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).     

Use of propolis and ultragriseofulvin to inhibit aflatoxigenic fungi

Propolis ethanolic extract (PEE) at 3 and 4 g/L and ultragriseofulvin (UG) at 0.75 and 1 g/L reduced the percentage of conidia germination in twoAspergillus flavus isolates. PEE at 1–4 g/L decreased the mycelial dry mass ofA. flavus isolates by 11–80%, and aflatoxin B₁ production by 34–100%. UG concentrations of 0.25–1 g/L reduced the growth and aflatoxin B₁ production of the isolates by 16–88 and 48–98%, respectively. Any increase in PEE and UG concentration was accompanied by a clear decrease in the per cent conidia germination, growth and aflatoxin B₁ production. At equal concentration, UG was about 4-times more effective than PEE.     

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.     

The relationship betweenPleurotus ostreatus andAspergillus flavus and the production of aflatoxin

The relationship betweenPleurotus ostreatus andAspergillus flavus in common mixed culture on various substrates was investigated. It was found thatP. ostreatus, similarly to some other higher fungi, can liquidate coloniesof A. flavus. This fungus does not produce aflatoxin and chromatographically similar compounds. On straw, corn cobs, millet and wheatA. flavus produced aflatoxin after a 3-week cultivation. A subsequent cultivation of P.ostreatus led to detoxication of straw and corn cobs but millet and wheat were not detoxicated. Cultivation of P.ostreatus in the presence of 40–100 μg of aflatoxin B₁ per g substrate did not result in detoxication of the material even after 34 d but the results showed that the aflatoxin concentration decreased to about one-fourth of the added amount.     

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.     

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.     

Detection of 3-nitropropionic acid and cytotoxicity inMucor circinelloides

Mucorales are regarded as the aetiological agents of Mucormycosis. Their capabilities to produce mycotoxins are not profoundly investigated, in contrast to those of the fungi from the generaPenicillium, Aspergillus, orFusarium. The aim of this study was to isolate and identify fungi of the order Mucorales and investigate mycotoxins production. Twelve samples of visibly moulded grass silage and eight samples of damaged whole crop maize silage were analysed. Malt extract agar plates were used for sub cultivation. Three fungal species of the order Mucorales were isolated from grass silage, which were identified by their macro-and micro-morphology asAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer. The cytotoxicity ofMucor circinelloides extract was analysed using the cytotoxicity test (MTT assay) and the result, showed a low cytotoxicity. Additionally extracts fromAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer were tested for mycotoxin-production using an LC/MS/MS-based multimycotoxin method. 3-nitropropionic acid was detected in the culture extract ofMucor circinelloides. Presented at the 30^th Mykotoxin Workshop Utrecht, Netherlands, April 28–30, 2008.     

Simultaneous analysis of beauvericin and moniliformin in fungal cultures and in cereal grain samples

Species ofFusarium subglutinans andFusarium proliferation have been found to produce two mycotoxins, beauvericin and moniliformin, under labolatory conditions as well as in infected ears. A method for simultaneous extraction, analysis and quantitation of both metabolites was elaborated. Recoveries were 85–97 % and 78–94 % for the first and the latter mycotoxin, respectively. Detection limit of beauvericin on high- performance thin- layer chromatography plates (Merck 5633) after exposure to iodine vapours was 3 μg/g and by high- performance liquid chromatography method 0.07 μg/g while moniliformin was analyzed at concentration level 1 μg/g by thin- layer chromatography and 0.05 μg/g by high- performance liquid chromatography method.     

Pollen,mould mites and fungi: improvements to mass rearing ofTyphlodromus doreenae andAmblyseius victoriensis

Populations ofTyphlodromus doreenae Schicha on 18×14 cm arenas using pollen ofTypha orientalis Presl. as food, increased from an estimated 50–150 to 1,000–1,600 motiles per arena in the presence of mould mites,Tyrophagus putrescentiae (Schrank) and the fungusRhizopus stolonifer (Ehrenberg ex. Fr.) Lind.T. putrescentiae in association withR. stolonifer, developed pigmentation and were preyed on byT. doreenae which also became pigmented. UnpigmentedT. putrescentiae were not attacked byT. doreenae which remained pale coloured.T. doreenae died when supplied with the fungus alone. Bean plants dusted with pollen ofT. orientalis from a talcum powder dispenser supported a large population (7–12 stages/leaf) ofAmblyseius victoriensis (Womersley). These improvements to mass rearing ofT. doreenae andA. victoriensis are discussed with respect to their potential for commercialisation.     

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     

Formation of biologically active substances by rhizosphere bacteria and their effect on plant growth

Nine out of seventeen strains of bacteria with a pronounced effect on seed germination and on seedling growth, isolated from root surfaces and rhizosphere soil of maize, were selected for a study on the formation of biologically active substances. β-Indole acetic acid (45–72 μg/1.000 ml) was produced by four strains, gibberelline-like substances (1.0–60.0 μg/1.000ml) by all strains, biotin and pantothenic acid by the majority of strains and nicotinic acid by five strains. Amino acids were formed by all strains but in low amounts. Four strains produced growth inhibitors. The highest amounts of biologically active substances were found in cultures ofPseudomonas fluorescens andBacillus brevis. The various cultures ofPseudomonas fluorescens differed in their capability to produce biologically active substances. The majority of bacterial cultures or their supernatants significantly stimulated the germination of seeds and some of them significantly affected the growth of plants. Inoculation of maize seeds with strainsPseudomonas fluorescens andChromobacterium violaceum significantly increased the yield of dry matter of plants.     

Photonastic and thermonastic opening of capitulum in dandelion,Taraxacum officinale andTaraxacum japonicum

The capitula ofTaraxacum officinale andT. japonicum open in response to temperature rise at lower temperatures (thermonasty), and in response to light at higher temperatures (photonasty), as was the case inT. albidum. The capitula ofT. officinale could respond to the same temperature rise more sensitively than those ofT. albidum orT. japonicum. The minimum temperature for photonastic opening is as low as 13 C forT. officinale, while that forT. albidum andT. japonicum is about 18 C. That is why the capitula ofT. officinale opened earlier than those ofT. albidum andT. japonicum in the morning in April under natural conditions. The capitulum continued to be open for about 13–14 hr inT. officinale and about 8–11 hr inT. japonicum and inT. albidum both under natural conditions in April and even under constant light-temperature conditions, suggesting that the time of capitula-closing in these three species is not controlled by changes in environmental factors (light and/or temperature).     

High frequency plantlet regeneration from cotyledonary node cultures of <Emphasis Type="Italic">Aegle marmelos</Emphasis> (L.) Corr.

High frequency plantlet regeneration was achieved in cotyledonary nodes of Aegle marmelos. Cotyledonary nodes from 1 mo. old in vitro grown seedlings of A. marmelos were cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) (0–8.8 μM), kinetin (KIN) (0–9.4 μM), and indole-3-acetic acid (IAA) (0–1.14 μM) either alone or in combinations. The highest regenerative response was observed on medium containing 6.6 μM BA + 1.14 μM IAA where approximately 86.6% of the cultures responded with an average shoot numbers of 487.5 per explant in 7-wk time. Cultures maintained on KIN-supplemented medium showed very poor response. In vitro responded shoots were transferred to root induction medium consisting of half-strength MS supplemented with auxins IAA, indole-3-butyric acid (IBA), or α-naphthalene acetic acid (NAA). Rooting was best in medium supplemented with 14.7 μM IBA. Rooted plantlets were acclimatized and transferred to the field with 80% survival rate.     

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.     

Karyological and taxonomical notes on three species of the genus<Emphasis Type="Italic">Epipactis (Neottioideae,Orchidaceae)</Emphasis> in the central-western Iberian Peninsula

Studies on the karyotypes and chromosome numbers of species ofEpipactis from the central-western Iberian Peninsula show that the species harbour enormous chromosome variability, have very asymmetric karyotypes and possess extraordinary diversity of aneuploidy. This paper provides the first report of a chromosome number forE. fageticola (2n=36, 40 + 0–2 B), as well as the first counts for Portuguese populations ofE. helleborine (2n=18, 32, 38) and first counts for Iberian populations ofE. tremolsii (n=20, 30, 2n=16, 24, 32, 34, 36, 38 + 1B, 40 + 1B, 52, 60). Among populations ofE. tremolsii there is a significant differentiation in ecology and somatic chromosome number, suggesting that there may be two different taxa in the region studied. Chromosomes are large to small, ranging in length from 10.8 μm to 1.8 μm. Karyotype asymmetry is of type 3C inE. fageticola andE. tremolsii and 2C inE. helleborine andE. tremolsii.     

Examination of fungal growth and aflatoxin production on marihuana

Under favorable growth conditions,Aspergillus flavus andA. parasiticus produced aflatoxins on marihuana. Cultures ofA. flavus ATCC 15548 produced both aflat oxin B₁(AFB₁) and G₁(AFG₁). The production of AFG₁ was substantially greater than that of AFB₁. Cultures ofA. flavus NRRL 3251 andA. parasiticus NRRL 2999 produced only AFB₁. All natural flora cultures tested negative for aflatoxins. NoAspergilli sporulations were observed in these cultures. In the cultures inoculated with known toxigenic fungi, the highest mean level for total aflatoxins was 8.7 g/g of medium. Marihuana appears not to yield large quantities of these mycotoxins but sufficient levels are present to be a potential health hazard for both the user and the forensic analyst who is in daily contact with such plant material. Careful processing, storage, and sanitation procedures should be maintained with marihuana. If these conditions are disregarded due to the illicit status of marihuana, the potential for mycotoxin contamination must be considered.     

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.     

A rapid identification method for aflatoxin-producing strains ofAspergillus flavus andA. parasiticus by ammonia vapor

The colony reverse of aflatoxin (AF)-producing strains ofAspergillus flavus andA. parasiticus turned pink when their cultures were exposed to ammonia vapor. The color change was visible for colonies grown on media suitable for AF production such as potato dextrose, coconut, and yeast extract sucrose agars after 2 d incubation at 25°C. Of the 120 strains ofA. flavus, A. parasiticus, and two related species inA. flavus group:A. oryzae andA. sojae tested in this study, only the AF-producing strains ofA. flavus andA. parasiticus showed the pink pigmentation. The color change occurred immediately after the colony was contacted with ammonia vapor. This method was useful for rapid screening the AF-producing strains ofA. flavus andA. parasiticus.     

On the mechanism of action of tetracycline antibiotics

It was found that low oxytetracycline (OTC) concentrations inhibited malic dehydrogenase (MDH) and lactic dehydrogenase (LDH) inStaphylococcus aureus andEscherichia coli (1–5 μg/ml for MDH and 10 μg/ml for LDH). Inhibition of these enzymes occurred almost instantaneously and could be demonstrated after 3–4 minutes. No MDH activity was found in OTC-resistant variants of these microorganisms, but LDH activity was not lowered. The inhibitory effect of OTC is specific for bacterial MDH and LDH. The same enzymes of mammalian origin are not inhibitedin vitro even by high OTC concentrations (100 μg/ml).