Invitro and in vivo anthelmintic efficacy of peppermint (Mentha x piperita L.) essential oil against gastrointestinal nematodes of sheep.

Nowadays, the exclusive use of commercial anthelmintics for the treatment of gastrointestinal nematode infections in ruminants is less sustainable due to anthelmintic resistance, as well as the problem of drug residues in animal products and the environment. Therefore, an integrated therapeutic approach is needed, including the search for alternatives to synthetic anthelmintic drugs. The aim of this study was to evaluate the possibility of using the essential oil of peppermint (Mentha x piperita L.) in the control of gastrointestinal nematodes in sheep. For this purpose, the in vitro and in vivo anthelmintic efficacy of this oil and the toxic effects on the hosts were examined. In the in vitro egg hatch test, ovicidal activity varied from 21.0-90.3% depending on the concentration of essential oil used (0.0125, 0.025, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 mg/mL). To some extent, anthelmintic efficacy was confirmed in the in vivo fecal egg count reduction test at a mean dose of 150 mg/kg, with an average reduction of nematode eggs of 26.9 and 46.0% at Days 7 and 14 after treatment, respectively. Furthermore, no toxic effects of applied oil were observed on sheep behavior, kidney, or liver function. The main compounds identified by gas chromatography-mass spectrometry analyzes were menthol (32.6%), menthone (22.0%), menthyl-acetate (10.0%), and isomenthone (9.39%). Due to their complex chemical compositions, numerous bioactive ingredients, and natural origin, herbal formulations represent a potentially valuable alternative for the control of gastrointestinal nematodes in sheep. In this context, the results of the present study showed that peppermint essential oil is one of the promising candidates. Further studies should be performed to collect more data on the safety profile of M. piperita EO in treated animals to find the most appropriate formulation for use in field conditions and to test it against resistant gastrointestinal nematode populations.

Anthelmintic Properties of Essential Oils to Control Gastrointestinal Nematodes in Sheep-In Vitro and In Vivo Studies.

Herbal products such as essential oils may play a promising role in the treatment of infections caused by gastrointestinal nematodes (GINs). The aim of this study was to evaluate the in vitro potential of 11 essential oils (EOs) and one binary combination of isolated EO compounds, as well as the in vivo anthelmintic efficacy of two EO formulations. Four GIN genera were identified in the coproculture examination: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia. The in vitro egg hatch test (EHT) was performed at six different concentrations (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL) for each EO, whereas in the in vivo faecal egg count reduction test (FECRT), each EO sample was diluted in sunflower oil and orally administrated at a dose of 100 mg/kg to the different group of animals. In the EHT, the EOs of Origanum vulgare, Foeniculum vulgare, Satureja montana, Satureja hortensis and two types of Thymus vulgaris were the most effective. The dominant compounds of these EOs were carvacrol, thymol, anethol, p-cymene and γ-terpinene, indicating their importance for the anthelmintic activity. In the FECRT, both T. vulgaris EO type 1 and linalool:estragole combination show an anthelmintic potential with a mean effect on FECR of approximately 25%. The results suggest the possible role of tested EOs as anthelmintic agents in sheep farms, although further in vivo tests are needed.

A Potential Anthelmintic Phytopharmacological Source of Origanum vulgare (L.) Essential Oil against Gastrointestinal Nematodes of Sheep.

The development of anthelmintic resistance in sheep gastrointestinal nematodes (GINs) requires novel strategies for the sustainable control of these parasites. This study aimed to evaluate the anthelmintic efficacy of the Origanum vulgare (L.) essential oil (EO) against sheep GINs and to evaluate the possibility of its use in control practice. The in vitro egg hatch test was conducted at eight different concentrations (50, 12.5, 3.125, 0.781, 0.195, 0.049, 0.025, and 0.0125 mg/mL) of the tested EO. For the in vivo fecal egg count reduction test, the EO of O. vulgare was administrated orally at a mean single dose of 150 mg/kg to sheep from two farms in Southern Italy, whereby potential toxic effects to the hosts were also evaluated. In the egg hatch test, the inhibition of egg hatchability varied from 71.3% to 93.7%, depending on the concentration used. The high anthelmintic potential was confirmed in the fecal egg count reduction test with an average reduction of nematode eggs in feces of 43.2% and 60.1% on days 7 and 14 after treatment, respectively. In addition, no toxic effects were noticed during the clinical examination of sheep or by observing blood count and liver or kidney function test results. The obtained results suggest the strong activity of the O. vulgare EO against sheep GINs, probably due to a high percentage of carvacrol (76.21%), whereby it can be considered safe for sheep at the dose tested in vivo. Therefore, it is suitable for use in veterinary practice as a part of an integrated strategy for the control of sheep GINs.

Repellent activity of Tanacetum parthenium (L.) and Tanacetum vulgare (L.) essential oils against Leptinotarsa decemlineata (Say).

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is one of the most destructive pest species to have developed resistance to most chemical insecticides. We determined the composition and evaluated the potential of Tanacetum parthenium L. and Tanacetum vulgare L. (Asteraceae family) essential oil (EO) application as an alternative eco-friendly control strategy against L. decemlineata. We assessed the antifeedant activity for L. decemlineata larvae and adults by estimating the damage to potato leaves treated with three concentrations of EOs dissolved in ethanol (0.125, 0.25 and 0.5%). Results showed that T. parthenium EO was more effective against larvae, and T. vulgare was more effective against adults. In an olfactometer assay, the time required to choose an untreated leaf disc did not depend on the Tanacetum species, or life stage examined. However, the concentration of EO exhibited a significant effect on the behaviour of both developmental stages. At higher EO concentrations, both third instar larvae and adults require less time to choose an untreated leaf disc. Additionally, T. parthenium EO provoked more rapid movement away from the treated leaf disc than T. vulgare, especially at the highest concentration. Successful modification of L. decemlineata behaviour by the two Tanacetum oils suggests that they possess the potential for use in potato protection.

Nematicidal Activity of Essential Oils on a Psychrophilic Panagrolaimus sp. (Nematoda: Panagrolaimidae).

Essential oils (EOs) have historically been used for centuries in folk medicine, and nowadays they seem to be a promising control strategy against wide spectra of pathogens, diseases, and parasites. Studies on free-living nematodes are scarce. The free-living microbivorous nematode Panagrolaimus sp. was chosen as the test organism. The nematode possesses extraordinary biological properties, such as resistance to extremely low temperatures and long-term survival under minimal metabolic activity. Fifty EOs from 22 plant families of gymnosperms and angiosperms were tested on Panagrolaimus sp. The aims of this study were to investigate the in vitro impact of EOs on the psychrophilic nematode Panagrolaimus sp. in a direct contact bioassay, to list the activity of EOs based on median lethal concentration (LC50), to determine the composition of the EOs with the best nematicidal activity, and to compare the activity of EOs on Panagrolaimus sp. versus plant parasitic nematodes. The results based on the LC50 values, calculated using Probit analysis, categorized the EOs into three categories: low, moderate and highly active. The members of the laurel family, i.e., Cinnamomum cassia and C. burmannii, exhibited the best nematicidal activity. Aldehydes were generally the major chemical components of the most active EOs and were the chemicals potentially responsible for the nematicidal activity.

When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe.

Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties.

Phytophthora ramorum Occurrence in Ornamentals in Serbia.

In a survey to determine the presence of Phytophthora ramorum in Serbia, ornamentals from garden centers, nurseries, and private and public gardens, as well as imported plant material, were inspected. In total, 577 plant, soil, and potting media samples were tested using various detection methods: lateral flow diagnostic test, enzyme-linked immunosorbent assay, conventional polymerase chain reaction, and isolation, followed by identification based on growth characteristics in culture and morphological features. P. ramorum was not detected in any of the 162 soil or potting media tested by the baiting method. P. ramorum was detected in 12 Rhododendron samples from one private garden in Zemun (City of Belgrade District) exhibiting symptoms of leaf necrosis and blight and petiole necrosis, and in three samples of Pieris spp. from one garden center exhibiting symptoms of leaf necrosis. Eight Phytophthora isolates were obtained from the positive Rhododendron plants and three isolates from Pieris plants, and all were identified as P. ramorum on the basis of their uniform morphological and growth characteristics. P. ramorum conformation was also made by sequencing of the internal transcribed spacer regions for a single isolate taken from one infected rhododendron and one pieris plant. Serbian isolates were determined as A1 mating type, due to formation of a few typical sexual structures when crossed with the A2 mating type of P. cinnamomi and P. cryptogea. Pathogenicity test on nonwounded detached leaves of 19 popular ornamentals, as well as the most frequently imported ones, revealed that 10 host species were susceptible, including Robinia pseudoacacia, which is widely distributed in Serbia. During this study, Cotoneaster horizontalis and C. dammeri were determined to be new experimental hosts of P. ramorum. This article provides evidence of P. ramorum introduction into Serbia. Although P. ramorum has not been detected in Serbian production nurseries, its presence outdoors might cause severe damages on susceptible common urban plants in public green and natural ecosystems.

Incidence and Distribution of Iris yellow spot virus on Onion in Serbia.

In a survey to determine the presence and distribution of Iris yellow spot virus (IYSV) in greenhouse ornamentals and onion field crops in 14 districts of Serbia as well as on imported ornamental plants, 1,574 samples were collected and analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). IYSV was not detected in nearly 1,200 plant samples collected from 39 genera of ornamentals grown in greenhouses in Serbia or imported from other countries during 2005 to 2007. The virus was detected in samples from an onion seed crop in the Sirig locality (South Backa District) that showed symptoms resembling those caused by IYSV and in samples without IYSV-like symptoms from an onion bulb crop in the Obrenovac locality (City of Belgrade District). Mechanical transmission of IYSV isolates was difficult, and only the isolate 605-SRB could infect four plant species, but not in all replications. No virus transmission could be demonstrated in 5,000 tested seeds originating from IYSV-infected onion crops. For further confirmation of IYSV, the nucleotide sequence of its nucleocapsid (NC) gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) in symptomatic onion samples as well as in symptomless leaves of Nicotiana benthamiana. Four previously developed primers were tested to determine their suitability for routine detection of Serbian IYSV isolates. Phylogenetic analysis showed clustering of isolates 605-SRB and 622-SRB from the onion seed crop and isolate 283-SRB from the onion bulb crop into two distant clades. The analysis indicated that Serbian isolates of IYSV do not share a recent common ancestor and that they represent two distinct lineages of IYSV in Serbia. Considering that onion is one of the most important and traditionally grown vegetable crops in Serbia, IYSV represents a potentially devastating pathogen in this country.