Effect of chemical modification of hydrodistillation on yield, composition and biological activity of Rosmarinus officinalis essential oil

Studies on the chemical modifications of Rosmarinus officinalis essential oil hydrodistillation process (HD) by using 5% citric acid (CA-HD) and 5% trisodium citrate (TSC-HD) as a water phase were performed. Composition of essential oils obtained in conventional and modified conditions was analyzed by gas chromatography with mass selective detector method (GC-MS) and compared. Antioxidant activity of all essential oils was determined spectrophotometrically by using DPPH radical scavenging method. It was found that applied modifications of hydrodistillation process enhanced yields and antioxidant activity and the best results were obtained using 5% citric acid as a modifier. Effect of this modification on fungicidal activity of essential oils against 8 various fungi strains (Alternaria alternata, Botrytis cinerea, Fusarium culmorum, Phythophtora cactorum, Rhizoctonia solani, Phythophtora infestans, Sclerotinia sclerotiorum and Ascosphaera apis) was also determined and in most cases enhanced activity was observed.

Biologically Active Extracts from Different Medicinal Plants Tested as Potential Additives against Bee Pathogens

Honey bees (Apis mellifera) perform pollination service for many agricultural crops and contribute to the global economy in agriculture and bee products. However, honey bee health is an ongoing concern, as illustrated by persistent local population decline, caused by some severe bee diseases (e.g., nosemosis, AFB, EFB, chalkbrood). Three natural recipes are in development based on the bioactive compounds of different plants extract (Agastache foeniculum, Artemisia absinthium, Evernia prunastri, Humulus lupulus, Laurus nobilis, Origanum vulgare and Vaccinium myrtillus), characterised by HPLC-PDA. The antimicrobial activity of these recipes was tested in vitro against Paenibacillus larvae, Paenibacillus alvei, Brevibacillus laterosporus, Enterococcus faecalis, Ascosphaera apis and in vivo against Nosema ceranae. A mix of 20% blueberry, 40% absinthium, 10% oakmoss, 10% oregano, 10% Brewers Gold hops, 5% bay laurel and 5% anise hyssop extract showed the strongest antibacterial and antifungal activity. Combing several highly active plant extracts might be an alternative treatment against bee-disease-associated parasites and pathogens, in particular to replace synthetic antibiotics.

First Record of Ascosphaera apis Isolated from Commercial Pollen from Northern Argentinian Provinces

In recent years, increased deforestation and replacement of areas intended for intensive cultivation have caused pathogens and parasites of Apis mellifera bees to emerge in regions where they had not previously been reported. Such is the case of the advancement of Ascosphaera apis, a fungus that causes a drop in colonies’ bee larvae population and great economic losses to beekeeping activity during severe cases. In northern Argentina, few studies have focused on this important entomopathogen. This is the first study on A. apis isolated from commercial pollen in Entre Ríos, Jujuy, and Misiones provinces, where its prevalence in bee colonies has not been reported. We identified this entomopathogen using macroscopic, microscopic, and molecular analyses, focusing on its botanical origin as a possible transmission route. We found the pathogenicity of each strain in A. mellifera bees to below 30%. The results of this study contribute to the promotion of sanitary measures that may reduce this disease’s progression in local apiaries.

The Effect of Artificial Media and Temperature on the Growth and Development of the Honey Bee Brood Pathogen Ascosphaera apis

Ascosphaera apis is a causative agent of chalkbrood, which is one of the most widespread honey bee diseases. In our experiments, the influence of several artificial media and cultivation under different temperatures was evaluated. Concretely, the radial growth of separated mating types was measured, reproductive structures in a Neubauer hemocytometer chamber were counted simultaneously, and the morphometry of spore cysts and spore balls was assessed. The complex set of experiments determined suitable cultivation conditions. A specific pattern between reproductive structure size and temperature was found. The optimal temperature for both mating types was 30 °C. SDA and YGPSA media are suitable for fast mycelial growth. Moreover, the effect of bee brood on fungus growth and development in vitro was investigated by modification of culture medium. The newly modified medium PDA-BB4 was most effective for the production of the reproductive structures. The result suggests that honey bee brood provides necessary nutrients for proper fungus development during in vitro cultivation. As there is no registered therapeutic agent against chalkbrood in most countries, including the European Union, the assessment of A. apis growth and development in different conditions could help to understand fungus pathogenesis and thus control chalkbrood disease.

Probiotic Properties and Potentiality of Lactiplantibacillus plantarum Strains for the Biological Control of Chalkbrood Disease

Ascosphaera apis is an entomopathogenic fungus that affects honeybees. In stressful conditions, this fungus (due not only to its presence, but also to the combination of other biotic and abiotic stressors) can cause chalkbrood disease. In recent years, there has been increasing attention paid towards the use of lactic acid bacteria (LAB) in the honeybees’ diets to improve their health, productivity and ability to resist infections by pathogenic microorganisms. The screening of 22 strains of Lactiplantibacillus plantarum, isolated from the gastrointestinal tracts of honeybees and beebread, led to the selection of five strains possessing high antagonistic activity against A. apis. This study focused on the antifungal activity of these five strains against A. apis DSM 3116 and DSM 3117 using different matrices: cell lysate, broth culture, cell-free supernatant and cell pellet. In addition, some functional properties and the antioxidant activity of the five L. plantarum strains were evaluated. All five strains exhibited high antagonistic activity against A. apis, good surface cellular properties (extracellular polysaccharide (EPS) production and biofilm formation) and antioxidant activity. Although preliminary, these results are encouraging, and in future investigations, the effectiveness of these bacteria as probiotics in honeybee nutrition will be tested in vivo in the context of an eco-friendly strategy for the biological control of chalkbrood disease.

In Vitro Activity of Several Essential Oils Extracted from Aromatic Plants against Ascosphaera apis

The use of natural substances such as essentials oils against bee pathogens is of great interest as an alternative to traditional methods based on synthetic compounds like antibiotics and fungicides, in order to minimize the risk of having toxic residues in hive products and to prevent the development of resistance phenomena. This study evaluated the inhibitory, fungicidal and sporicidal activity of ten essential oils extracted from aromatic plants against Ascosphaera apis, the etiological agent of chalkbrood, an invasive honey bee mycosis. The most effective essential oils were Thymus herba-barona, Thymus capitatus and Cinnamomum zeylanicum, which showed values of minimum fungicidal concentration and minimum sporicidal concentration ranging from 200 to 400 ppm. Carvacrol was the main component of Thymus capitatus and Thymus herba-barona oils, whereas cinnamic aldehyde prevailed in Cinnamomum zeylanicum oil. Further in-apiary studies will allow the evaluation of side effects on bees and residues in hive products.

Molecular detection of bee pathogens in honey

Honey bees are affected by a wide variety of parasites and pathogens, some of them were previously found in bee honey. Considering that spore-forming microorganisms are expected to remain latent in raw honey, presence of microsporidia, spore-forming bacteria, and viruses protected by peptide structures might represent a threat for bees. It is widely known that parasites and pathogens are one of the main factors implied in the worldwide decline of wild pollinators and colony losses of managed bees, and their detection in honey could be used to prevent the spread of diseases among colonies. Honey from 57 apiaries of Argentina located in Buenos Aires, Córdoba, Corrientes, Entre Ríos, Formosa, La Rioja, Neuquén, Río Negro and Santa Fe provinces was collected between March and October, 2012. DNA was extracted from the pellet obtained by centrifugation of 10 g of honey and pathogen DNA was amplified by qPCR, and products were purified, sequenced and analysed using BLAST software. Honey from every apiary contained DNA of at least one pathogen, with a high occurrence of Apis mellifera filamentous virus (AmFV) and Apicystis bombi, although Nosema ceranae, Paenibacillus larvae, and Ascosphaera apis were also detected. Here, we report the presence of DNA of several bee pathogens in honey from commercial apiaries, and provide a fast and efficient screening method that could be useful to indirectly estimate pathogen presence in apiaries.