Comparative study of immunobiological activities ofPseudomonas aeruginosa andBrucella melitensis lipopolysaccharides

The toxic activity ofBrucella melitensis andPseudomonas aeruginosa lipopolysaccharides as well as their behavior as immunogens, mitogens, and interferon inducers have been studied. Although their toxicities were very similar, the former molecule was incapable of eliciting a primary immune response in mice. Rabbit hyperimmunization gave titers half of those obtained withP. aeruginosa lipopolysaccharide. Optimal mitogenic responses of spleen cell cultures were obtained using 10–50 μg/ml and 50–100 μg/ml ofPseudomonas andBrucella lipopolysaccharide, respectively, giving the latter a lower stimulation of³H-thymidine uptake. Interferon titers induced in chickens byBrucella lipopolysaccharide were three times lower than those obtained withPseudomonas lipopolysaccharide.     

Effects of 5-fluoro-2′-deoxyuridine on bacterial growth its use for DNA labelling

Experiments on the action of 5-fluoro-2′-deoxyuridine on growth ofEscherichia coli B, CECT 101;Pseudomonas fluorescens, CECT 318;Pseudomonas savastanoi, CECT 93;Micrococcus luteus, ATCC 4698;Bacillus cereus, CIP 52.58;Bacillus macerans, ClP 52.58 andBacillus subtilis, ATCC 6633, are described. The inhibition of growth is reversed by thymine plus uracil in all cases except inPseudomonas strains in which uracil alone is active, and in which no exogenous thymine is taken up, not even in the presece of 2′-deoxyguanosine. Growth conditions for improved labelling of bacterial DNA are discussed in the light of the results.     

Effect of aeration on ethylene production by soil bacteria and soil samples cultivated in a closed system

Summary Ethylene production was studied in shaken cultures ofPseudomonas putida andPseudomonas fluorescens isolated from soil and in unsterile garden soil samples moistened to 60% of the water holding capacity. The highest ethylene accumulation in bacterial cultures was reached under conditions of delayed aeration,i.e. when the culture was closed and the aeration started after the oxygen content decreased to 4%. The ethylene production rose immediately after the beginning of aeration. Under these conditions ethylene production was inP. fluorescens 2–3 times and in glucosecultivatedP. putida 6 times higher than in the fully aerated cultures. Methionine stimulated ethylene production byP. fluorescens, whereas glucose proved to be more suitable forP. putida. This strain was incapable of growth on methionine as the sole carbon source. Samples of nonsterile garden soil produced the highest amounts of ethylene under anaerobic conditions. Artificial inoculation of soil samples byP. putida resulted in an increase of ethylene formation in samples with delayed aeration. Addition of glucose or glucose with methionine stimulated ethylene production in all soil samples.     

Effect of berberine on growth of the callus cultures of several species

A bacteriostatic concentration of berberine much inhibited growth of the callus cultures ofLithospermum erythrorhizon, Datura inooxia andCarthamus tinctorius, but little inhibited the callus growth ofMacleaya cordata. On the other hand, the high concentration of berberine tended to stimulate the callus growth ofCoptis japonica var.japonica. Among callus cultures of the five species described above, 4-desoxypyridoxine inhibited growth of the callus cultures ofD. innoxia andC. tinctorius. In these two callus cultures, recovery effects of some of the vitamin B₆ group (10 μg/ml) on the inhibition of callus growth by berberine (100 μg/ml) or 4-desoxypyridoxine (50 μg/ml) were observed.     

Bacterization of peanut withPseudomonas fluorescens for biological control ofRhizoctonia solani and for enhanced yield

Severity of stem-rot disease of peanut caused byRhizoctonia solani was reduced by 54.9 and 68% in plants of two cultivars treated in the greenhouse with antagonistic strains ofPseudomonas fluorescens. These strains were selected based on theirin vitro toxicity to mycelial growth and sclerotial germination ofR. solani. In field experiments, bacterization of peanuts withP. fluorescens resulted in taller plants (by 25.7%) and increased yields (by 59.0%).     

Organogenesis andin vitro flowering ofEchinochloa colona. Effect of growth regulators and explant types

Echinochloa colona regeneration via organogenesis in callus cultures derived from leaf base and mesocotyl expiants andin vitro flowering were achived. Shoot bud regeneration was achieved on Murashige and Skoog’s (MS) basal medium supplemented with 6.66 μM 6-benzylaminopurine (BAP), 2.68 μM 1-naphthalene acetic acid (NAA) and 3 % (m/v) saccharose. Regenerated shoots were rooted on half strength basal MS medium with 2 % (m/v) saccharose devoid of growth regulators. About 90 -95 % of rooted plantlets survived in the greenhouse.In vitro flowering was induced in the regenerated shoots derived from callus on half strength MS medium supplemented with 4.4 μM BAP, 74.07 μM adeninesulphate, 0.72 μM gibberellic acid, and 3 % (m/v) saccharose. The frequency ofin vitro flowering was 80 – 90 % in three repeated experiments. Fertile seeds were recovered fromin vitro grown plantlets which were subsequently germinated into plants. Acknowledgement: The authors wish to thank to the Department of Environment and Forests, Government of India for financial assistance to undertake this investigation.     

Seasonal variation in allelopathic potential of<Emphasis Type="Italic">Artemisia princeps</Emphasis> var.<Emphasis Type="Italic">orientalis</Emphasis> on plants and microbes

We investigated seasonal variations in allelopathic potential ofArtemisia princeps var.orientalis. Aqueous and meth-anol extracts and volatile substances were prepared in the laboratory from samples collected monthly (April through October). Their impacts were then assessed on the germination and seedling growth ofLactuca sativa andAchyranthes japonica. The allelopathic potential varied with the time of sample collection and the concentration tested. For example, germination ofL. sativa was not inhibited by the aqueous extract but seedling growth (shoots and roots) was, with its seasonal effect being significant. ForA. japonica, seed germination was not inhibited at lower concentrations (except for August samples). However, at higher concentrations and in certain months (especially July), germination was more negatively affected. The degree of seedling growth inhibition also differed by month and by extract concentration, with roots being impacted more than shoots. Volatile substances also had a time-dependent influence on the germination and seedling elongation ofA. japonica. In a separate experiment, the ethyl-acetate and water fractions of a crude methanol extract were prepared monthly fromA. princeps var.orientalis. Here, we examined their antimicrobial activities against three gram-positive bacteria (Bacillus cereus, Bacillus subtilis, andStaphylococcus aureus), two gramnegative bacteria (Escherichia coli andPseudomonas fluorescens), and one lactic acid bacterium,Lactobacillus plantar urn. The ethyl-acetate fraction that was sampled in September was remarkably potent againstB. cereus andB. subtilis, whereas the water fraction collected in August and September showed great antimicrobial activity against the grampositive and -negative bacteria. In contrast,L. plantarum was not inhibited by the water fraction, regardless of the sampling month. Likewise, the ethyl-acetate and water fractions collected in April and October had the lowest levels of antimicrobial activity.     

Proline-based modulation of 2,4-diacetylphloroglucinol and viable cell yields in cultures of Pseudomonas fluorescens wild-type and over-producing strains

The antifungal compound 2,4-diacetylphloroglucinol (DAPG) is produced in the rhizosphere of wheat by pseudomonad populations responsible for the natural biological control phenomenon known as “take-all decline.” Studies were conducted to elucidate the impact of DAPG and its co-product 2,4,6-trihydroxyacetophenone (THA) on the production of Pseudomonas fluorescens for biological control. Increasing DAPG from 0.1 g/l to 0.5 g/l and THA from 0.05 g/l to 0.5 g/l significantly inhibited the growth and lowered the yield of viable bacteria in liquid cultures. On further examination of these metabolites applied in seed coatings, levels of DAPG and THA exceeding 0.05 mg/g seed significantly reduced wheat germination percentages. The three-way interaction of DAPG, THA, and culture medium ingredients was significant, and greatest seed germination loss (40–50%) was observed when 0.5 mg DAPG and 0.25 mg THA were combined in a coating of 0.5 ml culture medium per gram of seed. Based on the results of Biolog GN microplate, flask, and fermentor screens of C sources, proline was found to optimize the viable cell yields of the P. fluorescens strains tested. The combination of proline with glucose and urea as C and N sources in growth media could be optimized to minimize DAPG production and maximize the vitality of P. fluorescens Q8R1-96 and Q69c-80:miniTn5:phl20 (DAPG over-producer). In production cultures, the proline supply rate offers a potentially useful means to optimize the biological control agent yield and quality.     

Embryo culture and taxane production inTaxus spp

Summary We have previously shown (Flores and Sgrignoli, 1991) that immature embryos ofTaxus brevifolia andT. X media are capable of precocious germination and can grow into seedlings in vitro. The cultural and environmental parameters for embryo germination and conversion into seedlings have been optimized and extended toT. baccata andT. cuspidata. A 14-h photoperiod improved embryo germination and growth into seedlings. A pregermination cold treatment of the seeds had a positive effect on both the onset and percentage of germination. Embryos from cold-treated seeds germinated earlier and at a higher frequency than those from control seeds. Boron was necessary for embryo germination, and levels of this micronutrient were established for optimal growth and germination ofT. brevifolia andT. X media cv. Hicksii embryos. Gupta and Durzan’s medium was superior to White’s for embryo germination and root formation. Naphthaleneacetic acid stimulated root formation in embryo-derived seedlings. We also found that immature embryos could be induced to form callus with embryogenic potential. Taxol and related taxanes were detected in embryo- derived seedlings.     

Effects of external stimuli on environmental bacterial strains harboring analgD-lux bioluminescent reporter plasmid for the study of corrosive biofilms

An alginic acid biosynthesis bioluminescent reporter plasmid, pUTK50, was transconjugated into environmental strains ofPseudomonas putida, Pseudomonas fluorescens, andStenotrophomonas maltophilia. Bioluminescent transconjugates were selected from each strain for investigation of environmental stress factors that promote alginic acid exopolymer biosynthesis in developing biofilms. Environmental stimuli associated with increased levels of alginate synthesis, in a previously developed organism,P. aeruginosa FRD1, were applied to the environmental strains. Increased salt concentrations and higher ratios of nitrate vs ammonium ions as the limiting nitrogen source induced bioluminescence in FRD1 and the environmental strains. However, for environmental strains ofP. putida, P. fluorescens andS. maltophilia, polysaccharides were detected with low uronic acids content and different structural components. When tested within a biofilm,S. maltophilia O46 demonstrated exceptional adhesive and corrosive properties while alginic acid synthesis was not high. In most of the environmental strains, periods of increased bioluminescence were induced by external stimuli, but exopolysaccharides other than alginic acid were expressed. It is hypothesized that the environmental strains have homologous but nonidentical promoter sequences which are responsive to certain environmental stimuli and may control genes necessary for the production of alternative exopolysaccharides.     

Phosphonic acid analogue of forfenicine — synthesis, antibacterial activity and degradation byPseudomonas fluorescens

Summary Phosphonic acid analogue of forfenicine, amino (p-formylbenzyl)-phosphonic acid, was synthesized and evaluated as antibacterial agent. As indicated by disc diffusion test this compound was found to inhibit significantly the growth ofBacillus subtilis andStaphylococcus aureus and moderately the growth ofEscherichia coli. Resistance ofPseudomonas fluorescens to the action of the aminophosphonate may result from the ability of the strain to degrade this compound.     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

Synthesis of shorter active analogues of Bactenecin7: The effect of change of N-terminal configuration on antimicrobial activity

Summary Bactenecin7, a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa=hydrophobic residues). A series of peptides, Xaa-Pro-Arg-Pro (Xaa=D-Ala, D-Leu, D-Val, D-Phe and D-Lys) were synthesized to investigate the effect of change ofN-terminal configuration on antimicrobial activity. The conformational preferences of these peptides in water and TFE were examined by circular dichroism. All the synthetic peptides with D-amino acid substitution atN-terminal showed potent antifungal activity againstAspergillus niger, Aspergillus flavus andFusarium moniliforme at the concentration level of 8–10 μg ml⁻¹. But the same tetrapeptides were unable to kill or suppress the growth of gram-negative and gram-positive bacteria such asEscherichia coli HB101,Pseudomonas aeruginosa, Klebsiella pneumoniae andStaphylococcus aureus even at the concentration level of 400 μg ml⁻¹. The present study reveals that the change of configuration at theN-terminal of tetrapeptide has negative impact on antibacterial activity but enhanced antifungal activity.     

Mechanism of action of aflatoxin B1

The inhibitory effects of aflatoxin B₁ were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0.05% w/v) or Tween-80 (0.05 % v/v) addition accentuated the aflatoxin B₁ growth inhibition inSalmonella typhi andEscherichia coli at different pH values. The inhibition of lipase production was only 5–20 % inPseudomonas fluorescence ca. 25–48% inStaphylococcus aureus andBacillus cereus at different aflatoxin B₁ concentrations (4–16μg/ml).However, inhibition of α-amylase induction was complete in1Bacillus megaterium whereas the inhibition was partial inPseudomonas fluorescence (27–40%) at 32μg aflatoxin B₁ concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B₁ concentration     

Microdistribution of sulfate-reducing bacteria in sediments of a hypertrophic lake and their response to the addition of organic matter

To clarify the ecological significance of the association of sulfate-reducing bacteria (SRB) with sediment particle size, SRB utilizing lactate (l-SRB), propionate (p-SRB) and acetate (a-SRB) were examined with different sizes of sediment particles in a hypertrophic freshwater lake using the anaerobic plate count method. The numbers ofl-SRB anda-SRB were 10⁴–10⁵ colony forming units (CFU) per ml in the 0–3 cm layer and 10²–10³ CFU ml⁻¹ in the 10–13 cm layer while the numbers ofp-SRB were one or two orders lower than those ofl-SRB anda-SRB. A sediment suspension was fractionated into four fractions (<1, 1–10, 10–94 and >94 μm). The highest proportions ofl-SRB anda-SRB were found in the 10–94 μm fraction: 66–97% forl-SRB and 53–98% fora-SRB. The highest proportion ofp-SRB was found in the >94 μm fraction (70–74%). These results indicate that most SRB were associated with sediment particles. One isolate from an acetate-utilizing enrichment culture was similar toDesulfotomaculum acetoxidans, a spore-forming sulfate-reducing bacterium. When lactate and sulfate were added to sediment samples,l-SRB anda-SRB in the <10 μm-fraction grew more rapidly than those in whole sediment for the first 2 days. This result suggests that nutrients uptake by free-living and small particle-associated (<10 μm) SRB is higher than that by SRB associated with larger particles.     

Effects of sudden temperature shifts on pure cultures of four strains of freshwater bacteria

Three psychrotrophic and one mesophilic strains were isolated from winter water samples of different freshwater biotopes and identified asCytophaga johnsonae (C-21),Cytophaga sp. (M-17),Pseudomonas fluorescens (KD), andEnterobacter cloacae (BS-2). Temperature shift-up experiments with emphasis on low temperatures were carried out with aerated pure batch cultures in glucose mineral medium. The effects of sudden temperature increases on growth rates and substrate conversion were investigated. All three psychrotrophic strains in the temperature increase experiments at low temperatures showed differing reactions within the linear zone of the Arrhenius plot. TheC. johnsonae (C-21) shift-up cultures adjusted the growth rate immediately to the rate of the temperature adapted cultures, whereasCytophaga sp. (M-17) shift-up cultures showed a lower andP. fluorescens (KD) a higher growth rate. The mesophilicE. cloacae (BS-2), likeC. johnsonae (C-21), adjusted immediately to the new growth rate. Substrate conversion increased in all experiments immediately after the shift-up. The extracellular substrate conversion byP. fluorescens (KD) of glucose to gluconate and 2-ketogluconate was particularly affected by the sudden temperature increase.     

The allelopathic potential ofCoridothymus capitatus L. (Labiatae). Preliminary studies on the roles of the shrub in the inhibition of annuals germination and/or to promote allelopathically active actinomycetes

Summary Suppression of annuals at various intensities was observed around some shrubs ofCoridothymus capitatus growing on kurkar formation in the coastal hills of Israel. The phenomenon was clearly observed as annuals-free belts of 15–20 cm around ‘aggressive’ shrubs. Quantitatively, density of annuals decreased by 16 fold in the annual-free belts as compared to a distance of 60–80 cm from the canopies of the shrubs. Their dry matter was decreased by 5.4 fold around the shrubs. Suppression rate of emergence of planted seeds of annuals (Plantago psyllium andErucaria hispanica) early in the season was 45% higher around ‘aggressive’C. capitatus than that around ‘non-aggressive’ ones. In the laboratory, seed germination of the annuals was strongly suppressed by diffusates and volatiles from shoots, as well as from their water extracts and their essential oils. Incubation of fresh shoots ofC. capitatus in soil collected from around ‘non-aggressive’ shrubs, for 7 days, increased population levels of actinomycetes by 9.6 fold and by 36.7 fold when soil was collected from around ‘aggressive’ shrubs. Isolates of some soil-borne actinomycetes inhibited germination of the test plantsLactuca sativa andAnastatica hierochuntica on agar plates (4–98%). The preliminary results indicate a possible synergistic inhibitory effect induced by essential oils of the aromatic shrub and the phytototic activity of actinomycetes.     

Incidence of mycoflora and mycotoxins in some edible fruits and seeds of forest origin

Twelve fungi namelyAlternaria alternata, Aspergillus flavus, A niger, A ochraceus, Actinomucor repens, Capnodoium spp., Curvularia lunata, Fusarium pallidoroseum, F solani, F verticillioides, Penicillium citrinum and Rhizopus stolonifer were recorded from samples ofAegle marmelos, Aesculus indica, Buchanania lanzan andPinus gerardiana. In case ofPrunus amygdalus only Rstolonifer was recorded. A significant variation in pattern of mycoflora incidence was observed in terms of source and season. Fungal infestation in most of the substrates was found to be highest during monsoon. Aflatoxins were the most common mycotoxins elaborated by different isolates ofA flavus obtained fromA marmelos, B lanzan andP gerardiana. The amount of aflatoxins produced by the toxigenic isolates ofA flavus was in the range of traces to 0.9–26.0 μg/ml inA marmelos, 0.8–17.5 μg/ml inP gerardiana and 0.65–13.2 μg/ml inB lanzan. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi. Aflatoxins were detected almost in all the samples analyzed for mycotoxin contamination. However, traces of zearalenone were detected inA marmelos. The concentration of aflatoxin B₁ was in the range of 0.13–0.75 μg/g inA marmelos, 0.09–0.60 μg/g inP gerardiana and 0.01–0.20 ug/g inB lanzan. Mycotoxins were not detected inAesculus indica andPrunus amygdalus.     

Responses ofGanoderma lucidum to heavy metals

The lelvels of seven heavy metals and their toxicity towardGanoderma lucidum under various cultivation conditions were assessed. The contents of Mn, Cu, Zn, Cd, Hg, Pb and U in the fruitbodies of cultivatedG. lucidum, and sawdust substrates were determined to be at trace levels for U, 0.01–0.1 μg/g for Cd and Hg, and 1–5 μg/g for Pb, 10–120 μg/g for Mn, Cu and Zn. The effects of heavy metals, on the growth of mycelia ofG. lucidium in pure cultures were examined over a wide range of concentrations (10–3,000 μg/ml), and their toxicities were found to decrease in the order: Hg>Cd>Cu>U>Pb>Mn=Zn. The translocation and accumulation of Zn from contaminated substrates (at 10 μg/g) in fruitbodies were investigated by using⁶⁵Zn tracer, andG. lucidum was found to take up Zn with an efficiency of >60%, leading to accumulation of >100 μ/g, in fruitbodies and >80 μ/g Zn in basidiospores.     

Comparative in vitro activity of moxalactam (LY127935), other beta-lactam antibiotics,and aminoglycosides

Moxalactam (LY127935), a novel beta-lactam antibiotic, was compared with semisynthetic penicillins, cephalosporins, and aminoglycosides by the agar dilution method against 5,317 recent clinical isolates of facultative and anaerobic bactria. At 0.5 μg/ml, moxalactam inhibited 90% of all Gram-negative bacilli tested except forPseudomonas aeruginosa (81% inhibited by 32 μg/ml) andAcinetobacter calcoaceticus (88% inhibited by 32 μg/ml). More than 90% ofBacteroides fragilis andStaphylococcus aureus were inhibited by 4 μg/ml and 8 μg/ml, respectively. Moxalactam was at least 16-fold more active by weight than cephalothin, cefamandole, and cefoxitin forEscherichia coli, Klebsiella pneumoniae, andEnterobacter species, and 2- to 4-fold more active than cefoxitin forB. fragilis. Moxalactam was 4-fold less active than cefamandole and cephalothin forS. aureus and 2- to 4-fold less active than piperacillin forP. aeruginosa. Moxalactam was as active or more active than the aminoglycosides for all facultative Gram-negative bacilli except forP. aeruginosa. Moxalactam was inhibitory (minimal inhibitory concentration <16 μg/ml) for 20/27 gentamicin-resistant isolates and 8/13 amikacin-resistant organisms. Moxalactam’s in vitro activity against Gram-negative bacilli is markedly superior to presently available cephalosporins and, except forP. aeruginosa, is comparable to the aminoglycosides.