Sandra Fuselli

An in vitro Evaluation of Tagetes minuta Essential Oil for the Control of the Honeybee Pathogens Paenibacillus larvae and Ascosphaera apis, and the Parasitic Mite Varroa destructor

Abstract Biological activity was evaluated of the Tagetes minuta essential oil in different in vitro laboratory experiments on the mite (Varroa destructor), honeybees (Apis mellifera), the bacterium that causes the American Foulbrood (Paenibacillus larvae), and the fungus that produces chalkbrood (Ascosphaera apis). Two methods of complete exposure were used for mite lethality test: by spraying in Burgerjons tower with 10 mg of active ingredient in solution with distilled water and emulsion, and in unmodified Petri dishes (60 x 20 mm) with oil (different concentrations) diluted in 1 mL of ethanol. Ratio selection was obtained as: LD50 of Apis mellifera/LD50 of V. destructor. Determination of Minimal Inhibitory Concentrations (MIC): to P. larvae was tested at concentrations of 25, 50, 100, 150, 200, 250, 350, 450, 500, 600, 650, 700, 800 and 1000 ppm. An A. apis strain was grown on agar MY20 supplemented with variable concentrations (between 0–800 ppm) of T. minuta oil was evaluated. Results obtained in tests of total exposure showed that the concentration was able to kill 50% of mites in 24 h (DL50) and was estimated to be 4.37 mg/cage. The efficacy after spray treatment reached 56%. The ratio selection was 3:11. Against P. larvae, the oil showed MIC values ranging from 700–800 μL/L depending on the tested bacterial strains. Tagetes minuta oil in agar MY20 inhibited mycelial growth of A. apis above concentrations of 200 ppm (p = 0.0001). Oil concentrations of 700 and 800 ppm achieved maximum growth inhibition of A. apis (67% of growth inhibition on average). Tagetes minuta oil demonstrated in vitro antibacterial, antifungical and miticide activity, although this oil shows a moderate inhibitor effect compared with other essential oils of native plants from Argentina. However, this oil presents a ratio selection that would allow it to be used in field conditions with a good safety margin. It is possible that this oil can be used in combination with others, in integrated pest management strategies in bee colonies.

Efficacy of Indigenous Plant Essential Oil Andean Thyme (Acantholippia seriphioides A. Gray) to Control American Foulbrood (AFB) in Honey Bee (Apis mellifera L.) Hives

Abstract Headspace volatiles of an oil of an autochthonous Argentinean plant, Andean thyme (Acantholippia seriphioides A. Gray) and its in vitro antimicrobial activity against thirteen strains of Paenibacillus larvae, causal agent of American foulbrood (AFB), was determined. This disease affects honey bees (Apis mellifera L.) colonies worldwide causing great economical loses. The antimicrobial activity of the essential oil was evaluated using the broth microdilution method. Seven compounds representing 93.1% of the total of this oil were identified in the headspace of the oil using SPME-GC/MS analysis. The main components were thymol (29.2%), p-cymene (23.3%), carvacrol (23.3%) and γ-terpinene (11.0%). One way ANOVA for minimum inhibitory concentration (MIC) data and minimal bactericide concentration (MBC) data, indicated highly significant differences between the P. larvae strains. Newman-Keuls test determined two homogeneous groups (α=0.05) corresponding to Argentinian and Italian strains with different responses to Andean thyme oil. The use of essential oils in the control of microbial strains allows an alternative scope for the control of this serious disease affecting honey and its by-products (wax, pollen and propolis). Such strategy can meet consumer demand for a diminution or absence of other antimicrobial chemical substances, which can be substituted by the addition of natural substances.

Laboratory Evaluation of Heterothalamus alienus Essential Oil Against Different Pests of Apis mellifera

Abstract The Heterothalamus alienus oil was investigated in laboratory for control of different pests that affect the colonies of bees, Apis mellifera, against Varroa destructor mite, the bacterium that causes the American Foulbrood, Paenibacillus larvae and the fungus that produces the chalkbrood, Ascosphaera apis. The oil composition was analyzed by GC and GC/MS, the main components of the oil were β-pinene (44.4%) and trans-muurola-4(14),5-diene (9.2%). The concentration to kill 50% of the mites in 24 h (LC50) was 0.59 mg/cage. Inferior and superior limits were the following ones: 0.34 mg/cage and 1.01 mg/cage, LC50 was estimated for 48 h, and 72 h showed a slight increase with respect to the record of the 24 h. Paenibacillus larvae strains were Gram positive and catalase negative, the oil presented MIC values of 800–900 mg/L and MBC of 1000–1200 mg/L. Disks of impregnated flter paper with H. alienus oil around colonies of A. apis in growth inhibited the micelial growth signifcantly by 51% in the frst experiment (seven days) and by 31% in the second experiment (eight days).

Susceptibility of the Honeybee Bacterial Pathogen Paenibacillus larvae to Essential Oils Distilled from Exotic and Indigenous Argentinean Plants

Abstract Antimicrobial properties and chemical composition of eight exotic and indigenous Argentinean essential oils to control American Foulbrood (AFB) were determined. The oils tested were extracted from Artemisia absinthium, Artemisia annua, Lepechinia floribunda, Aloysia polystachya, Verbena officinalis, Wedelia glauca, Satureja odora and Hetherothalamus alienus. The components of the essential oils were identified by SPME-GC/MS analysis. The antimicrobial activity of the oils against Paenibacillus larvae were determined by broth microdilution method. Two-way ANOVA for minimum inhibitory concentrations (MICs) data and minimal bactericide concentrations (MBCs) data, indicated no significant differences between the thirteen P. larvae strains tested and highly significant differences between the oils. The antimicrobial assays showed that the oils of A. absinthium, A. annua and L. foribunda inhibited the bacterial strains at the lowest concentrations tested, MICs and MBCs averages of 393.6 mg/L to 416.7 mg/L and 517.9 mg/L to 624.4 mg/L respectively, indicating the highest inhibitory capacities. Their antimicrobial activities were attributed to the kind and the variation in percentage of the components. α-Thujone (62.3%) was largely predominant in the oil of the A. absinthium meanwhile artemisia ketone (36.3%) and 1,8-cineole (31.5%) constituted the bulk of the oil of A. annua. 1,8-Cineole (27.5%) was also very important in the composition of the oil of L. floribunda, which also possessed camphene (16.6%) and camphor (12.9%).

Effects of Lactobacillus Johnsonii AJ5 Metabolites on Nutrition, Nosema Ceranae Development and Performance of Apis Mellifera L.

Abstract The European honey bee (Apis mellifera L.) is known to be affected by such stress factors as pathogen load, poor nutrition and depressed immunity. Nosema ceranae is one of the main parasites that affect colony populations. The relationship between the stress factors and honey bee-bacteria symbiosis appears as an alternative to enhance bee health. The aim of this study was to evaluate the effect of the oral administration of bacterial metabolites produced by Lactobacillus johnsonii AJ5 on nutritional parameters, the N. ceranae development and the performance of A. mellifera colonies. Laboratory assays were performed and demonstrated that the bacterial metabolites did not have a toxic effect on bees. Field trial showed an increase of colonies population over time. Also, a decreasing trend of fat bodies per bee was detected in all colonies but there were no evident changes on abdomen protein content at the end of the assay. Lastly, N. ceranae prevalence showed a tendency to reduce with the organic acids. Future studies should be performed to increase our knowledge of the physiological effects of bacterial metabolites on the health of bee colonies.