Sensitivity to metalaxyl of Phytophthora infestans populations in potato crops in south-west England in 1980 and 1981

A laboratory test was developed to assess the sensitivity of field populations of Phytophthora infestans to metalaxyl. Discs of potato leaf tissue were floated upon solutions of the fungicide at different concentrations and inoculated with spores. The extent of symptom development was noted after incubation under standard conditions for 5–6 days. In preliminary experiments growth of isolates of P. infestans obtained from culture collections was severely inhibited in discs treated at 2 μg/ml. By contrast the development of an isolate obtained from a crop in Eire in which blight control with metalaxyl had failed, and known to be markedly less sensitive in vitro, was unaffected in discs treated at 100 μg/ml. During the summer of 1980, 234 samples of P. infestans were obtained from 20 sites in south-west England, 10 of which had received sprays containing metalaxyl and 10 of which had not. All samples were sensitive to metalaxyl applied at 2 μg/ml. In 1981, 35 sites within the same area, 30 of which had received sprays containing either metalaxyl or ofurace (a related fungicide), were similarly surveyed. Most of the 79 samples of P. infestans examined proved sensitive and at all sites the amount of blight was small. However, at three sites, including one not treated with acylalanine fungicides, strains were found which were unaffected by 100 μg/ml metalaxyl in leaf disc tests. These findings are discussed in relation to the development of resistant blight in other areas and to the use of fungicide mixtures.     

Changes in the resistance of tobacco leaf to Erysiphe cichoracearum DC. induced by topping,cytokinins and antibiotics*

Treatments that produced different rates of growth in attached tobacco leaves and leaf discs, also affected the growth of powdery mildew on them. Topping (removal of flower head) increased the resistance of upper leaves, and attached leaves of topped and intact plants were more susceptible than leaf discs from them incubated on water. Hyphal growth increased on leaf discs incubated on water at increasing light intensities, as did dry and fresh mass of healthy discs. On kinetin, dry and fresh mass also increased with light intensity but hyphal growth decreased slightly. Discs incubated at all light intensities on kinetin had less hyphal growth than those on water at the lowest intensity (< 50 lx). Floating leaf discs on chloramphenicol at 500 μ/ml restricted fungal growth but not quite as much as kinetin (10 μ/ml); kinetin inhibited the fungus in the presence of chloramphenicol. Actinomycin D (2.μ5 μ/ml) and puromycin (5.0 μml) had little effect on the fungus.     

Variation in the glucosinolate content of oilseed rape (Brassica napus L.) leaves

The glucosinolate content of oilseed rape {Brassica napus) leaves was monitored over the growth period 30–70 days after planting, and a comparison made between a single-low cultivar (low in erucic acid), Bienvenu, and a double-low cultivar (low in erucic acid and glucosinolate), Cobra. In older, fully-expanded leaves the glucosinolate concentration was very low (< 0.3 μmol/ml tissue water) and did not alter during the course of the experiment. In developing sixth leaves glucosinolate content increased rapidly and reached a maximum concentration (4–5 μmol/ml tissue water) 40 days after planting (6 days after leaf emergence). The concentration then declined, to about 1 μmol/ml after 60 days although the total glucosinolate content in leaves continued to increase until 50 days; much of the reduction in concentration was simply a result of leaf expansion. No major differences were seen between the two varieties in total glucosinolate content or in the individual compounds present. Cv. Cobra developed more quickly than cv. Bienvenu so direct comparison between leaves of the two cultivars was complex. When comparing the glucosinolate content of oilseed rape leaves, between cultivars or between treatments, it is vital to ensure that carefully matched leaves of comparable developmental age are selected.     

Reversible,long-term inhibition of ovulation with a gonadotropin-releasing hormone antagonist in the marmoset monkey (Callithrix jacchus)

The effects of weekly injections of a gonadotropin-releasing hormone (GnRH) antagonist (GnRHa) ([N-acetyl-DβNal1-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8D-Ala10] NH2 GnRH) on pituitary and ovarian function were examined in the marmoset monkey, Callithrix jacchus. In experiment 1, five cyclic females were given weekly injections of vehicle (50% propylene glycol in saline) for 6 weeks followed by GnRHa for 20 weeks, animals receiving either 200 μg GnRHa/injection (n = 2) or 67 μg GnRHa/injection (n = 3) for 10 weeks, after which the treatment was reversed. Bioactive luteinizing hormone (LH) and progesterone (Po) were measured in blood samples (0.2–0.4 ml) collected twice weekly until at least 8 weeks after the last GnRHa injection. GnRHa treatment, timed to begin in the midluteal phase, caused a rapid decline in LH and Po and luteal regression after a single injection (both doses). Po levels were consistently low (<10 ng/ml), and ovulation was inhibited throughout 200 μg treatment in all animals. Short periods of elevated Po (>10 ng/ml) were, however, occasionally seen during 67 μg treatment, indicating incomplete ovarian suppression. Mean LH levels were significantly lower during GnRHa treatment compared with the period of vehicle injection (all animals 200 μg; three animals 67 μg), and there were significant differences in LH levels between GnRHa treatments (200 μg vs. 67 μg) in four animals. Four animals resumed normal ovarian cycles after the end of GnRHa treatment (15/16 days, three animals; 59 days, one animal); the fifth animal died of unknown causes 32 days after the last GnRHa injection. In a second experiment, pituitary responsiveness to exogenous GnRH was tested 1 day after a single injection of vehicle or antagonist (200 or 67 μg). Measurement of bioactive LH indicated that pituitary response to 200 ng native GnRH was significantly suppressed in animals receiving the antagonist, the degree of suppression being dose related. A third experiment examined the effect of four weekly injections of 200 μg GnRHa on follicular size and granulosa cell responsiveness to human follicle-stimulating hormone (hFSH) in vitro. Follicular development beyond 1 mm was inhibited by GnRHa treatment (preovulatory follicles normally 2-4 mm) although granulosa cell responsiveness to FSH during 48 hr of culture was not impaired. These results suggest that the GnRHa-induced suppression of follicular development and ovulation was mediated primarily by an inhibition of pituitary gonadotropin secretion and not by a direct action at the level of the ovary.     

纳米硒通过抗氧化应激调节大脑NO含量改善睡眠剥夺小鼠认知功能

研究了纳米硒对睡眠剥夺(SD)小鼠(Mus musculus)认知功能的影响,并探讨其作用机制。将120只雄性昆明小鼠随机分成两批,第一批24只分为3组:对照组(NC)、亚硒酸钠组(SE)和纳米硒组(NS),分别给予硒浓度为4μg/ml的亚硒酸钠和纳米硒溶液每只0.5ml/d,NC组给等体积蒸馏水,连续30d,第31天测定SE和NS两组小鼠的血硒及全血GSH-Px活性,评价两种硒源的生物利用性;第二批96只分为4组:对照组(N-SeC),纳米硒低、中、高剂量组(L、M、H),L、M和H组分别给予硒浓度为2μg/ml、4μg/ml、8μg/ml的纳米硒溶液每只0.5ml/d,N-SeC组给予同体积蒸馏水,连续30d。第二批小鼠每组又各自分为4小组:SD对照组(SDC)及SD18h、SD36h、SD54h组,采用单平台水环境法(SPM)制作小鼠SD模型。在SD后,N-SeC、L、M和H组利用Y-型迷宫试验测定认知能力,同时测定小鼠大脑GSH-Px、NO、MDA含量。结果表明,纳米硒对GSH-Px活性的提高优于传统硒源亚硒酸钠,但血硒无显著差异;与SDC组比较,SD降低了小鼠的认知能力及大脑GSH-Px活性,提高了NO和MDA含量;与N-SeC比较,纳米硒使SD小鼠的认知功能得到改善,大脑GSH-Px活性提高,MDA和NO含量下降。上述结果表明,纳米硒能够改善SD小鼠的认知功能,这可能与其提高大脑GSH-Px活性并降低了自由基对大脑神经的损害有关。     

Leaf Disc Inoculation, a Fast and Precise Test for the Screening of Metalaxyl Tolerance in Sunflower Downy Mildew

A new method, based on leaf disc inoculation, was developed for the screening of metalaxyl tolerance in field isolates of Plasmopara halstedii . High-pressure liquid chromatography (HPLC) was used to determine the fungicide concentration in the inoculation medium and in the incubated leaf tissue over the test period. These measurements revealed that the fungicide concentration inside the leaf tissue within 24 h had adjusted to the concentration in the outer medium and remained constant for the time of cultivation over a period of more than 11 days. In contrast to whole seedling tests with application of the fungicide via seed dressing, the leaf disc method allows precise quantification of the effective fungicide concentration at the site of infection and is less space and time consuming. Metalaxyl tolerance of P. halstedii isolates was gradually determined according to the sporulation of the pathogen on sunflower leaf discs in the presence of increasing fungicide concentrations. Isolates collected in South Germany showed no tolerance and sporulation was prohibited when tests were carried out at 0.02  μ g (a.i.)/ml of metalaxyl or more. In contrast, a tolerant French isolate developed sporangia on leaf discs incubated in a metalaxyl solution of 100  μ g (a.i.)/ml.     

Variation in the sensitivity to metalaxyl of Pythium spp. isolated from carrot and other sources

Over a 3-yr period 261 isolates of 17 species of Pythium were tested for sensitivity to metalaxyl at concentrations of 5, 50 or 100 μ/ml. A wide range of responses was observed, from isolates where growth ceased at 5 μg/ml to those where growth at 100 μg/ml was similar to that of the untreated controls. In further tests isolates of 11 different species had ED50's < 1 μg/ml. A lower sensitivity was detected in isolates of six Pythium spp. where values in the range 1–10 μg/ml were obtained. This lower sensitivity was not related to previous known use of metalaxyl. Three isolates of Pythium dissotocum from sites where the fungicide had been used repeatedly had ED50's > 100 μg/ml and were considered resistant. The resistance was stable over a 2-yr period and isolates were cross-resistant to furalaxyl, benalaxyl, ofurace, cyprofuram and oxadixyl. Increasing concentrations of metalaxyl reduced or prevented the production of zoospores by four species of Pythium, although when zoospores were produced, this was followed by the normal processes of encystment and germination. Culturing P. dissotocum on different sub-lethal concentrations of metalaxyl for 18 wk did not induce a high level of resistance to the fungicide.     

Prolonged leaf senescence in Clusia multiflora H.B.K.

 Leaf aging and senescence in Clusia multiflora H.B.K. was investigated by artificial treatments, such as floating leaf discs on water in darkness, or darkening leaves attached to the parent plant in situ in trees living in a tropical cloud forest. In both cases several parameters modified by age were evaluated such as nitrogen levels, chlorophyll content, succulence and carbohydrates levels. A prolonged senescence (nearly 3 months in floating leaf discs) was observed, contrasting with species such as Heliocarpus americanus (5 days) and Cecropia palmatisecta (20 days), characterized by low values of leaf weight per area, but similar to species with high leaf weight per area and with high levels of organic acids such as Clusia minor and Fourcroya humboldtiana, where acids may act as a reserve of C and energy. After 30 days in darkness C. multiflora leaf samples collected in the field did not show differences in comparison to non-darkened opposite leaves with respect to chlorophyll, titratable protons and carbohydrates, and leaves performed photosynthesis after 2 months in darkness. The effect of age in leaves was evaluated in a gradient of leaves, sampled at different positions from the apex and ranging in age from 15 days to 2 years old. The study of senescence in tropical wild plants is uncommon, but it is important knowledge for understanding foliar development, and response to internal rather than environmental regulation in climates where seasons are not strongly marked as is the case in the tropical mountain forest, where C. multiflora constitutes an important component in the early successional vegetation. Received: 21 October 1996 / Accepted: 12 November 1996     

Lower marine fungi (labyrinthulomycetes) and the decay of mangrove leaf litter

This paper deals with the association of members of the Labyrinthulomycetes (Thraustochytriales and Labyrinthulales) with decaying or decayed leaves at an intertidal mangrove at Morib, Malaysia. Representatives of both orders of these obligately marine unicellular eukaryotes of unresolved taxonomic affinities (Chamberlain & Moss, 1998) were consistently isolated from leaves at all stages of decay from the recently fallen to those in an advanced stage of decay, but not from either green or senescent yellow leaves attached to trees. Baiting experiments using -irradiated leaf discs of Sonneratia and Rhizophora spp. immersed in the aquatic environment of the mangrove, revealed that leaf material was colonised by both labyrinthulids and thraustochytrids within 24 hours of immersion at the test site and these organisms were isolated from the leaf material throughout the 14 day study period. In vitro experiments using axenic cultures of three thraustochytrid genera inoculated onto sterile discs of Sonneratia leaves and incubated for 14 days caused loss of both biomass and structural integrity of the leaf material. Freeze fracture, followed by scanning electron microscopy of leaves inoculated with a thraustochytrid and a strain of Labyrinthula, revealed that penetration of the leaf occured after 4 days and that the thraustochytrid was associated with localised degradation of internal leaf tissues. Cellulase production by an isolate of Schizochytrium aggregatum was detected. The results of all the above investigations are discussed with reference to the role of members of the Labyrinthulomycetes in nutrient cycling in the mangrove.     

HPLC determination of a chemically modified nonantimicrobial tetracycline: Biological implications

Chemically modified tetracycline (4-de-dimethylamino tetracycline), like commercially available tetracyclines, is known to inhibit experimentally induced pathologic collagen breakdown. A method for measurement of chemically modified tetracycline in small volumes (50 μl) of rat serum was developed using reversed-phase HPLC; this was necessary because this tetracycline analog lacks antimicrobial activity and, therefore, cannot be measured with standard bioassays. This method uses the same solution for extraction and elution thus providing a simple and rapid assay for both drugs. Using this technique, the concentration of chemically modified tetracycline and tetracycline were determined in rat serum at different times after oral administration. The serum concentration of chemically modified tetracycline was much higher than that for tetracycline, and its serum half-life was greater. The IC₅₀ of chemically modified tetracycline and tetracycline, as inhibitors of collagenase from rat polymorphonuclear leukocytes, was determined and found to be 4.1 × 10⁻⁸m (0.02 μg/ml) and 2.4 × 10⁻⁴m (120 μg/ml), respectively. Based on the serum levels of these drugs after oral administration, and their IC₅₀ values, chemically modified tetracycline is potentially a far more potent inhibitor of excess collagenase activity than tetracycline, during pathologic conditions, and may have the added advantage of not producing some of the typical complications of long-term antibiotic therapy.     

The relationship between the entry of isoproturon into Alopecurus myosuroides and its effect on growth and CO2 fixation

Alopecurus myosuroides (blackgrass) was grown in nutrient and with isoproturon added to the solution when plants had three leaves. The transpiration stream coefficient factor was calculated over a period of 96h after treatment to be approximately 1. On this basis isoproturon entry into A. myosuroides was estimated using data for water loss through plants and there was a linear relationship between herbicide entry and plant weight 14 days after treatment. Treatment for at least 7 days with isoproturon at a dose of 1 μg a.i./15 ml nutrient solution (≡ 0.32 × 10^-6M) before returning plants to fresh nutrient was necessary for damage to be still evident after 14 days. This time period corresponded with the reduction of CO₂ exchange to zero as measured by infra-red gas analysis. Some recovery occurred from 6 h treatment with 120 μg a.i. isoproturon/15 ml nutrient solution when assessed after returning plants to untreated nutrient for 14 days. A quicker depletion in CO₂ uptake by blackgrass occurred when both the primary and secondary roots were treated compared with secondary alone but eventually the levels reached were similar. The ‘CALF’ model predicted much higher concentrations of isoproturon than appeared necessary to damage A. myosuroides. This suggests a major influence of climate on the plant and this and other interactions are discussed.     

Anaerobic cellulolytic rumen fungal populations in goats fed with and without Leucaena leucocephala hybrid, as determined by real-time PCR

The effect of Leucaena leucocephala hybrid-Bahru (LLB), which contains a high concentration of condensed tannins, on cellulolytic rumen fungal population in goats was investigated using real-time PCR. The fungal population in goats fed LLB was inhibited during the first 10 days of feeding, but after 15 days of feeding, there was a tremendous increase of fungal population (157.0 μg/ml), which was about fourfold more than that in control goats (39.7 μg/ml). However, after this period, the fungal population decreased continuously, and at 30 days of feeding, the fungal population (50.6 μg/ml) was not significantly different from that in control goats (55.4 μg/ml).     

Laboratory tests for leaf resistance to Botrytis squamosa in onions

Two laboratory methods of testing for resistance to Botrytis squamosa in Allium cepa, A. fistulosum and hybrid material derived from them were developed. In one method, seedlings were sprayed with conidiospores, incubated for 3 days at 15°C (2 days at 100% r.h.) and the numbers of lesions per leaf assessed. This test was destructive. The second method was non-destructive, the plants being retained for breeding purposes. Leaf segments (25 × 15 mm) or leaf discs (14 mm diameter) were inoculated individually with a drop (0·014 ml) of spore suspension, and maintained at 15°C and 100% r.h. with a 12 h photoperiod. The length of the latent period, denned as the number of days for 50% of the leaf portions to produce lesions bearing visible mycelium, was determined for each onion line in a test. The reciprocal (1/t) of the latent period was used to test for significant differences between lines by analysis of variance. With both test methods, spores were applied at concentrations that discriminated relative resistance most readily. In both seedling and leaf segment tests A. fistulosum was more resistant than A. cepa to B. squamosa. The leaf disc test indicated that cultivars of A. fistulosum differed in their resistance. In hybrid material resistance was intermediate or more like that of A. fistulosum.     

Pulsatile infusion of luteinizing hormone-releasing hormone: Effects on luteinizing hormone secretion and ovarian function in prepuberal gilts

Ten intact and hypophysial stalk-transected (HST), prepuberal Yorkshire gilts, 112–160 days old, were subjected to a pulsatile infusion regimen of luteinizing hormone-releasing hormone (LHRH) to investigate secretion profiles of luteinizing hormone (LH) and ovarian function. A catheter was implanted in a common carotid artery and connected to an infusion pump and recycling timer, whereas an indwelling external jugular catheter allowed collection of sequential blood samples for radioimmunoassay of LH and progesterone. In a dose response study, intracarotid injection of 5 μg LHRH induced peak LH release (5.9 ± 0.65 ng/ml; mean ± SE) within 20 min, which was greater (P < 0.001) than during the preinjection period (0.7 ± 0.65 ng/ml). After HST, 5 μg LHRH elicited LH release in only one of three prepuberal gilts. Four intact animals were infused with 5 μg LHRH (in 0.1% gel phosphate buffer saline, PBS) in 0.5-ml pulses (0.1 ml/min) at 1.5-h intervals continuously during 12 days. Daily blood samples were obtained at 20-min intervals 1 h before and 5, 10, 20, 40, 60 and 80 min after one LHRH infusion. Plasma LH release occurred in response to pulsatile LHRH infusion during the 12-day period; circulating LH during 60 min before onset of LHRH infusion was 0.7 ± 0.16 ng/ml compared with 1.3 ± 0.16 ng/ml during 60 min after onset of infusion (P < 0.001). Only one of four intact gilts ovulated, however, in response to LHRH infusion. This animal was 159 days old, and successive estrous cycles did not recur after LHRH infusion was discontinued. Puberal estrus occurred at 252 ± 7 days in these gilts and was confirmed by plasma progesterone levels. These results indicate that intracarotid infusion of 5 μg LHRH elicits LH release in the intact prepuberal gilt, but this dosage is insufficient to cause a consistent response after HST.     

Columbin inhibits cholesterol uptake in bloodstream forms of Trypanosoma brucei-A possible trypanocidal mechanism

The diterpenoid furanolactone (columbin) from Aristolochia albida inhibited growth of culture forms of Trypanosoma brucei. In vitro analysis of the compound at 5–250 μg/ml showed complete lysis of the parasites within 10–20 minutes post incubation. At 50 μg/ml, columbin killed about 50% of the parasites which initially appeared swollen under phase contrast microscopy. Also the total amount of cholesterol diminished dose-dependently in the presence of 10–100 μg/ml of columbin after a 3-day incubation period.

In vivo analysis of the compound in T. brucei-infected mice revealed that 25 mg/kg administered for 3 consecutive days, completely cleared the parasites from the peripheral circulation. However, columbin could not clear parasites in the cerebrospinal fluid.     

Rapid determination of tetracycline and lumecycline in human plasma and urine using high-performance liquid chromatography

A rapid and accurate method for the determination of tetracycline in human plasma and urine is presented. Determination of tetracycline in plasma is based on precipitation of plasma proteins with trifluoroacetic acid, followed by injection of the centrifuged plasma sample onto a μBondapak C₁₈ column. Acetonitrile in phosphate buffer pH 2.2 is used as mobile phase. Only tetracycline, and no trace of lumecycline can be detected in plasma and urine after administration of lumecycline, indicating that lumecycline is completely degraded to tetracycline, lysine and formaldehyde in the gastrointestinal tract prior to absorption.Determination of tetracycline in urine was performed by injection of urine diluted with phosphoric acid onto a μBondapak Phenyl column. The precision of determination of tetracycline in plasma, expressed as the relative standard deviation, was < 3% at tetracycline concentrations of 0.05 and 3.7 μg/ml. Urine determinations were made with a precision of < 1.5% at tetracycline concentrations of 0.5 and 6.7 μg/ml.     

Induction of ethylene biosynthesis in tobacco leaf discs by cell wall disesting enzymes

Cellulysin induces ethylene production in tobacco leaf discs by initiating the formation of 1-aminocyclopropane-1-carboxylic acid. Induction occurred within 30 to 60 min of incubation and was inhibited by aminoethoxyvinylglycine, and the antiproteases, PMSF and soybean trypsin inhibitor. Cycloheximide (CHI) at 2.8 μg/ml and chloramphenicol (CAP) at 100 μg/ml did not inhibit this induction although incorporation of the label from (3,4-¹⁴C)methionine into the acid-insoluble fraction was inhibited by 57%. At 14 μg/ml CHI, and CAP, ethylene production was inhibited by 25% while protein synthesis was inhibited by 75%. We suggest that either the low amounts of protein synthesis that appear to be insensitive to CHI is sufficient to induce ethylene biosynthesis or that Cellulysin activates a preexisting but inactive form of ACC synthase to promote ethylene biosynthesis. Also, induction of ethylene production by microbial enzymes that digests plant cell walls may be an initial protective response of plants that serves to combat microbial infection.     

Some effects of benomyl and carbendazim on Aphis fabae and Acyrthosiphon pisum on field bean (Vicia faba)

Field bean plants were treated with benomyl in a glasshouse, then nymphs of Aphis fabae and Acyrthosiphon pisum were caged on the second pinnate leaf. Soil drenches at concentrations of 150 and 75 μg benomyl/ml or above increased mortality of A. fabae and A. pisum respectively; 250 μg a.i./ml increased mortality and decreased progeny production of alate A. fabae. The effect on mortality persisted for at least 16 days after treatment. Foliar sprays increased mortality at concentrations of 75 μg a.i./ml and above (A. fabae). Field populations of A. pisum were reduced when bean plants were drenched or sprayed at a concentration of 250 μg a.i./ml and A. fabae populations were reduced by drenches but not by foliar sprays. A commercial formulation of carbendazim (Bavistin) increased aphid mortality whereas the formulation medium did not. Under the experimental conditions, benomyl affected the distribution of both species on young bean plants but did not induce a repellent effect; aphids preferred untreated leaves. Mortality and preference tests, and a field experiment, indicated that A. pisum was affected more than A. fabae.     

Quantitative Analysis of Polymyxin B Released from Polymyxin B-Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 10⁸ cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl₂ solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.