Behavioural response of the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to oxamyl

Infective juveniles of the entomopathogenic nematode Steinernema carpocapsae show a low level of locomotory activity that is presumed to limit their usefulness as biological insecticides. A 30 μg ml^-1 solution of the carbamate pesticide oxamyl reduced the proportion of nonmobile nematodes by nearly two thirds to 35%, while stimulating a 7.5-fold increase in sinusoidal movement. This increase in activity did not result in a corresponding increase in host-finding. Oxamyl treatment did not enhance infective juvenile pathogenicity to Galleria mellonella larvae. At higher concentrations, oxamyl caused aberrant nematode movement and partial paralysis. Heterorhabditis bacteriophora infective juveniles maintain a high level of locomotory activity. Treatment with 30 μg ml^-1 oxamyl increased the proportion of sinusoidal over nonsinusoidal movements, but infective juvenile host-finding and pathogenicity were significantly reduced. Higher rates impaired movement and induced complete paralysis. We conclude that oxamyl is incompatible with S. carpocapsae and H. bacteriophora. The concept of chemically activating infective juveniles to increased locomotory activity and thereby achieving enhanced efficacy is inconsistent with our results.     

Susceptibility of different developmental stages of the root-knot nematode, Meloidogyne incognita, to the nematicide oxamyl

Oxamyl (6.25 μg ml^-1 soil water) was applied to cucumber roots containing Meloidogyne incognita at different stages of nematode development. Oxamyl was more effective in reducing the proportion of juveniles which developed into females when applied soon after infection to second stage juveniles than when applied later (to third and fourth stage juveniles). Early application of oxamyl also significantly reduced the proportion of females with egg masses, whereas late application had no such effect. However, the number of eggs per egg mass and the size of the young adult females was significantly reduced by all oxamyl treatments - the earlier the application the greater the effect. These results support the hypothesis that actively feeding second stage juveniles of Meloidogyne spp. are more susceptible to systemic nematicides than are the non-feeding third and fourth stage juveniles.     

Behaviour of the systemic nematicide oxamyl in plants in relation to control of invasion and development of Meloidogyne incognita

A single foliar application of oxamyl (12.5 μg) in acetone significantly reduced invasion of cucumber seedlings by Meloidogyne incognita juveniles for at least 21 days but did not affect the early stages of development of juveniles which had already invaded the roots. In contrast, application of oxamyl to the roots significantly reduced both invasion and development of juveniles. Concurrent studies using radiolabelled oxamyl showed that the amount of toxicant in the roots after 3 days was 13 times greater following root application than after foliar treatment. It is probable that oxamyl concentrates at the sites of nematode attack as an overall concentration of only 3 ng oxamyl g^-1 root was sufficient to prevent invasion. Much greater concentrations than this were required to affect the nematode in vitro. Oxamyl appeared to be lost from the roots into the soil principally in the form of its non-toxic oxime and it is suggested that the site of action following foliar application is at the root surface or outer cortex. Studies on the invasion behaviour of M. incognita juveniles on agar showed that the action of oxamyl had a sensory component.     

Inhibition of development of Meloidogyne incognita by root and foliar applications of oxamyl

Oxamyl (2.5 μg cm^-3sand) applied to the roots of cucumber seedlings significantly reduced the number of vermiform second stage juveniles of Meloidogyne incognita which developed into swollen forms for at least 30 days. However, when cucumber seedlings containing nematodes were removed from oxamyl-treated sand 10 days after the pesticide was applied and placed in clean sand a large proportion of the juveniles had become swollen after a further 18 days. This suggested the effect of oxamyl on nematode development was reversible. A single foliar application of oxamyl (25–100 μg) in distilled water was also found to significantly reduce development of second stage juveniles to saccate forms However, the use of various adjuvants with a threshold foliar treatment (25 μg oxamyl) failed to significantly improve its activity.     

Differential control of potato cyst-nematodes, Globodera rostochiensis and G. pallida by oxamyl and the yields of resistant and susceptible potatoes in treated and untreated soils

In sandy loam infested with Globodera rostochiensis (2–95 eggs g^-1 soil) the yield of Desiree potatoes was decreased by 8·2 t ha^-1 for every increment of 20 eggs g^-1 soil. Oxamyl incorporated in the seedbed at 5 kg ha^-1 before planting prevented significant loss of yield and damage to the tubers and minimised nematode increase. Cara and Maris Piper potatoes, which were resistant and tolerant to G. rostochiensis usually responded less to oxamyl than did susceptible cultivars. In a range of cultivars, yield responses to oxamyl treatment of soil infested with G. rostochiensis often differed from those in soil infested with G. pallida. In field experiments, oxamyl controlled G. pallida less than G. rostochiensis. In pots, such differential control of the two species by oxamyl was not observed.     

Elimination of apple stem grooving virus by chemotherapy and development of an immunocapture RT-PCR for rapid sensitive screening

Ombuin (7,4′-dimethyl quercetin) (10 μg ml^-1, for 12 wk), glycyrrhizin/quercetin (80 μg ml^-1and 10 μg ml^-1respectively, for 18 wk), ribavirin (10 μg ml^-1, for 12 wk) and quercetin/ribavirin (10 μg ml^-1each, for 9–12 wk) reduced the titre of apple stem grooving virus (ASGV) when applied in vitro to infected tissue cultures of Nicotiana occidentalis obliqua Wheeler, and/or Malus domestica. ASGV was not detectable in both plant species after the quercitin/ribavirin treatment when tested by ISEM, herbaceous host indexing, RT-PCR, and immunocapture RT-PCR. A sensitive immunocapture RT-PCR procedure for the detection of ASGV was developed for the screening of treated samples to assess antiviral activity.     

Efficacy of avermectins against a plant parasitic nematode

Avermectins (AVM) B_1a/1b and B_2a-23-one rapidly impaired the locomotor activity of Meioidogyne incognita second stage juveniles. On agar plates, concentrations as low as 0–0003 μg AVM B_2a-23-one cm^-3 significantly reduced the number of juveniles in cucumber roots and the proportion of juveniles which developed to a saccate stage. The effects of AVM on hatching and movement of nematodes appeared to be at least partly reversible.     

Control of the rootknot nematode meloidogyne javanica by Bacillus cereus

Exposure of Meloidogyne javanica second‐stage juveniles to the bacterium Bacillus cereus in soil inhibited the penetration of the juvenile nematodes into tomato roots. Culture filtrate of the bacterium grown on nutrient broth and tryptic soy broth revealed nematocidal activity on M. javanica juveniles and eggs. Loss of the nematocidal activity of the media by lowering pH, boiling or dialysis raised the possibility that the active ingredient in the culture filtrate was ammonia, released during the breakdown process of peptides in the media by bacterial activity. Free ammonia (NH₃) concentrations in the nutrient broth and tryptic soy broth culture filtrates measured after 48 h were 140 and 190 µg ml^?1 respectively. Exposure of second‐stage juveniles to 9.3 µg ml^?1 ammonia for 40 h in vitro was lethal to 95% of the nematode population. In a nitrate medium, nitrite accumulated up to 250 µg ml^?1 during the growth of the bacterium, and its culture filtrate revealed nematocidal activity. The nematocidal activity of the bacterium increased when the bacterium was applied with various proteinaceous supplements to soil. Soil treated with the bacteria and peptone showed an earlier nematocidal activity than either the bacteria or peptone applied alone, and also had a higher level of ammonia than the individual treatments. However, the level of ammonia was lower than the lethal level for second‐stage juveniles recorded in vitro. The nematocidal activity exhibited by the bacterium‐proteinaceous amendment combination is not fully understood; the ammonia released during protein degradation by the bacterium may contribute significantly to the recorded nematocidal activity.     

Emergence of juvenile potato cyst-nematodes Globodera rostochiensis and G. pallida and the control of G. pallida

Speed of emergence of juveniles from cysts in potato root diffusate (PRD) in vitro differed between Globodera rostochiensis and G. pallida and between populations within each species. Early emergence in vitro was slower in most populations of G. pallida than in most populations of G. rostochiensis. Fewer G. rostochiensis juveniles emerged from 4 or 6 month old than from 4 yr old cysts. More G. rostochiensis emerged in solutions of sodium metavanadate at concentrations of 10^-2 and 10^-3 M than in PRD and as many G. pallida emerged in the same solutions as in PRD. In plots of bare fallowed sandy loam, emergence of G. pallida was stimulated by 10^--³ M sodium metavanadate. The emergence of two populations of C. pallida in PRD was stimulated by the addition of benomyl at 0.1 ppm (3.4 × 10^--⁷ m). In microplots, cv. Cara potatoes grown for 8 wk decreased four populations of G. pallida by up to 93%. During a 4 wk period in PRD, more than 20 juveniles per gravid female emerged from five of 25 populations of G. pallida. In root observation boxes in which cv. Désirée was grown, oxamyl applied to the top 15 cm of a peaty loam soil greatly increased G. pallida in soil 1545 cm deep. In another peaty loam, but not in a sandy loam, the same treatment appeared to increase the nematode in soil 15–30 cm deep. Oxamyl incorporated in the uninfested top 15 cm of all three soils largely prevented nematode increase from juveniles migrating upwards from untreated heavily infested soil 15–30 cm deep. These experiments suggest that inadequate control of G. pallida increase on susceptible potatoes by an oximecarbamate nematicide of short persistence, such as oxamyl, is primarily due to the slow rate of juvenile emergence in most populations of G. pallida, with a second generation and the upward migration of juveniles from deeper untreated soil later in the growing season as potential contributory factors.     

Control of Globodera rostochiensis in Relation to Method of Applying Nematodes

Soaking potato tuber pieces for 15 min in 8,000 μg/ml of oxamyl just before planting reduced the number of Globodera rostochiensis cysts that developed on potato roots, but this treatment was phytotoxic. Five foliar applications of 1.12 kg a.i./ha of oxamyl or carbofuran at 10-day intervals beginning when 90% of the plants had emerged suppressed increase in G. rostochiensis densities. Similar foliar applications of phenamiphos were ineffective in controlling G. rostochiensis. Soil applications (in the row at planting) of aldicarb, carbofuran, phenamiphos, ethoprop, and oxamyl at 5.6 kg a.i./ha reduced the numbers of white females that developed on potato roots, but only those treatments involving aldicarb and oxamyl suppressed G. rostochiensis population increase. Combined soil and foliar treatments did not provide any advantage over soil treatment alone, as soil applications of 5.6 kg a.i./ha alone were equal to, or better than, combined soil (3.4 kg a.i./ha) and foliar (2.2 kg a.i./ha) applications in controlling G. rostochiensis.     

Investigation of the antibacterial activity of 3-O-octanoyl-(-)-epicatechin

Aims: To measure antibacterial activity of the semi-synthetic flavonoid 3-O-octanoyl-(–)-epicatechin and investigate the mechanism of action. Methods and Results: MICs determined by the broth microdilution method were 50 μg ml⁻¹ for β-lactam sensitive and resistant Staphylococcus aureus, and 100 μg ml⁻¹ for vancomycin sensitive and resistant enterococci. In time-kill studies, 100 μg ml⁻¹ 3-O-octanoyl-(–)-epicatechin reduced colony forming unit numbers of antibiotic sensitive and methicillin-resistant Staph. aureus below detectable levels within 120 min. Bacterial aggregation was not observed when cells exposed to 3-O-octanoyl-(–)-epicatechin were examined by light microscopy. It was also shown that 50 μg ml⁻¹ 3-O-octanoyl-(–)-epicatechin is capable of reducing colony forming unit numbers of high cell density Staph. aureus populations by 80-fold within 60 min incubation, and inducing leakage of 50% of their internal potassium within just 10 min. Conclusions: 3-O-Octanoyl-(–)-epicatechin is active against Gram-positive bacteria, has bactericidal activity against both antibiotic sensitive and resistant strains, and is likely to exert its primary antibacterial effect by damaging the cytoplasmic membrane. Significance and Impact of the Study: 3-O-Octanoyl-(–)-epicatechin has significant antibacterial activity and additional structural modification and/or formulation studies may allow this to be potentiated.     

Bio-analytically fluorimetric method for estimation of ertapenem in real human plasma and commercial samples; application to pharmacokinetics study

Ertapenem (EPM) has been recently approved by the United States Food and Drug Administration (US-FDA) as an antimicrobial drug. EPM has a broad spectrum of action against different bacterial strains and is most commonly prescribed in Egypt for the treatment of Klebsiella pneumonia. In this study, EPM was estimated using a sensitive and selective spectrofluorimetric method for human plasma and pharmaceutical vials. The measured fluorescence (at 540 nm) was obtained from reaction of EPM with 0.05% w/v benzofurazan (NBD-Cl) using 0.1 M borate buffer pH 8.8 after excitation at 460 nm. The fluorometric linear range was stable from 10 to 350 ng ml⁻¹. The lower limit of detection and the lower limit of quantitation were found to be 2.13 and 6.47 ng ml⁻¹ respectively. Many factors such as pH, temperature, heating time, and NBD-Cl concentration were optimized. The presented work was validated according to International Council for Harmonisation guidelines and bio-analytically validated using FDA recommendations. The significant finding of this study, sensitivity, was successfully applied in Egypt for a pharmacokinetic application and commercial vials. Pharmacokinetic parameters were studied and the result, recorded as C_max of EPM, was found to be 83.60 μg ml⁻¹ after infusion of 0.5 g of Invanz® for 30 min. AUC_0-∞ was found to be 320 ± 30.2 μ.h ml⁻¹.     

The effects of deep cultivation and oxamyl on control of potato cyst-nematode, Globodera rostochiensis

Soil compaction associated with frequent cultivation of potatoes was partly removed with a deep winged-tine coulter. This increased the yield of tubers of cv. Cara in 1987 and 1988 and of cv. Désirée in 1987 in soil which was heavily infested with potato cyst-nematode, Globodera rostochiensis (Woll.), and which was treated with oxamyl at 5.6 kg ha^-1. In 1988, in soil not treated with oxamyl, deep cultivation significantly decreased the yield of cv. Cara. In both years, oxamyl decreased numbers of G. rostochiensis eggs in the soil following cv. Désirée potatoes but not following cv. Cara which were resistant to the nematode.     

Bioactive 3,8‐Epoxy Iridoids from Valeriana jatamansi

Twelve 3,8‐epoxy iridoids, including four new compounds, jatamanins R–U ( 1 – 4 ), and eight known compounds ( 5 – 12 ), were obtained from the roots and rhizomes of Valeriana jatamansi. The structures were elucidated from analysis of spectroscopic data. The absolute configurations of 1 – 4 were determined by comparison of experimental and literature ECD spectra. Moreover, the compounds were evaluated for cytotoxic effects against glioma stem cells, inhibition of NO production, activity against influenza A virus and reversal of multidrug resistance of HepG2/ADR cells. Compounds 9 and 12 showed significant cytotoxic potency against GSC‐18# (IC₅₀=1.351 and 4.439 μg ml^?1, respectively) and GSC‐3# (IC₅₀=10.88 and 6.348 μg ml^?1, respectively) glioma stem cells, while compound 12 was also slightly less potent against GSC‐12# (IC₅₀=13.45 μg ml^?1) glioma stem cell growth. In addition, compounds 9 and 12 displayed obvious inhibition of NO production (IC₅₀=4.6 and 15.8 μm , respectively).     

Effects of rotation length and oxamyl on potato yield and the potato cyst-nematode, Globodera rostochiensis, in a sandy loam soil

In sandy loam infested with golden potato cyst-nematode, Globodera rostochiensis, oxamyl at 5.6 kg a.i. ha^-1 incorporated in the top 15 cm of the soil just before planting potatoes greatly reduced nematode population increase on susceptible cv. Désirée grown six, seven or eight years after the last susceptible potato crop, but did not significantly increase tuber yields. In four-course and two-course rotations, oxamyl also controlled increase of G. rostochiensis and greatly increased yields of both cv. Désirée and resistant cv. Maris Piper. Oxamyl maintained tuber yields in a four-course rotation at the same level as in a six to eight-course rotation. Decline of G. rostochiensis in the soil was much faster under barley in some two-course rotations than under barley in four-course rotations.     

Mode of action of fosthiazate used for the control of the potato cyst nematode Globodera pallida

Fosthiazate (Nemathorin 10G Ishihara Sangyo Kaisha Ltd, Japan) is a new nematicide approved for use on potatoes Solanum tuberosum L. in die UK for die control of die potato cyst nematodes Globodera rostochiensis (Woll). Skarbilovich and G. pallida (Stone). Fosdiiazate delayed and suppressed hatch of die potato cyst nematode Globodera pallida in bom in vitro laboratory tests and a glasshouse pot experiment. In vitro hatch was temporarily inhibited by fosdiiazate concentrations above 0.09 μg ml^-1 and increasing me fosdiiazate concentration further prolonged the duration of hatch inhibition. Analysis of fosthiazate soil concentrations, using high-pressure liquid chromatography, during me glasshouse experiment showed mat hatch was suppressed in the soil at concentrations above 0.5 mg kg^-1. Other factors such as the paralysis of hatched nematodes in the soil solution are also involved.     

Content of cysteine, reduced and oxidized glutathione in spermatozoa of representatives of Acipenseriformes (Acipenser baerii and A. ruthenus) as well as teleosts (Perca fluviatilis and Sander lucioperca)

Glutathione belongs to a vital intra‐ and extra‐cellular protective antioxidant and is found almost exclusively in its reduced form. The ratio between its reduced and oxidized within cells is often used as a marker of cellular toxicity. The objectives of the study were to (i) determine both the reduced (GSH) and oxidized glutathione (GSSG) and cysteine (Cys) in the sperm of the Acipenser baerii and Acipenser ruthenus, as well as in Perca fluviatilis and Sander lucioperca, and (ii) to demonstrate the differences in concentration levels between representatives of acipenseriform and teleost species. High performance liquid chromatography with electrochemical detection was employed. The average content of the thiols determined in the sperm samples were as follows: Acipenser baerii (cysteine 55 ± 8 μg ml^?1; GSH 126 ± 19 μg ml^?1; GSSG 49 ± 7 μg ml^?1), Acipenser ruthenus (cysteine 62 ± 9 μg ml^?1; GSH 768 ± 115 μg ml^?1; GSSG 180 ± 16 μg ml^?1), Sander lucioperca (cysteine 251 ± 38 μg ml^?1; GSH 185 ± 28 μg ml^?1; GSSG 93 ± 14 μg ml^?1), Perca fluviatilis (cysteine 281 ± 42 μg ml^?1; GSH 496 ± 74 μg ml^?1; GSSG 138 ± 21 μg ml^?1). Based on the results obtained it can be concluded that this method is sensitive and selective for the determination of these compounds in real samples. Results revealed differences in cysteine content between species of the two systematic categories but also showed that ratios between GSH and GSSG can vary between species while potentially predict oxidative stress in fish sperm.     

Effect of the nematicide oxamyl on the multiplication of Globodera rostochiensis and G. pallida and on the haulm growth and yield of six potato cultivars with different levels of resistance and tolerance

The effect of a pre-planting application of oxamyl on the yields of six potato cultivars was studied in co-operative field trials in 1981. Two sites were ‘uninfested’ with potato cyst nematodes (PCN), two were lightly infested (<25 eggs/g soil) and six were moderately to heavily infested (three with Globodera rostochiensis and three with G. pallida). At the uninfested and lightly infested sites oxamyl had little effect on mid-season haulm weights or on final tuber yields. At sites moderately to heavily infested with G. rostochiensis the haulm growth of all cultivars tended to be increased by a similar amount on plots treated with oxamyl, Pentland Dell being least responsive. Yield was increased by different amounts, the increases being least for cvs Cara and Maris Piper and most for Corsair and Pentland Dell. At the sites moderately to heavily infested with G. pallida Cara was again tolerant, its yield being increased little by oxamyl compared with the other cultivars. Maris Piper gave the largest yield increase. Final populations of PCN on non-resistant cultivars were reduced by oxamyl at some sites but not at others. Resistant cultivars also decreased the final numbers of PCN at most sites. Two cultivars derived from Solanum vernei with different degrees of resistance, appeared to be almost equally effective in controlling G. rostochiensis and G. pallida.     

In vitro anti‐enterovirus 71 activity of gallic acid from Woodfordia fruticosa flowers

Aims: The anti‐enterovirus 71 (EV71) activity of six Nepalese plants’ extracts and gallic acid (GA) isolated from Woodfordia fruticosa Kurz (family; Lythaceae) flowers were evaluated in Vero cells. Methods and Results: The anti‐EV71 activity of tested compounds was evaluated by a cytopathic effect reduction method. Our results demonstrated that flowers’ extracts of W. fruticosa exerted strong anti‐EV71 activity, with a 50% inhibitory concentration (IC₅₀) of 1·2 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived therapeutic index (TI) was more than 83·33. Rivabirin showed no antiviral activity against EV71. Furthermore, GA isolated from W. fruticosa flowers exhibited a higher anti‐EV71 activity than the extract of W. fruticosa flowers, with an IC₅₀ of 0·76 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived TI was 99·57. Conclusions: This study demonstrated that flower extracts of W. fruticosa possessed anti‐EV71 activity and GA isolated from these flowers showed stronger anti‐EV71 activity than that the extracts. Significance and Impact of the Study: Our results suggest that the GA from W. fruticosa flowers may be used as a potential antiviral agent.     

Molecular typing, serotyping and cytotoxicity testing of Campylobacter jejuni strains isolated from commercial broilers in Puerto Rico

Aims: Thirty Campylobacter jejuni strains isolated from fecal samples (n = 94; 32%) from 13 positive farms (n = 17; 76%) from commercial broiler chickens in Puerto Rico were analysed by molecular methods. Methods and Results: Isolates were identified with multiplex polymerase chain reaction assays, tested for their antimicrobial susceptibility and characterized with pulsed‐field gel electrophoresis (PFGE), multilocus sequence typing (MLST), serotyping and bacterial cytotoxicity in mammalian cells. Isolates exhibited high resistance to vancomycin (minimum inhibitory concentration, MIC of >256 μg ml^?1) and trimethoprim (MIC of >32 μg ml^?1); few were resistant to clindamycin (MIC₉₀ 4 μg ml^?1), erythromycin (MIC₉₀ 8 μg ml^?1) and tetracycline (MIC₉₀ 8 μg ml^?1); but none was resistant to azithromycin (MIC₉₀ 4 μg ml^?1), ciprofloxacin (MIC₉₀ 1 μg ml^?1) or gentamycin (MIC₉₀ 4 μg ml^?1). Most strains restricted with SmaI, but a combination of SmaI–KpnI digestion was more discriminatory. MLST analysis yielded four sequence types (ST), and ST‐2624 was the predominant one. Phylogenetic analysis revealed a high degree of recombination for glnA and pgm genes. The predominant serotypes were O:3 and O:5. Most strains had lowest cytotoxicity potential with Caco‐2 cells, medium cytotoxicity with INT‐407 and Hep‐2 cells and high cytotoxicity with CHO cells. Conclusion: A low degree of antimicrobial resistance, 13 PFGE profiles, 4 ST and a large variability in cytotoxicity assays were found for these strains. Significance and Impact of the Study: This is the first characterization of C. jejuni strains isolated from broilers in Puerto Rico. The genetic diversity of these strains suggests that several techniques are needed for strain characterization.