Egg Hatching Inhibition Research Articles

Suppressive Effects of Crude Extracts of Bacillus sp. CT16 and Neobacillus sp. JC05 against Egg Hatch of Meloidogyne incognita

Root-knot disease caused by Meloidogyne incognita is major soil pathogen and cause severe economic damages to vegetable crops. In this study, we selected rhizobacteria for biocontrol of the root-knot nematode, M. incognita, and identified; performed bioassay of the bacterial extracts in cucumber seedlings. The crude extracts of strains CT16 and JC05 out of 180 strains inhibited egg hatching and increased juvenile mortality in vitro assay; based on 16S rRNA sequences analysis, the two strains were identified as Bacillus sp. CT16, and Neobacillus sp. JC05. After extracting the bacterial supernatants by using various organic solvents, n-butanol and n-hexane extracts of strain CT16 and n-butanol extract of strain JC05 showed inhibitory activity of egg hatching depending on concentrations. Subsequently, n-butanol extracts of two strains significantly suppressed formation of egg masses in cucumber seedling. Therefore, these results indicated that strains CT16 and JC05 could be used as potential biocontrol agents against M. incognita.

Anthelmintic, antimycobacterial, antifungal, larvicidal and cytotoxic activities of acetone leaf extracts, fractions and isolated compounds from Ptaeroxylon obliquum (Rutaceae)

Ethnopharmacological relevancePtaeroxylon obliquum (Thunb.) Radlk, sneezewood, is a commonly used medicinal plant in South Africa for the treatment of parasitic infections in animals, tuberculosis (TB) and related symptoms, and other microbial infections. Aim of the studyIn this study anthelmintic, antifungal, antimycobacterial, larvicidal and cytotoxic activities of the acetone leaf extract of P. obliquum, solvent-solvent derived fractions of the extract and isolated compounds (obliquumol and a mixture of lupeol and β-amyrin) were determined. Materials and methodsThe in vitro anthelmintic activity was tested against Haemonchus contortus ova and larvae using the egg hatch inhibition and larval development assays. The antifungal activity was investigated using a serial microplate dilution method against Aspergillus fumigatus, Cryptococcus neoformans and Candida albicans. Antimycobacterial activity was evaluated by a tetrazolium violet-based broth microdilution method against pathogenic Mycobacterium tuberculosis and Mycobacterium bovis. The larvicidal activity was evaluated against Aedes aegypti. Cytotoxicity was determined using human liver (C3A) and Vero African green monkey kidney cell lines. ResultsObliquumol (ptaeroxylinol acetate) was more effective than the crude acetone extract against H. contortus ova and larvae with an LC50 of 95 μg/mL against the larvae. Obliquumol had very good antifungal activity with minimal inhibitory concentrations (MIC) values from 2 to 16 μg/mL. Lupeol and β-amyrin mixture also had good activity against C. neoformans and C. albicans with an MIC of 16 μg/mL against both fungi. The crude extract and fractions had average to weak antimycobacterial activity against the two pathogenic mycobacteria with MICs of 313–625 μg/mL for the extract and fractions tested. Obliquumol had good activity with an MIC of 63 μg/mL against pathogenic M. tuberculosis ATCC 27115. The acetone crude extract was toxic against Vero cells with an CC50 of 14.2 μg/mL. Obliquumol and the mixture of lupeol and β-amyrin were not toxic against both Vero and C3A cells at the highest concentration tested of 200 μg/mL and had good selectivity index values against the opportunistic fungal pathogens. No tested samples had any larvicidal activity against A. aegypti at the concentrations tested. ConclusionThe activity noted against H. contortus may support the ethnoveterinary use of this plant against animal parasites in South Africa although aqueous extracts were not active. The activity of obliquumol against opportunistic fungal pathogens was also promising. The cytotoxicity of the acetone leaf extract, however, raises concerns about the traditional use of P. obliquum to treat a wide range of diseases although if an aqueous extract is used the toxicity concern may not be relevant.

Antinematode Activity of Abomasum Bacterial Culture Filtrates against Haemonchus contortus in Small Ruminants.

Simple SummaryHaemonchus contortus is an important gastrointestinal nematode parasite of the tropical and sub-tropical regions that cause haemonchosis in small ruminants like goats and sheep. It causes low production, reduced growth and may cause death of the infected animals. Due to the resistance development and environmental issues, the use of anthelmintics can be replaced with biological control, which is an environment friendly alternative. In the present study, three bacteria viz; Comamonas testosteroni, C. jiangduensis and Pseudomonas weihenstephanesis showed significant effect on nematode mortality and egg hatch inhibition. It was also observed that the anthelmintic activity of these bacteria was dose dependent, where 100% bacterial metabolite concentration showed the highest activity. It is suggested that these bacteria may included in the integrated nematode management.Haemonchosis is a parasitic disease of small ruminants that adversely affects livestock production. Haemonchus contortus is one of the most prevalent nematode parasites that infect the abomasum of small ruminants. This parasite reduces milk production, overall growth and sometimes causes the death of the infected animals. The evaluation of the biocontrol potential of some abomasum bacterial isolates against H. contortus is investigated in this study. Out of which, three isolates—Comamonas testosteroni, Comamonas jiangduensis, Pseudomonas weihenstephanesis—show significant effect against the nematode L3, adult, and egg hatch inhibition assays. Various concentrations of metabolites from these bacteria are prepared and applied in different treatments compared with control. In the case of adult mortality assay, 50% metabolites of C. testosteroni and P. weihenstephanesis show 46% adult mortality, whereas C. jiangduensis shows 40% mortality. It is observed that decreasing the concentration of bacterial metabolite, lowers nematode mortality. The minimum nematode mortality rate is recorded at the lowest filtrates concentration of all the bacterial isolates. The same trend is observed in egg hatch inhibition assay, where the higher concentration of bacterial culture filtrates shows 100% inhibition of H. contortus egg. It is concluded that the effect of bacterial culture filtrates against H. contortus is dose-dependent for their activity against nematode L3, adult, and inhibition of egg hatchment.

Integrated management of Meloidogyne incognita on tomato using combinations of abamectin, Purpureocillium lilacinum, rhizobacteria, and botanicals compared with nematicide

BackgroundAcceptable alternative eco-friendly tools in the present study were tested to control the root-knot nematode, Meloidogyne incognita, on greenhouse-cultivated vegetables. The nematicidal effect of rhizobacteria (Pseudomonas and Serratia), egg parasitic fungus (Purpureocillium lilacinum), abamectin (Streptomyces avermitilis), and 3 botanicals (colocynth, Citrullus colocynthis; moringa, Moringa oleifera; marigold, Tagetes erecta L.) singly or in combination was tested against M. incognita, in comparison with emamectin benzoate.ResultsIn vitro treatments revealed that egg hatching and juvenile mortality were influenced by the type of bioagents, plant species of botanicals, and exposure time. All the tested bioagents and botanicals displayed nematicidal potential via their ovicidal and larvicidal action on egg hatching and J2 mortality of M. incognita. Three and 5 days post-treatment, abamectin and emamectin benzoate were more effective than P. lilacinum, Serratia and Pseudomonas, and C. colocynthis in inhibiting egg hatching: 96.31 and 94.88%; 95.79 and 94.05%; 94.11 and 94.46%; 85.54 and 87.28%; 88.87 and 84.30%, respectively. On the other hand, after 10 days, P. lilacinum gave the highest inhibition percentage (99.00%), followed by abamectin (89.25%). However, the difference was insignificant compared with the inhibition percentage of rhizobacteria, Serratia and Pseudomonas (88.69%; p ≤ 0.05). Moreover, juvenile mortality was 100.0, 96.80, and 91.60% after 10 days of treatment, respectively. However, botanicals showed a lower effect on egg hatching and juvenile mortality. Under greenhouse conditions, potential antagonism towards M. incognita by application the mixture of biocontrol agents and botanicals was more effective in controlling M. incognita than single treatments.ConclusionsThe combination of abamectin and/or emamectin benzoate with P. lilacinum and rhizobacteria was the most effective against M. incognita, followed by rhizobacteria and P. lilacinum, not only in decreasing galls and reproduction of M. incognita but also in increasing plant growth of tomato parameters than the control. The application of various bioagents including abamectin might be a potential antagonism strategy against phytonematodes in protected agricultural areas.

In vitro Anthelmintic activity of Musa balbisiana Colla (Square banana) against Haemonchus contortus eggs

Objective: To evaluate the anthelmintic activity of aqueous and ethanolic extracts of Musa balbisiana Colla, against H. contortus eggs. Design / methodology / approach: The anthelmintic activity was evaluated using an egg hatching inhibition test. The aqueous extracts from leaves, peels and roots were obtained by infusion and subsequently lyophilized. Ethanolic extracts were obtained by maceration and later concentrated in a rotary evaporator. Spectroscopic, phytochemical, chemical and total polyphenol content analyzes were performed. The 50 % lethal concentration to inhibit H. contortus eggs from hatching was calculated following a Probit analysis. Results: The identified functional groups in the FT-IR analysis were hydroxyl (-OH) and methyl groups (CH3). The proximal analysis revealed significant differences in the dry matter percentage (P < 0.05). No significant differences were found in the protein content (P > 0.05). The egg hatching inhibition rates at the highest concentration 4.8 mg / mL were 100 % for the aqueous and ethanolic extracts from leaves, and 93.7 and 62 % for the shell and roots, respectively. Study limitations / implications: Further studies are required in in vivo systems. Findings / Conclusions: With a LC50 of 225 µg / mL and a 95 % confidence interval, with a range between 33 and 418.4 µg / mL, the aqueous extract from the leaves was the most active.

Screening the nematicidal potential of indigenous medicinal plant extracts against Meloidogyne incognita under lab. and greenhouse conditions

BackgroundThe root-knot nematode, Meloidogyne incognita, causes a high damage and yield decrease for many economic plants. The need for non-systemic effective new approaches and environmentally friendly methods for controlling the nematodes has directed research to some new and safe agrochemicals found in medicinal plants as new viable management options.ResultsIn laboratory experiments, solidago and periwinkle aqueous and ethanolic extracts achieved high J2 mortality (%) concerning different dilutions; however, aqueous extracts were more effective for mortality than ethanolic extracts. Also, there was a direct relationship between the nematicidal activity of these extracts with both concentration and time of application. Inhibition of egg hatching by Periwinkle extracts was higher than that of solidago. Moreover, the nematicidal activity of tested extracts against J2 decreased significantly with prolonged storage time at + 5 °C, while did not with stored frozen at – 5 °C for 12 months. Periwinkle and solidago extracts killed the non-target organisms, i.e., rotifers and free-living nematodes. Seventy-five and 90% of total phytochemicals recovered from periwinkle and solidago, respectively were nematostatic or nematicidal to nematode viability, egg hatch in vitro, and development and reproduction in vivo despite the method of application (foliar and soil drench). The antagonistic effects of solidago were more pronounced in soil drench than periwinkle concerning their concentrations and methods of application.ConclusionSolidago and periwinkle plant extracts showed important sources of effective control phytochemicals against M. incognita.

Bio efficacy of Cinnamaldehyde from <em>Cinnamomum verum</em> essential oil against <em>Culex quinquefasciatus</em> (Diptera: Culicidae)

This research aims to study the efficacy of Cinnamomum verum (Cv) extracts for ovicidal, larvicidal, and repellent activities against Culex quinquefasciatus mosquito vectors. The active components of C. verum or cinnamon oil by Gas Chromatography (GC) analysis showed the highest cinnamaldehyde at 83.53%. For ovicidal assay, C. verum essential oil at concentrations 12.5, 25 and 50 ppm at 72 h had 100% egg hatch inhibition and had a significant difference when compared to the control group (p<0.05). Larvicidal activity showed that concentrations of 25 and 50 ppm were highly effective in killing 100% mosquito larvae. Morphological changes in egg raft showed a faded color and eggs that seemed to have split from their raft while the larvae changed to a pale white wrinkled body with a destroyed inner tube of the body and were motionless. A Scanning electron microscope study showed that the eggshell and micropyle were wrinkled with the chitin peeled out. After treatment with cinnamon oil, larvae appeared to have a wrinkled body, the thorax and abdominal cuticle were also destroyed with high density of oil particles observed on mouth brushes and obstructing the spiracle. The repellent assay showed that cinnamon oil could repel both male and female mosquitos for up to 180 min. From the results, it was concluded that cinnamon oil had highly effective repellency against Culex quinquefasciatus adults and insecticidal activity on eggs and larvae stages evidenced by LC50 at 6.59±0.54, 9.07±0.67 and 36.91±7.56 ppm and its morphological changes indicated how the mosquito could not survive after cinnamon oil treatment hence this may be a useful alternative method that is green friendly for controlling mosquitoes in endemic areas.

A Systematic Review on Comparative Analysis, Toxicology, and Pharmacology of Medicinal Plants Against Haemonchus contortus.

Background: Haemonchus contortus is an important pathogenic nematode parasite and major economic constraint of small ruminants in tropics and subtropics regions. This review is an attempt to systematically address the; (a) efficacy of different plants against H. contortus by in vitro and in vivo proof; (b) toxicology, mechanism of action, and active phyto-compounds involve in anti-haemonchiasis activity; (c) and comparative analysis of plant species evaluated both in vitro and in vivo. Methods: Online databases (Google Scholar, PubMed, Scopus, and ScienceDirect) were searched and published research articles (1980–2020) were gathered and reviewed. Results: A total of 187 plant species were reported belonging to 59 families and 145 genera with Asteraceae and Fabaceae being frequently used. Out of the total plant species, 171 species were found to be evaluated in vitro and only 40 species in vivo. Twenty-four species were commonly evaluated for in vitro and in vivo anti-haemonchiasis activity. Among the reported assays, egg hatching test (EHT) and fecal egg count reduction (FECR) were the most widely used assays in vitro and in vivo, respectively. Moreover, sheep were the frequently used experimental model in vivo. After comparative analysis, Lachesiodendron viridiflorum, Corymbia citriodora, Calotropis procera, and Artemisia herba-alba were found highly effective both in vitro and in vivo. L. viridiflorum inhibited enzymatic activities and metabolic processes of the parasite and was found to be safe without toxic effects. C. citriodora was moderately toxic in vivo, however, the plant extract produced promising nematicidal effects by causing muscular disorganization and changes in the mitochondrial profile. Additionally, C. procera and A. herba -alba despite of their high anti-haemonchiasis activity were found to be highly toxic at the tested concentrations. C. procera caused perforation and tegumental disorganization along with adult worm paralysis. Nineteen compounds were reported, among which anethole and carvone completely inhibited egg hatching in vitro and significantly reduced fecal egg count, decreased male length, and reproductive capacity of female in vivo. Conclusion: This review summarized different medicinal plants owing to nematicidal activities against H. contortus eggs, larvae, and adult worms. Plants like L. viridiflorum, C. citriodora, C. procera, and A. herba-alba, while compounds anethole and carvone having promising nematicidal activities and could be an alternative source for developing novel drugs after further investigation.

In Vitro Antibacterial, Antifungal, Nematocidal and Growth Promoting Activities of Trichoderma hamatum FB10 and Its Secondary Metabolites.

Microbial natural biocides have attracted much more attention in recent years in order to avoid the unrestricted use of chemical biocides in the environment. The aim of this study is to analyze the antibacterial and antifungal activities of secondary metabolites and growth promoting, nematicidal, and soil enzyme activity mediated by Trichoderma hamatum FB10. The bactericidal and fungicidal activities were performed using cell-free extract. Results revealed that the selected strain exert antibacterial activity against Acidovorax avenae, Erutimacarafavora, and Xanthomonas campestris. The selected fungal strain FB10 showed antagonistic activity against fungal pathogens such as, S. sclerotiorum, Rhizoctonia solani, Alternaria radicina, Alternaria citri, and Alternaria dauci. Among the bacterial pathogens, A. avenae showed least MIC (30 ± 2.5 µg/mL) and MBC (70 ± 1.25 µg/mL) values. T. hamatum FB10 strain synthesized bioactive volatile secondary metabolite, which effectively inhibited the growth of bacteria and fungi and indicated the presence of 6-pentyl-alpha-pyrone as the major compound (67.05%). The secondary metabolite synthesized by T. hamatum FB10 showed nematicidal activity against M. incognita eggs. Egg hatch inhibition was 78 ± 2.6% and juvenile stage mortality rate was 89 ± 2.5% when the strain FB10 was treated with nematode. The cell free extract of T. hamatum FB10 showed protease, amylase, cellulase, chitinase, glucanase activities. T. hamatum FB10 inoculated with green gram increased 11% plant height, compared to the control. The fresh weight of the experimental group inoculated with T. hamatum FB10 increased 33.6% more compared to the control group. The green gram seedlings inoculated with T. hamatum FB10 increased 18% more dry weight than control group. Soil enzymes such as, urease, phosphatase, catalase and saccharase were improved in the soil inoculated with T. hamatum FB10. These biochemical components play potent role in soil fertility, energy conversion, and in soil organic matter conversion.

In vitro Evaluation of the Nutraceutical Potential of Theobroma cacao pod Husk and Leaf Extracts for Small Ruminants.

Some agroindustrial by-products could be used as nutraceutical materials for small ruminants helping with their nutrition while controlling their gastrointestinal nematodes. This study evaluated the potential invitro nutraceutical value of pod husks and leaves of three varieties of Theobroma cacao using two Haemonchus contortus isolates with different polyphenol susceptibility. Leaves and husks from three T. cacao varieties (AZT, CAL and CEY) were evaluated for their bromatological composition, invitro dry matter digestibility and polyphenol content. Acetone:water extracts (70:30) of each plant variety were evaluated using the egg hatch and larval exsheathment inhibition tests, using two isolates (FESC and PARAISO) of H. contortus. Effective concentrations 50% (EC50) were determined for both tests. The role of polyphenols was confirmed using polyvinylpolypyrrolidone. L3 exposed to CAL leaf extract were submitted to transmission electron microscopy. Both plant materials showed a good nutritional value to complement protein-rich diets for small ruminants. Extracts inhibited exsheathment of H. contortus L3 more effectively than the egg hatching, and the leaf extracts were more active than husk extracts in the L3 exsheathment inhibition. The FESC isolate was more sensitive to extracts. Polyphenols blocked exsheathment inhibition of leaf extracts. Structural damage was observed in the sheath and muscles of L3 exposed to CAL leaf extracts. The two T. cacao materials tested showed their potential to be used as ruminant feeds. Extracts affected H. contortus by blocking L3 exsheathment, particularly with the leaf extracts. The in vivo nutraceutical value should be confirmed in small ruminants.

Increased size and energy reserves in diapausing eggs of temperate Aedes aegypti populations

Many insects overwinter in diapause, a pre-programmed anticipated response to unfavorable environmental conditions, often induced by a short-day photoperiod. Diapause involves morphological changes and increased energy stores required for metabolic demands during winter. In diapausing mosquito eggs, the accumulation of lipids plays an important role, because these molecules are the primary fuel consumed during embryogenesis and pharate larvae metabolism, and have a key role in egg desiccation resistance. The supposed inability of the mosquito Aedes aegypti to lay diapausing eggs has been recently challenged by a study on a temperate population, which showed that the inhibition of egg hatching in response to short days is possible in this species. Thus, the aim of the present study was to assess the effects of parental photoperiod on embryonic diapause-related traits, such as the triglyceride content and size of eggs laid, of two populations whose localities of origin differ in their winter length. Two colonies were maintained for each population: one under a Short-Day Photoperiod (SD: 10 h:14 h – Light:Dark) and the other under a Long-Day Photoperiod (LD: 14 h:10 h – Light:Dark). The eggs obtained from each combination of population and light treatment were used for size measurement (length, width and volume) and for the quantification of triglyceride content. Egg size showed differences between photoperiod treatments, with larger width and volume in eggs from the SD treatment. Remarkably, eggs from the SD treatment accumulated twice as many triglycerides as those from the LD treatment. Also, the eggs derived from the population having the longer winter accumulated larger amounts of triglycerides. The higher lipid content is probably contributing to a better survival during the cold season in both populations. The photoperiod-induced response in egg size and amount of triglycerides observed in this study support the hypothesis that the Ae. aegypti populations studied are able to lay diapausing eggs, a fact that provides physiological bases for the further expansion of this species to colder regions.

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC50 varying from 0.61 to 0.26 mg P mL−1. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.

Potential Effect of the Nematicide Oxamyl and Surfactant Combinations on Root-Knot Nematode Meloidogyne incognita Infecting Tomato Plants

The adjuvants involved in pesticide formulation have a vital role in enhancing the physical properties of active ingredient delivered to the target site in plant-parasitic nematodes. The present study elucidates the potential effect of combinations of the nematicide oxamyl (Vaydate® 24 % SL) and surfactants on the root-knot nematode Meloidogyne incognita infecting tomato plants. Three surfactants namely, Agrimax-3H®, Silwet L-77®, and Sylgard 309® were used at their recommended rats (15 ml/100 L, 5 ml /100 L, and 250 ml/100 L, respectively) as well as the combinations of their respective ¼ R, ½ R, ¾ R with oxamyl at three rates i.e. the recommended (R, 48 mg a.i /plant); ¾ R, 36 mg a.i./plant and ½ R, 24 mg a.i./plant. The results revealed that surfactants caused approximately 50 and 25% hatching inhibition in egg masses and eggs, respectively while oxamyl exhibited the highest egg hatching inhibition by 90% throughout the trial period. The combination of oxamyl and Agrimax-3H showed significantly (P ≤ 0.01) more effectiveness against egg masses (71.17%) or free eggs (55.45% ) hatching as exposure time increase. Moreover, oxamyl potency increased to kill 95% of IJ2 whereas, other surfactants caused a 30% reduction. The mixtures of all tested adjuvants with oxamyl at the recommended rate raised oxamyl potency to kill 100 % IJ2 accompanied by a shortage of oxamyl latent period (LP). Whereas, Agrimax-3H surpassed other surfactants and shorted the LT50 of oxamyl to 0.82 days increasing its nematicidal effect. The addition of surfactants to oxamyl as protective treatment displayed a significant increase in tomato plant growth and suppressed galling of M. incognita while the combination between oxamyl and surfactants as curative treatments showed a vigor control efficacy within oxamyl and Agrimax-3H only and surpassed oxamyl formulation without significant difference.

Nematicidal potential of Citrus reticulata peel essential oil, isolated major compound and its derivatives against Meloidogyne incognita

Citrus reticulata (Kinnow) peel essential oil, isolated major compound limonene and its derivatives were evaluated for their in vitro nematicidal activity against Meloidogyne incognita. The essential oil was extracted from peels by hydrodistillation . Gas Chromatography-Mass Spectrometry analysis of essential oil revealed limonene as the major compound (51.42%) which was isolated from essential oil. Two derivatives of limonene viz. limonene tetrabromide and carvone were synthesized. Maximum egg hatch inhibition and juvenile mortality was shown by carvone at 1500 µg ml−1 after 96 hours of exposure followed by limonene, limonene tetrabromide and essential oil. The present studies showed that essential oil, major component limonene and its derivatives are effective against M. incognita and can be further explored for nematicidal properties. Further, substitution of carbonyl group in limonene as in carvone helps in enhancement of the nematicidal potential which is useful observation for exploration of these compounds in nematodes management

The carB Gene of Escherichia coli BL21(DE3) is Associated with Nematicidal Activity against the Root-Knot Nematode Meloidogyne javanica.

Biological nematicides have been widely used to lower the losses generated by phytoparasitic nematodes. The purpose of this study was to evaluate the nematicidal effects of Escherichia coli BL21(DE3) against Meloidogyne javanica and to identify nematicide-related genes. Culture filtrates of BL21(DE3) caused juvenile mortality and inhibited egg hatching in a dose-dependent manner. In the greenhouse, treatment of tomato seedlings with BL21(DE3) culture filtrates at 50 and 100% concentrations not only reduced the amount of M. javanica egg masses and galls, but improved plant root and shoot fresh weight. Culture filtrate analysis indicated that the nematicidal active ingredients of strain BL21(DE3) were non-proteinaceous, heat and cold resistant, sensitive to pH and volatile. To identify the genes associated with nematicidal activity, a BL21(DE3) library of 5000 mutants was produced using Tn5 transposase insertion. The culture filtrate of the MB12 mutant showed no nematicidal activity after 72 h of treatment and thermal asymmetrical interlaced PCR demonstrated that the carB gene was disrupted. Nematicidal activity was restored when the pH of the MB12 culture filtrate was adjusted to the original pH value (4.15) or following MB12 complementation with the carB gene, confirming a role for carB in mediating pH value and nematicidal activity. The outcomes of this pilot study indicate that BL21(DE3) is a potential microorganism for the continuable biological control of root-knot nematode in tomato and that carB affects the nematicidal activity of BL21(DE3) by modulating the pH environment.

Isolation and characterization of nodules endophytic bacteria Pseudomonas protegens Sneb1997 and Serratia plymuthica Sneb2001 for the biological control of root-knot nematode

Root-knot nematode disease, caused by Meloidogyne spp., is one of the most serious soil-borne diseases for vegetable industry worldwide. A total of 448 endophytic bacteria isolates were obtained from root nodules and screened for the potential biological control activity against M. incognita in the field and pot under the greenhouse conditions. Strain Sneb1997 and Sneb2001 were screened with high biocontrol efficacy against M. incognita and identified as Pseudomonas protegens and Serratia plymuthica by morphology analysis, physiological biochemical test and biomolecular assay, respectively. Furthermore, 24 h after treatment, P. protegens Sneb1997 and S. plymuthica Sneb2001 showed high lethal effect on the second stage juveniles of M. incognita with mortality rate were 87.78% and 92.67% and inhibited egg hatching significantly (P < 0.05) with the hatch inhibition rate of 87.76% and 82.86%, respectively. Meanwhile, P. protegens Sneb1997 was effective in improving seed germination and S. plymuthica Sneb2001 did not affect seed germination. Then, the stability and efficiency of M. incognita control by P. protegens Sneb1997 and S. plymuthica Sneb2001 was further assessed in greenhouse and in the field in 2018 and 2019. The results of pot experiment showed that the metabolites of P. protegens Sneb1997 and S. plymuthica Sneb2001 both significantly (P < 0.05) decreased the number of root galls and egg masses of root and P. protegens Sneb1997 significantly (P < 0.05) promoted the growth of tomato plants. The results of field experiments demonstrated that two tested strains could significantly (P < 0.05) reduce gall index and significantly (P < 0.05) increased the tomato plant biomass. Confocal microscopy and colonization assay demonstrated that both endophytes could colonize in tomato roots and stems with a high density for a long period of time. In summary, our results demonstrate that endophytic P. protegens Sneb1997 and S. plymuthica Sneb2001 can be used as potential biological control agents to control root-knot nematode disease.

Antihelminthic action of the Anethum graveolens essential oil on Haemonchus contortus eggs and larvae.

Gastrointestinal nematodes are responsible for great economic losses in sheep raising, and their control has long been carried out almost exclusively by the administration of anthelmintics, which have led to serious resistance problems. In the search for alternative control measures, phytotherapic research is highlighted. The aim of this study was to evaluate the action of Anethum graveolens (dill) essential oil on different stages of Haemonchus contortus life cycle, as well its cytotoxicity MDBK (Madin-Darby bovine kidney) cells. H. contortus larvae and eggs were obtained from infected sheep feces, and essential oil extracted from plant seeds through the Clevenger apparatus. 9.4, 4.7, 2.35, 1.17. 0.58 and 0.29 mg/mL concentrations were evaluated. The Egg Hatch Inhibition (HI), Larval Development Inhibition (LDI) and Larval Migration Inhibition (LMI) techniques were used. Thybendazole 0.025 mg/mL in HI and Levamisole 0.02 mg/mL in the LDI and LMI tests were used as positive controls, while distilled water and a Tween 80 solution were used as positive negative controls. The inhibition results obtained for the highest oil concentration were: HI 100%, LDI 98.58% and LMI 63.7%, differing (𝑝 <0.05) from negative controls. Main A. graveolens oil components present in 95.93% of the total oil were Dihydrocarvone (39.1%), Carvone (22.24%), D-Limonene (16.84%), Apiol (10.49%) and Trans-dihydrocarvone (7.26%). Minimum A. graveolens essential oil concentrations required to inhibit 50% (IC50) of egg hatching, larval development and larval migration were 0.006 mg/mL, 2.536 mg/mL and 3.963 mg/mL, respectively. Cell viability in MDBK (Madin-Darby bovine kidney) cells, when incubated with A. graveolens essential oil, was 86% for the highest (9.4 mg/mL) and 99% for the lowest concentration (0.29 mg/mL). A. graveolens essential oil, according to the results obtained in this study, is a promising alternative in sheep gastrointestinal nematode control.

Inhibition of Protease and Egg Hatching of Haemonchus contortus by Soybean Seed Exudates.

Gastrointestinal nematode infection of small ruminants causes losses in livestock production. Plant compounds show promises as alternatives to commercial anthelmintics that have been exerting selective pressures that lead to the development of drug-resistant parasites. Soybean (Glycine max) is an economical value crop, with a higher protein content compared to other legumes. The objective of this study was to evaluate whether the protease inhibitors exuded from the G. max mature seeds have anthelmintic activity against Haemonchus contortus. To obtain the soybean exudates (SEX), mature seeds were immersed in 100 mM sodium acetate buffer, pH 5.0, at 10 C, for 24 hr. Then the naturally released substances present in SEX were collected and exhaustively dialyzed (cutoff 12 kDa) against distilled water. The dialyzed seed exudates (SEXD) were heated at 100 C for 10 min and centrifuged (12,000 g, at 4 C for 15 min). The supernatant obtained was recovered and designated as the heat-treated exudate fraction (SEXDH). The protein content, protease inhibitor activity, and the effect of each fraction on H. contortus egg hatch rate were evaluated. The inhibition extent of SEX, SEXD, and SEXDH on H. contortus egg proteases was 31.1, 42.9, and 63.8%, respectively. Moreover, SEX, SEXD, and SEXDH inhibited the egg hatching with EC50 of 0.175, 0.175, and 0.241 mg ml-1, respectively. Among the commercial protease inhibitors tested, only EDTA and E-64 inhibited the H. contortus hatch rate (79.0 and 28.9%, respectively). We present evidence demonstrating that soybean exudate proteins can effectively inhibit H. contortus egg hatching. This bioactivity is displayed by thermostable proteins and provides evidence that protease inhibitors are a potential candidate for anthelmintic use.

Nematicidal activity of teleocidin B4 isolated from Streptomyces sp. against pine wood nematode, Bursaphelenchus xylophilus

Pine wilt disease (PWD) is a plant disease that causes serious damage to pine trees. PWD occurs when the host plant is infected with pinewood nematode (PWN), Bursaphelenchus xylophilus. In this study, a compound with nematicidal activity was isolated from actinomycetes and its efficacy was investigated in vitro. We screened and selected Streptomyces sp. 680560, which had nematicidal activity against B. xylophilus. Based on 16S rRNA sequence analysis, it showed 99.93% similarity with Streptomyces blastmyceticus NRRB-5480T . Furthermore, the active compound was isolated and identified as teleocidin B4. Teleocidin B4 at concentrations ranging from 6.25 to 100 μM had low nematicidal activity after 24 and 36 h against adult and stage juveniles (J2) of B. xylophilus, but after 48 h nematicidal activity exceeded 95%. The rate of inhibition of egg hatching for Teleocidin B4 6.25, 12.5, 25, 50, and 100 μM was confirmed to be dose-dependently inhibited after 48 h of treatment. Teleocidin B4 is not only toxic to hatched B. xylophilus, but also affects egg hatching. This study was carried out to isolate actinomycete metabolites from pine tree endophytes from various natural environments for control of PWD. A compound with nematicidal activity was isolated from a selected strain and its structure was identified as teleocidin B4. The nematicidal effect of the isolated active substance, teleocidin B4, was confirmed. This is the first report of the effect of teleocidin B4 on B. xylophilus, suggesting its possibility as a PWD control agent. © 2020 Society of Chemical Industry.

Evaluation of the efficacy of benzimidazoles against Ancylostoma caninum by egg hatch assay

Monitoring of drug susceptibility or resistance is important to maintain the efficacy of currently available therapeutic agents against parasites of veterinary importance. The egg hatch assay was performed to determine the in vitro efficacy of thiabendazole against Ancylostoma caninum eggs collected from naturally infected dogs in Krishna district, Andhra Pradesh. The results revealed a highly significant (P<0.01) inhibitory effect on egg hatching rates by thiabendazole in a dose dependant manner. Thiabendazole resulted in 100% inhibition of egg hatching at a concentration of 0.048 μg/mL. Further, the effective doses (ED50) required to induce 50% inhibition of egg hatching was 0.0049 μg/ mL, which is lower than the discriminating dose of thiabendazole (0.1 μg/mL). The results revealed that A. caninum in the study area were susceptible to thiabendazole indicating benzimidazole resistance was absent or insignificant in the study area.