Egg Hatching Inhibition Research Articles

Ovicidal and larvicidal potential of Rosmarinus officinalis to control gastrointestinal nematodes of sheep.

Gastrointestinal Nematode Infection (GIN) are the main constraint to the production of small ruminants. Studies of medicinal plants have been an important alternative in the effort to control these parasites. Therefore, the purpose of this study was to evaluate the in vitro ovicidal and larvicidal activity of essential oil of Rosmarinus officinalis. The oil was extracted, analyzed by gas chromatography and tested on GIN eggs and larvae in six concentrations, 227.5mg/mL, 113.7mg/mL, 56.8mg/mL, 28.4mg/mL, 14.2mg/mL and 7.1mg/mL. To determine the ovicidal activity, GIN eggs were recovered from sheep feces and incubated for 48h with different concentrations of the oil. For the evaluation of larval migration, third-stage larvae (L3) were obtained by fecal culture, and associated with the essential oil for 24h at the same concentrations, after which they were left for another 24 hours on microsieves, followed by the count of migrating and non-migrating larvae. The assays of R. officinalis oil showed a significant (p<0.05) 97.4% to 100% inhibition of egg hatching and a significant (p<0.05) 20% to 74% inhibition of larval migration. The main constituent revealed by gas chromatography was Eucalyptol. The results indicate that R. officinalis essential oil has ovicidal and larvicidal activity on sheep GINs.

Ovicidal and Larvicidal Activities of Saba senegalensis (A.DC) Pichon (Apocynaceae) Extracts and Fractions on Heligmosomoides bakeri (Nematoda, Heligmosomatidae)

Aims: To investigate ovicidal and larvicidal activities of an aqueous decoction (AD) and hydroethanolic macerate (HEM) extracts and fractions of the leaves of Saba senegalensis.&#x0D; Study Design: In vitro, the ovicidal and larvicidal activities of AD and HEM extracts and fractions of the leaves of Saba senegalensis on the eggs and larvae (L1) of Heligmosomoides bakeri.&#x0D; Place and Duration of Study: The experiment was conducted at the department of Medicine and Traditional Pharmacopeia-Pharmacy (MEPHATRA-PH) of Institute of Research in Health Sciences (IRSS) between June 2015 and December 2016.&#x0D; Methodology: The phytochemical groups of the extract and fractions of Saba senegalensis were determined by a colorimetric and Thin Layer Chromatography methods. The eggs were obtained from feces of mice deliberately infected and the larvae from the eggs were incubated at 25 ±2℃ for 72 hours. Eggs and larvae were exposed to increasing concentrations (100; 625; 1250; 2500; 3750 µg/mL) of the different extracts, 48 hours and 24 hours for the eggs and larvae respectively. Distilled water and DMSO 0.1% were used as negative controls while albendazole and levamisole were used as positive controls.&#x0D; Results: The phytochemical groups of interest are the tannins, saponins, flavonoids and triterpenes. The negative control had given 2.16% of egg hatch inhibition and 0% of larvae mortality mean while the positive control had given 100% in both cases. The extracts inhibited eggs hatching and affected larval survival. Pharmacological effects were concentration-dependent. The ovicidal and larvicidal activity of HEM is more interesting than that of AD with an Emax = 95.60% and an IC50 = 390 µg/mL. It is the same for the larvicidal activity with Emax = 100% and an LC50 = 900 µg/mL. However, the differences were not statistically significant.&#x0D; Conclusion: These results show the ovicidal and larvicidal properties of the S. senegalensis leaves.

Caenorhabditis elegans muscle Cys-loop receptors as novel targets of terpenoids with potential anthelmintic activity.

The anthelmintic treatment of nematode infections remains the pillar of worm control in both human and veterinary medicine. Since control is threatened by the appearance of drug resistant nematodes, there is a need to develop novel compounds, among which phytochemicals constitute potential anthelmintic agents. Caenorhabditis elegans has been pivotal in anthelmintic drug discovery and in revealing mechanisms of drug action and resistance. By using C. elegans, we here revealed the anthelmintic actions of three plant terpenoids -thymol, carvacrol and eugenol- at the behavioral level. Terpenoids produce a rapid paralysis of worms with a potency rank order carvacrol > thymol > eugenol. In addition to their paralyzing activity, they also inhibit egg hatching, which would, in turn, lead to a broader anthelmintic spectrum of activity. To identify drug targets, we performed an in vivo screening of selected strains carrying mutations in receptors involved in worm locomotion for determining resistance to the paralyzing effect of terpenoids. The assays revealed that two Cys-loop receptors with key roles in worm locomotion -Levamisole sensitive nicotinic receptor (L-AChR) and GABA(A) (UNC-49) receptor- are involved in the paralyzing effects of terpenoids. To decipher the mechanism by which terpenoids affect these receptors, we performed electrophysiological studies using a primary culture of C. elegans L1 muscle cells. Whole cell recordings from L1 cells demonstrated that terpenoids decrease macroscopic responses of L-AChR and UNC-49 receptor to their endogenous agonists, thus acting as inhibitors. Single-channel recordings from L-AChR revealed that terpenoids decrease the frequency of opening events, probably by acting as negative allosteric modulators. The fact that terpenoids act at different receptors may have important advantages regarding efficacy and development of resistance. Thus, our findings give support to the use of terpenoids as either an alternative or a complementary anthelmintic strategy to overcome the ever-increasing resistance of parasites to classical anthelmintic drugs.

Galloyl flavonoids from Acacia farnesiana pods possess potent anthelmintic activity against Haemonchus contortus eggs and infective larvae.

Acacia farnesiana (L.) Willd is a shrub legume used as condiment, medicinal plant and bioactive herbage. This species is used in traditional medicine of several countries to relieve the symptoms of gastrointestinal diseases, diarrhoea, stomach pain and typhoid as well as astringent, antidysenteric and anthelmintic. Some studies have shown that this plant displayed anthelmintic activity against several gastrointestinal nematode parasites of livestock, and also against parasites of human beings, such as malaria. This work describes the isolation and chemical identification of the anthelmintic compounds of Acacia farnesiana pods against eggs and infective larvae of the sheep parasitic nematode Haemonchus contortus. The bio-guided chemical fractioning of A. farnesiana pods using ethyl acetate against H. contortus eggs and infective larvae allowed for the identification of naringenin 7-O-(6″-galloylglucoside) (flavonol group) as the compound responsible for the anthelmintic activity against this important parasitic nematode. Anthelmintic activity was assessed using the egg hatching inhibition assay (EHI) and mortality tests. A complete hydroalcoholic extract (HA-E) at 12.5-50mg/mL, an aqueous fraction (Aq-F) at 3.12-25mg/mL and an ethyl acetate fraction (EtOAc-F) at 3.12-25mg/mL were analysed in the first selection phase. The purification of compounds through the chromatographic separation of the organic fraction resulted in nine less complex mixtures (C1F1, C1F2, C1F3, C1F4, C2F1, C2F2, C2F3, C2F4 and C2F5) that were assessed at 0.62-5mg/mL concentrations. In addition, thiabendazole (0.6mg/mL) and ivermectin (5 mg/mL) were used as positive controls. Likewise, distilled water and 4% methanol were used as negative controls. The bioactive compounds of EtOAc-F were obtained and characterised through chromatographic processes like open column chromatography, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) and gass chromatography-mass detection (GC-MS). Bioactive compounds were identified by spectroscopy (1H and 13C NMR) and mass spectrometric analysis. Additionally, the H. contortus eggs and infective larvae exposed to the bioactive compounds were observed through environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). Data were analysed based on a completely randomised design using ANOVA through a general linear model. The EtOAc-F fraction showed the highest ovicidal and larvicidal activities, at close to 100% at 3.12 and 6.25mg/mL, respectively. The treatments C1F2, C1F3 and C2F3 displayed the main ovicidal activity (80-100%) at 2.5mg/mL. The major compounds found in these sub-fractions were identified as galloyl derivatives and flavanones, including gallic acid (1), methyl gallate (2), ethyl gallate (3), naringin (4), naringenin 7-O-(4″, 6″-digalloylglucoside) (5), naringenin 7-O-(6″-galloylglucoside) (6) and naringenin (7). Likewise, the ESEM and CLSM images showed that the assessed compounds adhered to the eggshell and the external cuticle of the larvae. These results indicate that A. farnesiana pods contain nematocidal compounds and might be promising natural anthelmintic agents against H. contortus. This leguminous plant could be used as a nutraceutical food source for the control of gastrointestinal nematodes in small ruminants.

Culex quinquefasciatus Egg Membrane Alteration and Ovicidal Activity of Cipadessa baccifera (Roth) Plant Extracts Compared to Synthetic Insect Growth Regulators.

BackgroundInsecticide resistance among mosquito vectors for synthetic insecticides still remains a major problem for control efforts. This study assessed the ovicidal potential of crude solvent extracts from the medicinal plant Cipadessa baccifera comparatively to standard registered synthetic insect growth regulators (IGR) on freshly laid eggs of Culex quinquefasciatus.MethodFive plant extracts were prepared using different solvents. The batches of eggs were exposed to different concentrations of each solvent extract comparatively to synthetic IGR. The hatched eggs of Cx. quinquefasciatus were subjected to different concentrations. The first instars that emerged from the eggs were counted daily. The egg hatching inhibition was observed 24, 48 and 72 hrs post treatment. The desiccation median time (DT50 and DT90) was calculated.ResultsThe percent egg hatching inhibition was inversely proportional to the concentration of extracts. The morphological damage to the eggs was observed. Among five solvent extracts, acetone extracts showed the highest ovicidal activity. The changes in eggshell morphology were observed. The maximum ovicidal activity was observed in acetone extracts with DT50 value of 1.70 hrs (0.91–2.22). The methanol plant extract using gas chromatography-mass spectrometry identified 14 compounds.ConclusionThese results suggest that the acetone extracts of C. baccifera have the potential to be used as an ovicidal agent for controlling mosquito populations in aquatic stages. The biodegradability of the extracts has the advantage of being eco-friendly.

Activity and reproductive capability of Meloidogyne incognita and sunflower growth response as influenced by root exudates of some medicinal plants

Abstract Two experiments were separately conducted to test the effect of root exudates of seventeen medicinal plant species on hatching and survival of the root-knot nematode, Meloidogyne incognita, in-vitro and in-vivo on infected sunflower cv. Giza 102. In vitro tests, rosemary and marjoram root exudates were the best treatments for inhibiting egg hatch at all concentrations. Nematicidal and nematistatic effects of exudates were concentration-dependent, as the anesthetic effect was obvious especially at low concentrations. In vivo experiment, marjoram and rosemary root exudates significantly reduced the numbers of galls, egg-masses on roots, eggs/root and reproduction factor (Pf/Pi) in all times of application. Furthermore, immersing juveniles in marjoram exudates for 90 min before inoculation caused approximately 50% reduction in Meloidogyne incognita infection and fecundity. Treatments of rosemary at all times of application were the best in improving plant fresh and dry weights. GC/MS/MS analysis revealed that the most abundant components in the root exudates of marjoram and rosemary were flavonoids, phenolics and terpenoids which were suppressive to nematode survival, development and reproduction regardless to their time of application.

Nematicidal potential of Streptomyces antibioticus strain M7 against Meloidogyne incognita

Meloidogyne spp. are microscopic, obligatory endoparasites with worldwide distribution which cause severe damage to agricultural crops. The present study revealed the nematicidal activity of Streptomyces antibioticus strain M7 against Meloidogyne incognita. The culture supernatant of the isolate caused 100% J2 mortality after 24 h and inhibited egg hatching (only 3%). In addition, the nematicidal activity of actinomycins V, X2 and D purified from strain M7 was also checked. In vitro studies displayed 97.0–99.0% juvenile mortality and 28.0–44.0% egg hatching after 168 h at 240 µg/ml of actinomycin, with LD50 (lethal dose) values of 28–120 µg/ml. In vivo study further validated the nematicidal activity of strain M7, where nematode infested tomato plants treated with culture supernatant/cells/solvent extract showed reduction in root galls and egg masses per plant by 50.0–62.06% and 53.48–76.74%, respectively, and significantly enhanced the shoot length (54.67–76.39%), root length (36.45–64.88%), shoot fresh weight (111–171.77%), root fresh weight (120–163.33%), shoot dry weight (54.45–145.45%), and root dry weight (100–133.3%) over the nematode infested plants treated with water. Furthermore, tomato plants treated with cells/culture supernatant/extract of strain M7 without nematode infestation also showed significant increase in various plant growth parameters. Thus, the outcome of the study revealed the potential of S. antibioticus strain M7 and actinomycins produced from it to be developed as safe nematicidal agents to control the root knot nematodes, and to increase the crop yield.

Reclamation of rhizobacteria newly isolated from black pepper plant roots as potential biocontrol agents of root-knot nematodes

Rhizobacteria have been of great interest due to their promising use in biocontrol and promotion of plant growth. The aim of this study was to select active anti-nematode rhizobacteria, enhance their nematicidal activity by optimizing fermentation conditions, and then determine the active components of the fermented product. Among more than 100 isolates, 20 strains showed potent inhibition (81.67–99%) against root-knot nematodes from the genus Meiloidogyne. Almost all of these belong to the genus Bacillus (14 of 20 strains). The six most active strains (with max inhibition of 96.13–98.33%) were RB.DS.05, RB.DL.28, RB.DS.29, RB.DL.31, H11, and CC01; these strains were further investigated for their effect on nematode egg hatching. Of these, Bacillus subtilis RB.DL.28 demonstrated the greatest inhibition (82%). Chitinase and protease were closely associated with the inhibition of egg hatching, while it was newly discovered that natural compounds with thermal stability play a key role in killing J2 nematodes. Based on LC–MS analysis, two major constituents, sulfacetamide and metronidazole-OH, were identified from the filtrates of B. subtilis RB.DL.28. The results from this study suggest that the most active strain, B. subtilis RB.DL.28, and other newly identified rhizobacteria may be good candidates for biocontrol of Meiloidogyne spp. These results expand the catalogue of novel active anti-nematode rhizobacteria communities recently recorded in Vietnam.

In Vitro Anthelmintic Activity of Three Tropical Plant Extracts on Haemonchus contortus.

To evaluate the anthelmintic activity of Anacardium occidentale shell, Illicium verum fruit, and Artocarpus heterophyllus seed to substantiate their traditional use against helminths. In vitro anthelmintic activity of hydroalcoholic extracts of three plants was evaluated against eggs, infective larvae (L3), adult, and lactate dehydrogenase (LDH) of Haemonchus contortus of naturally infected sheep. The three extracts exhibited significant (P < 0.001) dose-dependent anthelmintic responses by inhibiting egg hatching and causing paralysis of larvae and mortality of worms. The extracts were most effective on egg and adult stage of H. contortus than on L3 stage at a higher concentration of 6 mg/mL. Anacardium occidentale shell exhibited maximum activity with 100% paralysis of L3 larvae. Probit analysis revealed that the extracts of A. Occidentale shell induced 50% egg hatch inhibition (LD50 = 0.0255 mg/mL), larval paralysis (LD50 = 0.196 mg/mL), and adult worm mortality (LD50 = 1.0365 mg/mL) at a lower concentration (LD50) compared with those of I. verum fruit and A. heterophyllus seed extracts. Further, all extracts significantly (P < 0.01) inhibited the LDH activity catalyzing the oxidation of lactate in adult H. contortus, with maximum level of inhibition caused by A. occidentale shell extract. Phytochemical screening of the extracts revealed the presence of alkaloids, flavonoids, tannins, saponins, and amino acids that could be responsible for the anthelmintic effects noticed. The results warrant further in vivo evaluation of these plants for potential use as anthelmintic agents.

Anthelmintic-like activity of polyphenolic compounds and their interactions against the cattle nematode Cooperia punctata

Polyphenolic compounds (PCs) have been proposed as one of the most bioactive group of secondary metabolites occurring in nature and have been associated to anthelmintic (AH)-like activity of plants against cattle nematodes. However, little is known regarding their synergetic / antagonistic interactions. This study assessed the in vitro AH-like activity of commercial PCs: quercetin, caffeic acid, rutin and coumarin, and their combinations against the egg hatching and larval exsheathment of Cooperia punctata; one of the most prevalent nematodes affecting grazing cattle in tropical regions. The molecules selected for the in vitro analysis were identified as bioactive phytochemicals of plants through bio-guided fractionation in previous studies. To estimate mean effective concentrations (EC50) five increasing concentrations were used for both Egg hatching inhibition assay (EHIA) and larval exsheathment inhibition assay (LEIA) (0.6–9.8 mg mL−1 and 0.15–2.4 mg mL−1, respectively). From the four molecules, only rutin did not affect egg hatching; while quercetin, showed no bioactivity against eggs or larvae (P > 0.766 and P > 0.621, respectively). Best-fit EC50 estimated through the EHIA was considered for PCs classification as bioactive (coumarin and caffeic acid) and non-bioactive (quercetin and rutin). Phytochemical interactions were subsequently assessed combining bioactive:non-bioactive PCs (8:2 ratio), and the nature of their interaction was classified using the fractional inhibitory concentration index (FICindex). Combinations had a highly synergistic interaction against larval exsheathment (FICindex < 0.5) except for coumarin:rutin against egg hatching (FICindex> 0.5). Quercetin and rutin acted as PCs AH-like activity enhancers, reducing EC50 of bioactive molecules in a range of 43%–64% and 68%–83% for EHIA and LEIA, respectively. A linear relationship between low molecular weight of molecules and ovicidal activity was observed; where, molecules with lower molecular weight displayed better-fit EC50 for ovicidal activity. Furthermore, coumarin and caffeic acid bioactivity against free-living stages of C. punctata makes them suitable candidates as markers for anthelmintic-like activity in bioactive forages. Combinations used through this investigation showed a potent anthelmintic-like activity against free-living forms of C. punctata, representing a first step towards the identification of promising alternatives for nematode control.

Effects of essential oils distilled from some medicinal and aromatic plants against root knot nematode (&lt;i&gt;Meloidogyne hapla&lt;/i&gt;)

Essential oils of medicinal and aromatic plants are important and promissing to manage the nematological problems in agriculture. In this study, five of the plants including Origanum onites , Salvia officinalis , Lippia citriodora , Mentha spicata and Mentha longifolia for egg hatching inhibition and four of the plants including Mentha piperita , Foeniculum vulgare , Coriandrum sativum and Ocimum basilicum for juvenile mortality were tested on Meloidogyne hapla under laboratory conditions. The oils were achieved by using water distillation method with a Clevenger apparatus. As the results of egg hatching trial, the highest egg hatching inhibition rate was found as 54% for O. onites. In addition, the other inhibition rates varied as 31.4%, 21.6%, 23.8%, 25.7% for the other plants, S. officinalis , M. longifolia , M. spicata and L. citriodora , respectively. Essential oil of each plant components were determined by gas chromatography (GC). Carvacrol was found as the main component (68.8%) of O. onites followed by Thujone 27.7% for S. officinalis , I-Menthone 76.9% for M. longifolia , Carvone 27.1% for M. spicata and Citral 19.3% for L. citriodora . For the juveile mortality, Mentha piperita showed the highest mortality rate as 93.2% and was followed by F. vulgare 72.9%, C. sativum 69.3% and O. basilicum 64.9%. The main component of the used plants were Carvone 39.3%, Anethole 40.2%, Linalool 81.3% and Linalool 54.6%, respectively. Keywords: Essential oil, Medicinal and aromatic plants, Meloidogyne hapla

Evaluation of the nematicidal potential of some botanicals against root-knot nematode, Meloidogyne incognita infected carrot: In vitro and greenhouse study

In this study effort was made to manage the root-knot nematode, Meloidogyne incognita by the utilization of selected plants such as Coccinia grandis, Commelina benghalensis, Leucas cephalotes, Phyllanthus amarus and Trianthema portulacastrum. During the in vitro experiment, the second juveniles (J2s) of M. incognita directly treated with aqueous extracts of the plant leaves at 1000, 2000, 3000, 4000 and 5000 ppm concentrations. As a result, the 5000 ppm concentration was found most toxic against the survival of second stage juveniles of M. incognita compared to other concentrations. Maximum mortality of 91% was observed in 5000 ppm extract of P. amarus after 72 h of exposure with LC50 value 2084.49 ppm while minimum mortality of 6.2% was found in 1000 ppm extract of T. portulacastrum after 24 h with LC50 value 9484.18 ppm. The percent mortality of juveniles calculated the LC50 values of all the treatments after 24, 48 and 72 h. Similarly, the in vitro tests showed a gradual decrease in the rate of egg hatching of M. incognita with the increasing concentrations of aqueous leaves extracts of plants, i.e., 1000 ppm to 5000 ppm and exposure period. Maximum inhibition in egg hatching was found at 5000 ppm (86.5%) after seven days in the extract of P. amarus while least was observed in T. portulacastrum at 1000 ppm (10.6%). The findings indicate that the plant extracts were found lethal to J2s and also inhibit egg hatching. In pots under greenhouse conditions, the plants treated with both the doses (50 and 100 g/pot) of fresh chopped leaves of the selected botanicals significantly experienced the reduced infestations in relation to root gall index, egg masses/root system and nematode population/250 g soil. The P. amarus treated at 100 gm / pot showed highest nematicidal efficacy with the maximum reductions in all the pathological parameters as compared to the untreated uninoculated control simultaneously resulted in maximum increase of growth and physiological parameters of carrot such as shoot and root lengths, shoot and root fresh weights, shoot and root dry weights, chlorophyll and carotenoid contents and nitrate reductase activity (NRA). The information derived from this study suggests that the leaves of P. amarus plant can be used for the management of M. incognita and could be a possible replacement for synthetic nematicides.

Development and optimization of a high-throughput screening method utilizing Ancylostoma ceylanicum egg hatching to identify novel anthelmintics.

Hookworms remain a major health burden in the developing world, with hundreds of millions currently afflicted by these blood-feeding parasites. There exists a vital need for the discovery of novel drugs and identification of parasite drug targets for the development of chemotherapies. New drug development requires the identification of compounds that target molecules essential to parasite survival and preclinical testing in robust, standardized animal models of human disease. This process can prove costly and time consuming using conventional, low-throughput methods. We have developed a novel high-throughput screen (HTS) to identify anthelmintics for the treatment of soil-transmitted helminths. Our high-throughput, plate reader-based assay was used to rapidly assess compound toxicity to Ancylostoma ceylanicum L1 larva. Using this method, we screened 39,568 compounds from several small molecule screening libraries at 10 μM and identified 830 bioactive compounds that inhibit egg hatching of the human hookworm A. ceylanicum by >50%. Of these, 132 compounds inhibited hookworm egg hatching by >90% compared to controls. The nematicidal activities of 268 compounds were verified by retesting in the egg hatching assay and were also tested for toxicity against the human HeLa cell line at 10 μM. Fifty-nine compounds were verified to inhibit A. ceylanicum egg hatching by >80% and were <20% toxic to HeLa cells. Half-maximal inhibitory concentration (IC50) values were determined for the 59 hit compounds and ranged from 0.05–8.94 μM. This stringent advancement of compounds was designed to 1) systematically assess the nematicidal activity of novel compounds against the egg stage of A. ceylanicum hookworms in culture and 2) define their chemotherapeutic potential in vivo by evaluating their toxicity to human cells. Information gained from these experiments may directly contribute to the development of new drugs for the treatment of human hookworm disease.

Anthelmintic Activity of Hymenodictyon pachyanta Stem Bark Extracts against Haemonchus Contortus

Abstract The development of host resistance to anthelmintics and the increasing cost of commercial anthelmintics have encouraged the need for the in vitro anthelmintic evaluation of crude extract and fractions of Hymenodictyon pachyanta plant as alternative drugs against Haemonchus contortus. H. contortus is one of the most prevalent and highly pathogenic parasitic nematodes in small ruminant farming globally. H. pachyanta stem bark is a prospective plant used by the local and indigenous farmers of Nsukka, Enugu state, Nigeria. The stem bark of H. pachyanta were collected, dried, pulverized and extracted with 80 % methanol. The purpose of this study was to investigate the in vitro anthelmintic effects of these crude extract and fractions against H. contortus in sheep and goats. The two extracts (crude and fractions) of H. pachyanta were tested by the egg hatch assay (EHA) and the larval development inhibition assays (LDIA) and to compared the results with albendazole (as the positive control). The concentrations for the crude extract and albendazole used for this study were 0.78, 1.56, 3.125, 6.25 and 12.5 mg.ml−1. The results demonstrated that the crude extracts, fractions and albendazole all at the concentration doses of 12.5 mg.ml−1 produced 100 % inhibition of egg hatching and larval development. Statistically, there was no significant difference (P &gt; 0.05) in the mean percentage inhibition of egg hatching and larval development inhibition of the crude extracts and fractions when compared with albendazole. However, a significant difference (P &lt; 0.05) was observed with n-butanol fraction which inhibited 96.17 % of egg hatchability. All of the extracts and albendazole showed ovicidal and larvicidal effects and were able to induce over 50 % of the egg hatching and mortality of larvae at the concentration ranges of 0.78—12.5 mg.ml−1. The results obtained from our study suggest that H. pachyanta had ovicidal and larvicidal activity against H. contortus and that the bioactive plants compounds responsible for this effect could be attributed to the presence of tannins, alkaloids and the saponins contained in the crude extracts.

A Convenient One-Pot Synthesis and Nematicidal Activity of Nicotinic Acid Amides

Seven nicotinic acid amides were synthesized by condensation of nicotinic acid adsorbed on silica gel with different aromatic amines. The synthesized compounds were characterized by 1H and 13C NMR and IR spectroscopy and screened for their nematicidal activity against root knot nematode Meloidogyne incognita by egg hatching and mortality test. All compounds exhibited significant nematicidal potential as compared to control. Maximum egg hatching inhibition potential was exhibited by N-(4-bromophenyl)nicotinamide while N-(2,4,5-trichlorophenyl)nicotinamide showed the maximum mortality potential.

Occurrence and functional diversity of bacteria in rhizosphere of citrus trees infested by Tylenchulus semipenetrans in a citrus-growing area of Tunisia

Tylenchulus semipenetrans is a major plant-parasitic nematode pest of citrus which readily infests sour orange rootstock Citrus aurantium. In order to examine the bacterial diversity and prevalence in the nematode infested citrus rhizosphere, 282 bacterial strains were isolated from roots of mature citrus trees, from egg masses of T. semipenetrans, and from nematophagous fungi in the orange producing area Cap bon in Tunisia. The rhizosphere of citrus had a high diversity of bacterial strains among the 20 agricultural parcels. According to morphological and biochemical characteristics, sixty bacteria strains were selected to explore the diversity of biological activity. None of the bacterial strains considered in this study have phytopathogenic activity. Each strain was identified and tested as a potential natural antagonist against fungi and against citrus nematode (egg hatching and juvenile mortality). Bacillus cereus, B. mycoides, B. pumilus and B. thuringiensis were the four most abundant species regardless the sampling location. Canonical discriminant analysis grouped the bacteria into five groups of biological activities: Group A, similar to water control, had not specific effect. Group B showed strong effect, and group C caused moderate growth inhibition of the nematophagous fungi Monacrosporium cianopagum CBM. Group C had stronger growth inhibition of the fungus Arthrobotrys conoides RGA than groups B and D. Groups D and E showed a strong activity on nematode egg hatch inhibition.

Adaptation to temperate climates: Evidence of photoperiod-induced embryonic dormancy in Aedes aegypti in South America

Dormancy is a developmental arrest in arthropods, in response to unfavorable conditions in temporally varying environments. In Aedes aegypti, the supposed inability of eggs to inhibit hatching has been used to explain the restriction of this species to tropical and subtropical regions. However, the geographic range of Ae. aegypti is constantly expanding towards temperate regions. Thus, the aim of the present study was to assess the ability of Ae. aegypti individuals from a temperate region (Buenos Aires City, Argentina) to enter photoperiod induced dormancy. To this end, we exposed both the parental generation and the eggs to short-day (SD: 10L:14D) and long-day (LD: 14L:10D) photoperiods, and studied the temporal variation in egg hatching. The experiment consisted of 28 treatment combinations of three factors: parental photoperiod (SD or LD), egg storage photoperiod (SD or LD), and age of eggs (14, 28, 42, 56, 70, 91, and 112 days). The results showed a lower hatching response with the SD parental photoperiod, and a trend to higher hatching with longer egg storage time in all photoperiod treatment combinations. The egg storage photoperiod showed no effect on egg hatching. In both parental photoperiod treatments, egg replicates of most ages from different females showed a large variability, with some replicates with lowest hatching response and others with highest hatching response. Our results show the ability of Ae. aegypti to inhibit egg hatching in response to a short-day photoperiod, which could allow the further expansion of this species to regions with colder winters.

Analysis of the activity and biological control efficacy of the Bacillus subtilis strain Bs-1 against Meloidogyne incognita

Root-knot nematodes are severe soil-borne pathogens that cause great damage to vegetable crops and economic losses all over the world annually. Therefore, there is an urgent need to seek novel nematicides or biological control agents. In this study, a bacterium strain, the Bacillus subtilis strain Bs-1, was obtained from rhizospheric soil, and had strong nematicidal effects and caused egg hatching inhibition and repellence of Meloidogyne incognita. Nematicidal activity was evaluated according to fermentation assays, and the mortality rate of second-stage juveniles (J2s) to the Bs-1 fermentation treatment was 100%. The results of ovicidal experiments indicated that fermentation of the strain Bs-1 also had a strong effect on decreasing the egg hatching rate. In both pot and field experiments, Bs-1 fermentation was able to significantly reduce root galls and promote the growth of cucumber. A repellence test of Bs-1 fermentation showed that a majority of J2s were obviously repelled by the Bs-1 fermentation and supernatant, but were attracted by cucumber root extracts. The gas chromatography-mass spectrometry (GC-MS) results showed that the volatile organic components (VOCs) produced by the strain Bs-1 were complex and diverse, such as ketones, acids, alkyls, sulfide and heterocyclic compounds and so on. These results revealed that the B. subtilis strain Bs-1 provides strong suppression against M. incognita and could be used as a potential biological control agent to suppress M. incognita.

Evaluation of Pochonia chlamydosporia (Goddard) Isolates for Suppression of Meloidogyne incognita, Root-Knot Nematode of Tomato

Restricted applications of chemical nematicides has directed to the development of ecofriendly alternatives. Culture filtrates in different concentrations (20, 40, 60, 80 and 100%) of the six isolates of Pochonia chlamydosporia were evaluated for their nematicidal activity. Significant variations were observed in the inhibitory activity of culture filtrates against egg hatching and second stage juveniles of M. incognita at different concentrations. The culture extract of PC-6 showed maximum inhibition of egg hatching followed by PC-1. On the other hand PC1 isolate caused high mortality of second stage juveniles followed by PC-6. Significant variation among the isolates was observed for parasitism of M. incognita juveniles and eggs. Among the tested isolates, PC-1 and PC-6 were selected for the further studies based on in vitro egg inhibition and larval mortality. Both PC1 and PC6 applied at the rate of 6 &amp;times; 103 chlamydospores per gram of soil per plant reduced the population of nematode and improved the growth and productivity of plant under screen house and field conditions. Different growth parameters like plant height, fresh shoot weight, dry shoot weight, fresh root weight and dry root weight were measured. Efficacy of isolates was different in winter and summer crop. No significant difference among the treatments was observed in summer crop. However in winter crop, considerable effect of both isolates was observed. Fungal application was effective at the rate of 6 &amp;times; 103 chlamydospores per gram of soil per plant in both winter and summer crops. Root coating with chlamydospores suspension significantly reduced number of galls on roots. Little effect of both isolates was observed with mycelial root coating. No lesions were seen on P. chlamydosporia inoculated roots of tomato plants.

Anthelmintic activity of Annona crassiflora leaves against Haemonchus contortus: part 1: in vitro inhibition of the hatchability and larval development

We evaluated the activity of Annona crassiflora leaves against Haemonchus contortus resistant to albendazol. Aqueous (AE), ethanolic (EE) and ethyl acetate (EAE) extracts were produced and the predominant presence of flavonoids was observed in HPLC-DAD chromatograms. Initially we evaluated the larval development inhibition (LDI) of dry A. crassiflora leaf powder or its AE directly in fecal quantitative cultures. The efficacies of the extracts, with or without tannins, on egg hatching inhibition (EHI) were investigated reveling that the EE was the most effective (LC90 = 8.96 mg/mL). However, after tannin removal, AE showed the highest activity (LC90 = 4.27 mg/mL). In the LDI test, the LC90 of AE was < 6.25 mg/g of fecal culture and to leaf powder of leaves was 69.14 mg/g. High efficacies of AE and EE for EHI were detected and the tannins were not the main active metabolites. The anthelmintic potential of this plant could be attributed to association between flavonoids and other metabolites.