant difference in the number of nymphs collected between the treatments and control 3 wk (F = 16.928, df = 2, P < 0.001) and 5 wk (F = 6.627, df = 2, P = 0.002) after the second application. During the 3 wk after the second application, 87.1 and 96.1% fewer ticks were collected from lower and higher rate-treated sites, respectively, and after 5 wk, tick reductions were 53.2 and 73.8%, respectively. Over one- third (36.4% of 173) of the nymphs collected from the treated sites developed mycosis from M. anisopliae. The application of M. anisopliae strain F52 could provide another tool for the integrated approach to managing ticks in the residential landscape.