Dose-responses of Bacillus cereus RS87 for growth enhancement in various Thai rice cultivars

To achieve the goal of reducing the levels of chemical fertilizers applied in rice production, there is a need to develop microorganisms with the capacity to enhance plant growth. Previous studies have demonstrated that Bacillus cereus RS87 promotes growth of various plants in greenhouse and field trials. The objectives of this study were to (i) evaluate the efficacy and determine the optimum concentration of B. cereus RS87 to enhance growth of various Thai rice cultivars, (ii) measure the chlorophyll content in leaves affected by strain RS87, and (iii) investigate the capacity of strain RS87 to solubilize phosphate and produce siderophores. Three concentrations of strain RS87 (log 6.0, log 7.0, and log 8.0 CFU/mL) were applied to each rice cultivar. Superior responses (i.e., enhanced development of roots and shoots of all rice cultivars) were observed using RS87 at log 8.0 CFU/mL compared with lower bacterial concentrations and the water-treated control treatment. In addition, log 8.0 CFU/mL of RS87 provided the greatest root length and plant height of all rice cultivars 45 days after planting in the greenhouse. Rice leaves treated with log 8.0 CFU/mL of RS87 yielded the highest total chlorophyll, specifically chlorophyll a and chlorophyll b, compared with the control. Strain RS87 also solubilized phosphate and produced siderophores. The results of these studies demonstrate that log 8.0 CFU/mL is the optimum concentration of strain RS87 for growth promotion of various Thai rice cultivars.     

品种、种子大小和施肥对冬小麦生物学特性的影响

试验设不同年代冬小麦品种、粒重、播种方式和施肥等4个因子,品种选用白芒麦（20世纪60年代）、咸农39（20世纪70-80年代）、小偃6号（20世纪90年代后期）、远丰998（近期）等不同年代的4个冬小麦品种,粒重分为2种截然不同重量的大粒和小粒,播种方式设小粒单播、大粒单播以及大小粒等比例混播等3种播种方式,施肥设不施肥（CK）、施氮（N）、施磷（P）和同时施氮磷（NP）等4种方式,共48个处理。以土垫旱耕人为土为供试土样,进行盆栽试验,研究不同品种、种子大小和施肥对冬小麦生物学特性的影响。结果表明,不同品种间、大小粒播种间、不同施肥间植株株高均存在极显著差异（p〈0.01）,且这些因子间存在显著的交互作用（p〈0.05）。品种间,苗期和越冬前以近期品种远丰998植株最高,灌浆期以早期品种白芒麦植株最高。株高稳定后以早期品种高,反映了育种的演变趋势。大小粒播种间,苗期和越冬前大粒株高均显著高于小粒株高,但灌浆期大小粒播种间株高差异基本消失,说明大粒种子植株在苗期生长具有一定优势。不同施肥处理间株高差异在苗期与越冬前表现一致,单施P和NP配施植株较高;灌浆期以NP配施植株株高明显高于其它施肥处理。不同品种、大小粒播种方式和施肥显著影响冬小麦分蘖和单株叶面积。白芒麦、咸农39和小偃6号的分蘖数基本一致,变化在4.37个/株-4.74个/株之间,远丰998最少,仅为2.95个/株;NP配施和施P能够显著增加分蘖数,其分蘖数几乎是不施肥（CK）和单施N的2倍;各品种大粒种子植株分蘖数均多于小粒种子植株。远丰998绿叶面积最大（45.72cm^2/单茎）,白芒麦最低（仅为26.97cm^2/单茎）;NP配施单株绿叶面积明显大于其它施肥处理。除远丰998大粒种子植株绿叶面积（50.42cm^2/单茎）显著大于小粒种子（41.01cm^2/单茎）外,其余品种大、小粒种子植株绿叶面积相当。就施肥处理而言,施肥对近期品种小粒种子株高、分蘖数和叶面积的促进作用相对较大,而对远期品种小粒种子植株的影响相对较小。     

Seed enhancement: getting seeds restoration‐ready

Seed enhancement technologies such as seed priming and seed coating, developed by the agricultural seed industry, are standard procedures for the majority of crop and horticultural seeds. However, such technologies are only just being evaluated for native plant seeds despite the potential benefits of such treatments for improving restoration effectiveness. Key approaches applicable to native seed include: (1) seed priming, where seeds are hydrated under controlled conditions, and (2) seed coating, in which external materials and compounds are applied onto seeds through a diversity of treatments. These technologies are commonly employed to accelerate and synchronize germination and to improve seed vigor, seedling emergence, establishment, and to facilitate mechanized seed delivery to site, through standardizing seed size and shape. Seed enhancement technologies have now been tested on native seeds to overcome logistical and ecological barriers in restoration. However, further research is needed to extend the application of seed enhancements to a broader array of species, ecosystems, and regions as well as to evaluate new and innovative approaches such as the incorporation of beneficial soil microorganisms and plant growth regulators in the coatings. As techniques in native seed enhancement develop, these approaches need to be capable of being scaled‐up to provide the tonnages of seed required for global restoration.     

IAA production during germination of Orobanche spp. seeds

Broomrapes (Orobanche spp.) are parasitic plants, whose growth and development fully depend on the nutritional connection established between the parasite and the roots of the respective host plant. Phytohormones are known to play a role in establishing the specific Orobanche-host plant interaction. The first step in the interaction is seed germination triggered by a germination stimulant secreted by the host-plant roots. We quantified indole-3-acetic acid (IAA) and abscisic acid (ABA) during the seed germination of tobacco broomrape (Orobanche ramosa) and sunflower broomrape (O. cumana). IAA was mainly released from Orobanche seeds in host-parasite interactions as compared to non-host-parasite interactions. Moreover, germinating seeds of O. ramosa released IAA as early as 24 h after the seeds were exposed to the germination stimulant, even before development of the germ tube. ABA levels remained unchanged during the germination of the parasites' seeds. The results presented here show that IAA production is probably part of a mechanism triggering germination upon the induction by the host factor, thus resulting in seed germination.     

Effect of the filling season on aquatic plants in Mediterranean temporary ponds

Aims Pond environmental conditions may differ among years with regards to the season in which ponds begin to fill. We experimentally evaluated how seedling emergence, plant growth and phenology differed among years in which filling occurred in winter, autumn or spring.Methods We collected sediments from a natural temporary pond and located them in aquariums. They were placed in a climatic chamber that simulated annual variation in field environmental temperatures and light conditions. Aquariums were assigned to one of three treatments, which differed in the date on which they were filled with water (autumn, winter and spring). We counted the number of seedlings of different species emerged and recorded data about the presence of flowers, seeds or spores every week. The experiment was finished in June, when we harvested the plants and estimated their biomass.Important findings In most species, seedling emergences were primarily related to time after filling, and thus synchronized their life cycles with the unpredictably timed wet phase of the ponds. Autumn filling resulted in the highest numbers of seeds/spores. However, winter filling promoted plant growth the most. In the spring filling treatment, more terrestrial plant seedlings emerged and fewer seeds/spores were produced. When ponds are flooded earlier, plants may produce a higher number of propagules. However, in years when inundation is delayed to spring and hydroperiods are short, seedling emergence deplete the seed bank and there is little to no seed production, while terrestrial monocots are able to colonize pond basin.     

Improvement of the growth and yield of lettuce plants by non-uniform magnetic fields

Influence of pre-sowing magnetic treatments on plant growth and final yield of lettuce (cv. Black Seeded Simpson) were studied under organoponic conditions. Lettuce seeds were exposed to full-wave rectified sinusoidal non uniform magnetic fields (MFs) induced by an electromagnet at 120 mT (rms) for 3 min, 160 mT (rms) for 1 min, and 160 mT (rms) for 5 min. Non treated seeds were considered as controls. Plants were grown in experimental stonemasons (25.2 m(2)) of an organoponic and cultivated according to standard agricultural practices. During nursery and vegetative growth stages, samples were collected at regular intervals for growth analyses. At physiological maturity, the plants were harvested from each stonemason and the final yield and yield parameters were determined. In the nursery stage, the magnetic treatments induced a significant increase of root length and shoot height in plants derived from magnetically treated seeds. In the vegetative stage, the relative growth rates of plants derived from magnetically exposed seeds were greater than those shown by the control plants. At maturity stage, all magnetic treatments increased significantly (p < 0.05)--plant height, leaf area per plant, final yield per area, and fresh mass per plant--in comparison with the controls. Pre-sowing magnetic treatments would enhance the growth and final yield of lettuce crop.     

Antigens of Bacillus cereus: A Comparison of a Parent Strain, an Asporogenic Variant and Cell Fractions

S ummary . The antigenic structure of a stable asporogenic variant of the M8 strain of Bacillus cereus has been compared with that of the parent strain. Ultrasonic extracts of cells of both parent strain and variant harvested at different ages have been analysed by immunoelectrophoresis against antisera prepared by injecting such extracts into rabbits.
Disintegrates of cells of the asporogenic variant were antigenically identical with disintegrates of vegetative cells of the parent strain. Disintegrates of cells in later stages of sporulation and of mature spores of the parent strain contained thermostable antigens which were never detected in the variant. Antigens of isolated cell walls, protoplasts and flagella were also studied.
Examination of esterase and catalase content of the two strains showed that although the variant had the same enzymes as the young vegetative cells of the parent strain it never developed the thermostable catalase found in disintegrated spores. Protein components of the two strains at different stages of growth and of the isolated cell fractions were studied by electrophoresis in polyacrylamide gels.     

Involvement of phytohormones in the development of interaction between wheat seedlings and endophytic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain 11BM

Wheat (Triticum aestivum L., cv. Kazakhstanskaya 10) seedling growth activation by the endophytic 11BM strain of the bacterium Bacillus subtilis Cohn and the involvement of phytohormones in this process were studied. At the concentration used, bacteria enhanced wheat root and shoot growth as compared to control. Biotests demonstrated auxin-, cytokinin-, and gibberellin-like activities of the endophyte, but the result depended on the way of experiment performing. When wheat seeds were treated with the spores of B. subtilis 11BM strain, the concentrations of IAA and ABA increased transiently in the roots and shoots of the seedlings. The involvement of IAA oxidase in the plant response to inoculation with bacteria was shown; it was accompanied by a decrease in enzyme activity, which occurred later than auxin accumulation. It was concluded that observed changes in the hormonal status of wheat plants under the influence of the endophytic bacterial strain may be one of the mechanisms for seedling growth stimulation.     

Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach

AIMS: To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium. Methods and RESULTS: The effects of acidity on the survival of B. cereus in a medium simulating human stomach content was followed on spores at pH 1.0-5.2, and on vegetative cells at pH 2.5-5.7. Gastric media (GM) were prepared by mixing equal volumes of a gastric electrolyte solution with J broth (JB), half-skim milk, pea soup and chicken. At pH 1.0 and 1.4, the number of spores slightly decreased in GM-JB and GM-pea soup and remained stable in GM-milk and GM-chicken. A rapid marked decrease (always higher than 2.0 log CFU ml(-1) in 2 h) in vegetative cell counts was observed at pH below 4.2, 4.0, 3.6 and 3.5 in GM-chicken, GM-JB, GM-milk and GM-pea soup, respectively. Between pH 5.0 and 5.3, B. cereus growth was observed in GM-JB (1.2 log CFU ml(-1) increase after 4 h) and in GM-pea soup (1.8 log CFU ml(-1) increase after 4 h). CONCLUSIONS: Bacillus cereus spores are very much more resistant to gastric acidity than vegetative cells. This resistance strongly depends on the type of food present in the GM. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the probability that viable B. cereus cells enter the small intestine, where they can cause diarrhoea, strongly depends on the form of the ingested cells (spores or vegetative cells), on what food they are ingested with, and on the level of stomach acidity.     

Effects of intraspecific and interspecific density on the demography of a perennial herb, Sanicula europaea

We examined the effects of intraspecific and interspecific competition on demographic processes in the perennial herb Sanicula europaea by manipulating the density of neighbouring plants. We followed the response in terms of survival, growth and reproduction and in terms of seedling recruitment. The demographic data from all phases of the life cycle enabled us to assess also the overall effects of treatments on population growth rate (λ) by transition matrix models. We also decomposed the differences in λ between control and treatments, using life table response experiments (LTRE). To study the effects of competition on recruitment in more detail and to evaluate the role of seed availability, we sowed seeds at different densities with or without vegetation removal.
Vegetative growth and flowering frequency of established individuals was not significantly affected by removal treatments, which suggest no, or a delayed response to released competition. Neighbour removal had no effect on seedling emergence but enhanced recruitment through a higher seedling survival. Conspecific and simultaneous conspecific and heterospecific removal of plants led to an increase in population growth rate (λ), whereas heterospecific removal alone led to a decrease. Emergence of seedlings and fate of vegetative established individuals contributed most to differences in λ between the control and the different treatments. Seed addition enhanced seedling emergence but, as seedling and juvenile survival were density dependent, densities of established individuals appear not to be seed limited.
In S. europaea removal treatments had different effects on established individuals and recruitment. This suggests that studies quantifying the effects of competition over the entire life cycle and performed in a natural environment are necessary to assess the importance of competition in perennial plant populations.     

Effect of Seed Treatment with Rhizobium leguminosarum on Pythium Damping-off, Seedling Height, Root Nodulation, Root Biomass, Shoot Biomass, and Seed Yield of Pea and Lentil

Field experiments were conducted in 2004 and 2005 to determine the effects of seed treatment with Rhizobium leguminosarum bv. viceae on damping‐off, seedling height, root nodule mass, root biomass, shoot biomass and seed yield of pea and lentil in a field naturally infested with Pythium spp. Compared with the untreated controls, treatment of pea seeds with R. leguminosarum bv. viceae strains R12, R20 or R21 significantly (P < 0.05) reduced incidence of damping‐off, promoted seedling growth and increased root nodule mass, root biomass and shoot biomass. Seed treatments with R12 or R21 also resulted in a significant (P < 0.05) increase in seed yield of pea. The strain R21 was most effective among the four strains of R. leguminosarum bv. viceae tested in peas. Although, the level of disease control by strain R21 was similar to seed treatment with the fungicide Thiram^TM, R21 was more effective in enhancing root nodule production and promoting plant growth. For lentil, treatment of seeds with R. leguminosarum bv. viceae strains R12 or R21 significantly (P < 0.05) reduced incidence of damping‐off compared with the untreated control. All of the four strains of R. leguminosarum bv. viceae tested increased lentil seedling height, root nodule mass and shoot biomass, and all except R20 increased root biomass. Seed yield was higher for the treatments of R12 and R21. The strain R12 was most effective among the four strains of R. leguminosarum bv. viceae tested in lentil. Although, strain R12 was as effective as Thiram^TM for control of damping‐off of lentil, it was more effective than Thiram^TM for the production of root nodules and promotion of plant growth. The study concludes that seed treatment with R. leguminosarum bv. viceae is effective in control of Pythium damping‐off of pea and lentil and that the efficacy of control is strain specific, strain R21 for control of the disease on pea and strain R12 for control of the disease on lentil.     

The effects of fluid drilling and seed covering medium on early carrot production under polyethylene mulch

Ungerminated seeds, fluid-drilled germinating seeds and fluid-drilled uniformly-germinated seeds were sown in furrows that were either uncovered or covered by soil and left unmulch ed or mulched with polyethylene sheet. The effects of these treatments on the emergence and growth of carrots for early harvest were examined. Leaving seeds uncovered or mulching them with polyethylene advanced seedling emergence and increased the percentage that emerged; leaving seeds uncovered also reduced the spread of emergence times and produced seedlings with larger cotyledons. Uniformly-germinated seeds emerged before germinating seeds and both emerged before ungerminated seeds. The best combination of treatments (i.e. mulched, uncovered uniformly-germinated seeds) reduced the mean number of days for emergence from the 52 required by traditionally sown seeds to 29. There was a more variable effect of seed treatments on the spread of emergence times and the percentage emergence. Earlier emergence generally led to roots reaching a marketable size earlier and more uniform emergence led to less variation in root weights at harvest.     

Effects of below- and above-ground herbivores on plant growth,flower visitation and seed set

Separate and combined effects of root and leaf herbivores on plant growth, flower visitation and seed set were tested in a factorial experiment using potted mustard, Sinapis arvensis, at an old fallow field. A 50% leaf removal by cabbageworms (Pieris rapae) when the seedlings had their first four leaves reduced plant height and shoot mass, and delayed the onset of flowering. Root herbivory by two wireworms (Agriotes sp.) over the whole experiment changed flower visitation; the number of flower visitors per plant was higher in plants with root herbivores than in plants without root herbivores. Combined leaf and root herbivory affected flowering period, number of fruits per plant and number of seeds per fruit. Plants attacked by leaf and root herbivores had a shorter flowering period and produced fewer fruits per plant than plants with root herbivores only. Although the experimental plants faced major herbivore-induced growth changes, plant reproduction (seed set and weight per plant) was similar in all treatments, documenting their ability to effectively compensate for leaf and root herbivory.     

新疆短命植物小拟南芥耐盐性的初步研究

利用种子萌发及石蜡切片等方法对新疆短命植物小拟南芥(Arabidopsis pumila)在盐胁迫下种子的萌发率及植株生长发育的变化进行了研究,结果显示:(1)盐胁迫对小拟南芥种子萌发具有抑制作用,当去除盐分后,部分种子能够重新萌发.(2)盐分影响幼苗的形态及内部结构,低盐(50 mmol/L)时,幼苗基生叶较大且数量增加;叶中栅栏组织细胞大,排列舒展;根中导管数量明显增多,植株生长健壮而旺盛;而较高盐(150 mmol/L)时,叶片变小增厚,色深绿,底部叶变黄白色,叶中栅栏组织细胞窄小,排列紧密;根中导管数急剧下降,植株生长受到明显抑制.(3)盐胁迫对成株期植株的高度、花序轴长、角果数等性状都有显著影响,但在NaCl浓度达500 mmol/L时,植株仍然存活且有一定的结实率,显示此时的耐盐性比萌发期和幼苗期有显著提高.研究表明,小拟南芥从幼苗至成株期耐盐性逐渐增强,该特征与其对自然生存环境的适应相关.     

Effects of application of Rhodopseudomonas sp. on seed germination and growth of tomato under axenic conditions

Purple nonsulfur bacteria were isolated from river sediments and their growth promoting capabilities on tomato were examined. Isolated strains KL9 and BL6 were identified as Rhodopseudomonas spp. by 16S rDNA sequence analysis. Rhodopseudomonas strain KL9 maximally produced 5.56 mM/ min/mg protein and 67.2 microM/min/mg protein of indole-3-acetic acid (IAA) and 5-aminolevulinic acid (ALA), respectively, which may be one of the mechanisms of plant growth enhancement. The germination percentage of tomato seed, total length, and dry mass of germinated tomato seedling increased by 30.2%, 71.1%, and 270.8%, respectively, compared with those of the uninoculated control 7 days after inoculation of strain KL9. The lengths of the root and shoot of germinated seedling treated with 3 mM tryptophan, a precursor of IAA, increased by 104.4% and 156.5%, respectively, 7 days after inoculation of strain KL9. Rhodopseudomonas KL9 increased 123.5% and 54% of the root and shoot lengths of germinated seedling, respectively, treated with 15 mM glycine and succinate, precursors of ALA. This plant growth promoting capability of purple nonsulfur bacteria may be a candidate for a biofertilizer in agriculture.     

Role of Pseudomonas putida indoleacetic acid in development of the host plant root system

Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens

The extractable protein antigens EA1 and EA2 of Bacillus anthracis were prepared from electrophoresis transblots of SDS extracts of vegetative bacteria of the Sterne strain. Hyperimmune guinea-pig antiserum against EA2 failed to react with B. anthracis cells in immunofluorescence (IF) tests. Guinea-pig antiserum against EA1 (anti-EA1) reacted strongly in IF tests with non-encapsulated vegetative cell of 10 of 12 strains of B. anthracis and with cells of strains of B. cereus and B. thuringiensis. The unreactive B. anthracis strains were delta-Vollum-1B-1 and Texas. Encapsulated cells of B. anthracis stained poorly except for small bright regions. Absorption of anti-EA1 with cells of B. cereus NCTC 8035 and NCTC 9946 removed activity towards all B. cereus strains tested, but only partly reduced cross-reaction with B. thuringiensis strains. Absorption of anti-EA1 with B. thuringiensis 4041 removed activity towards this strain and B. cereus strains. Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth. The reaction of anti-EA1 with Bacillus spores immobilized in clumps on microscope slides was attributed to contaminating vegetative debris because well-separated individual spores failed to react. A rapid IF test was developed allowing identification of B. anthracis sampled from overnight cultures on blood plates. When sodium dodecyl sulphate extracts of B. anthracis vegetative cells were analysed on immunoblots (Western blots) by reaction with anti-EA1, a number of bands were visualized in addition to the expected 91 kiloDalton EA1 band. Prior absorption of anti-EA1 with B. cereus or B. thuringiensis cells resulted in the disappearance of most or all of the brands in blots of these species, but had less effect on blots of the B. anthracis strains. All six B. anthracis strains that were blotted including delta-Vollum-1B-1 and Texas, could thus be distinguished from B. cereus and B. thuringiensis by their differential reaction with unabsorbed and absorbed anti-EA1.     

The effects of Korean propolis against foodborne pathogens and transmission electron microscopic examination

This study was performed to evaluate the effects of Korean propolis against foodborne pathogens and spores of Bacillus cereus and to investigate the antimicrobial activity against B. cereus structure by transmission electron microscopy (TEM). The antimicrobial effects of the Korean propolis were tested against foodborne pathogens including Gram-positive (B. cereus, Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (Salmonella typhimurium, Escherichia coli and Pseudomonas fluorescence) bacteria by agar diffusion assay. Gram-positive bacteria were more sensitive than were Gram-negative bacteria. The vegetative cells of B. cereus were the most sensitive among the pathogens tested with minimum inhibitory concentration (MIC) of 0.036 mg/μl of propolis on agar medium. Based on MIC, sensitivity of vegetative cells of B. cereus and its spores was tested in a nutrient broth with different concentrations of propolis at 37°C. In liquid broth, treatment with 1.8 mg/ml propolis showed bactericidal effect against B. cereus. B. cereus vegetative cells exposed to 7.2mg/ml of propolis lost their viability within 20 min. Against spores of B. cereus, propolis inhibited germination of spores up to 30 hours, compared to control at higher concentration than vegetative cells yet acted sporostatically. The bactericidal and sporostatic action of propolis were dependent on the concentration of propolis used and treatment time. Electron microscopic investigation of propolis-treated B. cereus revealed substantial structural damage at the cellular level and irreversible cell membrane rupture at a number of locations with the apparent leakage of intracellular contents. The antimicrobial effect of propolis in this study suggests potential use of propolis in foods.     

Germination and outgrowth of Schizosaccharomyces pombe ascospores isolated by Urografin density gradient centrifugation.

A simple method for the isolation of single ascospores of the fission yeast Schizosaccharomyces pombe was examined. Single spores in the 7-day-old sporulating culture of a homothallic strain were separated from remaining vegetative cells by isopycnic centrifugation in the linear gradient from 10 to 60% of Urografin solution at 700 X g for 20 min. Protein content of isolated spores was very low as compared with that of vegetative cells. The isolated spores germinated through the following steps when cultured in a liquid medium at 25--35 degrees C; loss of refractility (darkening) under a phase-contrast microscope, spherical growth (swelling), emergence of germ tubes, elongation of germ tubes, cell plate formation, and cell separation. The absorbance at 650 nm of the spore suspension initially decreased, accompanied by darkening of spores, and then increased with spherical growth. The germination rate of isolated spores reached almost 100%.