High Inoculum Concentration Research Articles

Effect of various culture treatments on virulence and aggressiveness expression of Phytophthora infestans

The effect of experiment date, inoculum concentration and number of passages through potato tissue on virulence spectrum and aggressiveness level of four <i>Phvtophthora infestans</i> isolates was examined in four tests. All these experimental variants significantly influenced the expression of virulence. Isolates chosen for study were characterized initially by complex wirulence and high aggresssiveness. The narro west spectrum of virulence was noted atJune and December in comparison with March and September. The inoculum concentration of 200 sporangia in 1 mm<sup>3</sup> influenced stronger frequency of virulence factors, that concentration of 50 sporangia in 1 mm<sup>3</sup>. The cultures passaged many times showed the wider virulence spectrum than single or double passaged isolates. The double passages of pathogen cultures influenced stronger expression factors of virulence than single one. The significant interaction between experiment dates and number of passages and experiment dales and inoculum concentrations were observed. Higher inoculum concentration and more abundant passages on potato tissues positively affected detection of particular virulence factors, especially v5 and v8. The cultures, both double and many times passaged on potato tuber slices, were significantly more aggressive in comparison with single passaged ones. The isolate MP 425 was the most stable in expression of its virulence and aggressiveness. The <i>P.infestans</i> cultures that lost infectivity on rye agar medium can recover their virulence and aggressiveness expression after double and frequent culturing on potato tissues.

Optimisation of inoculum concentration and incubation duration for an in vitro hindgut dry matter digestibility assay

The aim was to optimise inoculum concentration and incubation duration for a published in vitro hindgut digestibility assay using ileal digesta (sampled from the chicken or rat) pertaining to a mixed human diet as the substrate. The study also sought to investigate the digestibility of the inoculum itself and the importance of correcting for this in the in vitro hindgut digestion assay. For two assays, hindgut dry matter digestibility (DMD) generally increased with inoculum concentration. A sharp increase in DMD observed at high inoculum concentrations may have been related to problems with filtering the inoculum. An inoculum concentration of 160g/L was considered optimal based on close agreement of observed values with previously published in vivo hindgut dry matter digestibility for similar diets. One of the methods was chosen for optimisation of the duration of incubation. Ileal substrate organic matter digestibility (OMD) increased with increasing time of incubation for all diets. An incubation duration of 18h using a mean inoculum digestibility value for calculation purposes was considered optimal based on observed in vivo hindgut DMD values in humans, but there was little difference in estimated in vitro hindgut DMD between 18 and 24h incubation durations. Although considerably lower than the OM digestibility of the substrate (no less than 51% after 48h), the OM digestibility of the inoculum (13% after 48h) itself was of significance in calculating estimated digestibility. The optimised assay gave realistic hindgut OMD values ranging from 55% to 79% (Wheat Bran Diet and Pectin Diet, respectively) using an 18-h incubation duration.

OPTIMASI KONSENTRASI INOKULUM, RASIO C:N:P DAN pH PADA PROSES BIOREMEDIASI LIMBAH PENGILANGAN MINYAK BUMI MENGGUNAKAN KULTUR CAMPURAN

The purposes of this research were to obtain the best inoculum concentration, C:N:P ratio, and pH, and also to identify the ability of mixed culture of hydrocarbonoclastic bacteria in oil waste degradation. The isolats were used are Acinetobacter baumanni, Alcaligenes eutrophus, Bacillus sp1., Methylococcus capsulatus, Bacillus sp2., Morococcus sp., Pseudomonas diminuta, Xanthomonas albilineans, Bacillus cereus and Flavobacterium branchiophiia. Variation of inoculum concentrations were 10%, 15%, and 20% (v/v), C:N:P ratios were 100:10:1, 100:10:0,5, 100:5:1, and 100:5:0,5, and pH were 6,5, 7,0, 7,5. Observed parameters in optimization were Total Plate Count (TPC) the culture every 24 hours, Total Petroleum Hydrocarbon (TPH) and Chemical Oxygen Demand (COD) examined at the end of the bioremediation period. Best optimization result then analyzed with GC/MS. Optimization result indicated the best inoculum concentration was 10% with TPH degradation 61,79% and COD slope 61,75%. It is assumed that the low value of TPH degradation and COD slope at 15% and 20% inoculum concentration were caused by competition inside the bacterial population at that high inoculum concentration. The competition result in low growth and degradation. C:N:P ratio was 100:5:1 with TPH degradation 66,55% and COD slope 85,18%. It is assumed that the C:N:P ratio is equal, so it can enhance the bioremediation procces. The best pH was 7,5 with TPH degradation 73,24% and COD slope 86,28%. The process at the optimum conditions using inoculum as a mixed culture enhanced the bioremediation process with the result as follows, TPH degradation 93,06%, COD 90,73% for treatment. The chromatogram indicated that total hydrocarbon compound from nC9 – nC32 have been degraded by 43,413% – 63,117%. A good result of bioremediation was obtained from mixed culture inoculum at 10% concentration, C:N:P ratio of 100:5:1, and pH 7,5.<br /><br />Keywords: Bioremediation, hydrocarbonoclastic bacteria, inoculum concentration, C:N:P ratio, pH<br /><br />

Influence of Phytophthora capsici L. inoculation on disease severity, necrosis length, peroxidase and catalase activity, and phenolic content of resistant and susceptible pepper (Capsicum annuum L.) plants

This study explored the level of infection caused by different inoculum concentrations (10^2, 10^3, and 10^4 zoospores mL^-^1) of Phytophthora capsici in 3 pepper cultivars at days 2, 4, and 6. The effect that the infection induced on the peroxidase (POX), catalase (CAT), and phenolics of resistant and sensitive seedlings, as well as the defense mechanism against the pathogen, were also investigated. The resistance of PM-702 against the isolate used was high, whereas KM-Hot and DEM-8 displayed sensitive reactions. As a result of analyses of the leaves and stems from the 3 pepper cultivars, important changes in biochemical parameters were observed between resistant and sensitive cultivars after inoculation with the root rot pathogen P. capsici. The maximum increase of POX and CAT was observed in the resistant PM-702 cultivar. However, with a high inoculum concentration (10^4 zoospores mL^-^1), a loss of CAT activity was determined, especially in susceptible cultivars. The maximum increase in phenolics was detected in leaves of susceptible DEM-8 and stems of PM-702 on day 6 following infection. The results suggest that during Phytophthora root rot development there is a relation between the disease induced by P. capsici and the antioxidant system.

Downy mildew: is resistance linked to inoculum concentration?

Leaves of different Vitis vinifera cultivars, susceptible (‘Chasselas’ and ‘2185’), less susceptible (‘2142’) or resistant to downy mildew ('Solaris’ and ‘2091’), were inoculated with four different concentrations of an aqueous sporangia suspension of Plasmopara viticola (5 × 105, 2 × 105, 6 × 104 and 2 × 104 sporangia·ml -1 ). The infection rate of these samples was then examined by light microscopy and synthesis of stilbenes was analysed at infection sites. Infection rate increased parallel with inoculum concentration, but there was no correlation between the infection rate and resistance to P. viticola . Moreover, at the lowest inoculum concentration, the infection rate is similar for susceptible and resistant grapevine varieties. Quantification of stilbenes at 72 hpi showed that at the lowest inoculum concentration, the most susceptible grape variety synthesized the largest amount of stilbenes, whose level remained however below the ED50 values defined for each of them. Conversely, at the highest inoculum concentration, the most resistant varieties produced the highest amounts of the most toxic stilbenes against P. viticola . The critical role of the inoculum concentration used for artificial inoculation to evaluate grapevine resistance to downy mildew is discussed.

Organ identity and environmental conditions determine the effectiveness of nonhost resistance in the interaction between Arabidopsis thaliana and Magnaporthe oryzae

Mechanisms leading to nonhost resistance of plants against nonadapted pathogens are thought to have great potential for the future management of agriculturally important diseases. In this article, we report an investigation of nonhost resistance motivated by the advantages of studying an interaction between two model organisms, namely Arabidopsis thaliana and Magnaporthe oryzae. During the course of our studies, however, we discovered an unexpected plasticity in the responses of Arabidopsis against this ostensibly nonhost pathogen. Thus, we elucidated that certain experimental conditions, such as the growth of plants under long days at constantly high humidity and the use of high inoculum concentrations of M. oryzae conidia, forced the interaction in leaves of some Arabidopsis ecotypes towards increased compatibility. However, sporulation was never observed. Furthermore, we observed that roots were generally susceptible to M. oryzae, whereas leaves, stems and hypocotyls were not infected. It must be concluded, therefore, that Arabidopsis roots lack an effective defence repertoire against M. oryzae, whereas its leaves possess such nonhost defence mechanisms. In summary, our findings point to organ-specific determinants and environmental conditions influencing the effectiveness of nonhost resistance in plants.

Culturable bacterial microflora associated with nectarine fruit and their potential for control of brown rot

Microflora of fruit surfaces have been the best source of antagonists against fungi causing postharvest decay of fruit. However, there is little information on microflora colonizing surfaces of fruits other than grape, apple, and citrus. We characterized bacterial microflora on nectarine fruit surfaces from the early stage of development until harvest. Identification of bacterial strains was made using MIDI (fatty acid methyl ester analysis) and Biolog systems. Biolog identified 35% and MIDI 53% of the strains. Thus results from MIDI were used to determine the frequency of occurrence of genera and species. The most frequently occurring genera were Curtobacterium (21.31%), followed by Pseudomonas (19.99%), Microbacterium (13.57%), Clavibacter (9.69%), Pantoea (6.59%), and Enterobacter (4.26%). The frequency of isolations of some bacteria - for example, the major pseudomonads (Pseudomonas syringae, Pseudomonas putida, and Pseudomonas savastanoi) or Pantoea agglomerans - tended to decline as fruit developed. As Pseudomonas declined, Curtobacterium became more dominant. Time of isolation was a significant factor in the frequency of occurrence of different bacteria, indicating succession of the genera. Throughput screening of the bacterial strains against Monilinia fructicola on nectarine fruit resulted in the detection of strains able to control brown rot. The 10 best-performing antagonistic strains were subjected to secondary screening. Four strains reduced decay severity by more than 50% (51.7%-91.4% reduction) at the high pathogen inoculum concentration of 105 conidia/mL.

Detached leaf inoculation of germplasm for rapid screening of resistance to citrus canker and citrus bacterial spot

A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 104, 105, or 108 cfu ml−1 were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (108 cfu ml−1), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 105 cfu ml−1, populations 14 dpi were 105). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.

Histopathology and Bacterial Populations of Atypical Symptoms-Inducing Xanthomonas axonopodis pv. citri Strains in Leaves of Grapefruit and Mexican Lime

Histopathology of atypical symptoms-inducing Xanthomonas axonopodis pv. citri (Xac) strains XW47 (type Xac-A') and XW121 (type Xac-A') in citrus leaves was examined with light and electron microscopy, and compared with that of a typical Asiatic citrus canker strain XW19 (type Xac-A). Strain XW47 induces typical canker lesions on leaves of Mexican lime (Citrus aurantifolia) but causes flat necrotic lesions with water soaked margin on leaves of grapefruit (C. paradisi): whereas strain XW121 induces restricted and raised corky lesions without watersoaked margin on both citrus leaves. Light microscopic examinations showed that XW47 induced hyperplasia and hypertrophy of mesophyll cells and ruptures of the epidermis as those observed in Mexican lime leaves induced by XW19. However, neither hypertrophic cells nor eruption of the epidermis were observed in the brownish necrotic and hyperplastic mesophyll tissues induced by XW47 on grapefruit leaves. A distinct layer of lignified tissues around the brownish disintegrated erumpent tissues in which masses of fibrillar matrix agglutinated with bacterial cells was observed in the sections of a restricted and raised corky lesion induced by strain XW121. Bacterial populations of strain XW121 in mesophyll tissues were approximately 10^3 folds lower than those of XW19 and XW47 under a lower inoculum concentration, but increased to those of XW19 and XW47 under a higher inoculum concentration. Although XW19 and XW47 induced distinct symptoms on grapefruit leaves, no significant difference of bacterial growth was observed.

A seed bank pathogen causes seedborne disease: Pyrenophora semeniperda on undispersed grass seeds in western North America

The generalist pathogen Pyrenophora semeniperda is abundant in seed banks of the exotic winter annual grass Bromus tectorum in semiarid western North America and is also found in the seed banks of co-occurring native grasses. In this study, we examined natural incidence of disease caused by this pathogen on undispersed host seeds, that is, seeds that were never directly exposed to inoculum in the seed bank. We determined experimentally that at least 90% of undispersed B. tectorum seeds exhibiting the disease were likely infected after maturity by conidia borne superficially on the seed-covering structures. The fraction of undispersed seeds exhibiting disease under optimum conditions for infection in the laboratory varied from 0% to 22% for three grass species. Relatively high disease incidence on undispersed seeds for this dry-sporulating fungus was significantly correlated with low mean annual rainfall, dry conditions during the conidial dispersal period in early summer, and high concentrations of soilborne inoculum as evidenced by the presence of high densities of killed seeds bearing fungal stromata in the seed bank. These three variables explained 66% of the variation in seedborne disease incidence for B. tectorum seed collections made in 2005. By dispersing conidia onto host seeds while seeds are still on the plant, this pathogen achieves the potential for targeted dispersal along with seeds of its host. This may be particularly advantageous for P. semeniperda, which has large conidia (phragmospores) that may not be efficiently dispersed beyond the grass canopy boundary layer. Key words: conidia, Bromus tectorum, Drechslera campanulata, Elymus elymoides, Leymus cinereus, Pyrenophora semeniperda, spore dispersal. L'agent pathogène opportuniste Pyrenophora semeniperda abonde dans les banques de semences de variétés exotiques de brome non alternatif Bromus tectorum des régions semi-arides de l'ouest de l'Amérique du Nord ainsi que dans les banques de semences de graminées indigènes environnantes. Au cours de cette étude, nous avons examiné l'incidence naturelle de la maladie causée par cet agent pathogène chez des semences hôtes non dispersées, c'est-à-dire chez des semences qui n'avaient jamais été directement exposées à l'inoculum dans une banque de semences. Nous avons déterminé expérimentalement qu'au moins 90 % des semences non disséminées de B. tectorum qui affichaient des signes de la maladie avaient de toute évidence été infectées, après leur murissement, par des conidies qu'elles portaient à la surface des structures qui les recouvrent. La fraction des semences non disséminées qui affichait des signes de maladie à la suite d'exposition dans des conditions contrôlées optimales variait de 0 % à 22 %, et ce, pour trois espèces de graminées. Une incidence relativement élevé de la maladie chez les semences non disséminées, causée par ce champignon sporulé à sec, a été significativement corrélée avec la faible précipitation moyenne annuelle, les conditions sèches durant la période de dispersion des conidies en début d'été et les fortes concentrations d'inoculum terricole, comme le prouvent les forts taux de semences mortes de la banque, semences portant des stromates fongiques. Ces trois variables expliquaient 66 % de la variation de l'incidence de la maladie transmise par les semences de B. tectorum ramassées en 2005. En disséminant des conidies sur les graines hôtes pendant que celles-ci sont encore attachées au plant mère, l'agent pathogène atteint son potentiel de dispersion le plus élevé lorsque les graines sont elles-mêmes dispersées. Ceci peut être particulièrement avantageux pour P. semeniperda qui possède de larges conidies (phragmospores) qui autrement risqueraient de ne jamais franchir la couche limite formée par la couverture herbeuse. Mots-clés : conidies, Bromus tectorum, Drechslera campanulata, Elymus elymoides, Leymus cinereus, Pyrenophora semeniperda, dispersion des spores.

317 POSTER Antiproliferative effects of PM02734, a novel marine cyclic peptide compared with currently used Erb-B inhibitors, in a panel of human cancer cell lines characterised for Erb-B expression

Ramoplanin, a new depsipeptide complex, was found to be 4- to 8-fold more active than vancomycin against all Gram-positive species (500 strains). Similarly, ramoplanin was both more active and had an additional spectrum compared to mupirocin (formerly pseudomonic acid) against Bacillus spp., Corynebacterium jeikeium, enterococci and Listeria monocytogenes. Mupirocin-resistant Staphylococcus spp. strains (<1%) were identified in these organisms collected from over 40 medical centers in the United States. These strains (S. capitis and S. saprophyticus) were susceptible to ramoplanin at 0.25 μg/ml or less. The three tested antimicrobial agents were not effective against Gram-negative organisms (20 species tested). Ramoplanin was bactericidal and MICs were not adversely influenced by high (⩾107 CFU/ml) inoculum concentrations. Becuase ramoplanin has previously shown promise as a topical drug, these in vitro results further substantiate its wide spectrum, potency, and potential clinical usefulness as mupirocin-resistant staphylococci and other Gram-positive species become more prevalent.

Isolation of Dunaliella spp. from a hypersaline lake and their ability to accumulate glycerol

The purpose of the present work was to study the potential biotechnological use of Dunaliella species isolated from a hypersaline lake in Turkey. Dunaliella spp. grown in Johnson’s medium were isolated and their glycerol production was studied in a batch system in order to determine the optimal conditions required for the highest glycerol accumulation. In the experiments performed with four newly isolated Dunaliella spp., the maximum glycerol accumulation was obtained at 20% NaCl concentration, and pH 6 (for strains T1 and T2) and pH 9 (for strains T3 and T4). Biomass production by strain T2 was significantly higher that by the other strains but the highest glycerol production in broth was obtained by strain T1 followed by strain T2. Strain T1 showed high glycerol production, i.e. 452.57 μg/ml of culture broth at 20% NaCl concentration. The highest glycerol accumulation on both dry weight and cell basis was obtained with strain T1, followed by strains T3 and T4 (55.01, 50.16, and 40.23 μg/10 6 cells (or pg/cell), respectively) at 25% NaCl concentration. When the high initial inoculum concentration was used at 25% NaCl concentration, strain T1 had the shortest (approximately 10–15 days) lag period. This study shows that the isolated strains T1 and T2 can be used for glycerol production because of their high productivity.

Using Marker Pens on Patients: a Potential Source of Cross Infection with MRSA

Marker pens are widely used in surgery but pre-operative marking of patients may be a cause of bacterial cross-infection. Two experiments were performed to assess whether marking pens can be cause of cross-infection: (i) 26 indelible marker pens were collected from surgical wards for analysis; and (ii) 'fresh' as well as 'dry' (artificially dried by removing cap and exposing tip for 2 h) new permanent marker pens, and whiteboard marker pens were inoculated by dipping the tips into various concentrations of methicillin-resistant Staphylococcus aureus (MRSA). Each pen was inoculated onto 2 blood agar plates at 0 (immediately after inoculation) to 30 min at various intervals, 4 h and 24 h. The plates were incubated for 18 h at 35 degrees C in an incubator. Of 26 pens collected from the wards, 2 cultured Micrococci spp. (skin commensals). The constituents of new 'fresh' pen tips rapidly kill MRSA - in all cases by 4 h, but usually within minutes. At high inoculum concentrations, MRSA is not killed immediately. Dry marker pens harbour MRSA for at least 30 min and probably longer. Marker pens can act as fomites for nosocomial infection. The ethanol-based ink in permanent marker pens has a bactericidal action against MRSA that starts within seconds, and they are likely to be safe to use with a gap of at least 2 min between patients. Usually, harmless skin commensals are not pathogenic except in immunocompromised patients. Old or dried-out marker pens can harbour pathogens and should be discarded before attempted use on patients. We recommend disposable markers for the immunocompromised and patients with a known positive MRSA status.

Characterization and Inheritance of a New Source of Resistance to Fusarium oxysporum f. sp. melonis Race 1.2 in Cucumis melo.

Four physiological races (0, 1, 2, and 1.2) of Fusarium oxysporum f. sp. melonis, causal agent of Fusarium wilt in melons, have been described. Whereas resistance against each of the races 0, 1, and 2 is encoded by a single dominant R-gene, resistance against the fourth race, FOM 1.2, is polygenically inherited and was recently characterized in the cultivar Isabelle. In the present study, we report on an independently derived source of resistance to race 1.2, the Israeli breeding line BIZ. The disease response of BIZ seedlings was compared with two susceptible genotypes, Line 33 and PI 414723, and the partially resistant genotype, Isabelle, at increasing concentrations of inoculum. BIZ exhibited near-complete resistance to race 1.2, even at inoculum levels of 106 spores per ml and root wounding, indicating that such resistance is stronger than that in Isabelle. Although in previous studies the F1 hybrids between BIZ and the sensitive lines displayed full resistance in the field, in the present study they were susceptible under artificial inoculation conditions that involve high inoculum concentrations of 106 spores per ml and root wounding; under intermediate inoculum levels (105 and 5 × 105 spores per ml) they were partially resistant. Segregation of the resistance response in the F2 and back-cross-1 progeny from a cross between BIZ and PI 414723 supported a model in which two complementary, recessive genes are required to obtain full resistance. While the fungus was shown to colonize root tissue of both resistant and susceptible lines, colonization was markedly lower in the resistant plants, and the fungus was nearly absent from upper stem segments.

Augmented Expression of Polysaccharide Intercellular Adhesin in a DefinedStaphylococcus epidermidisMutant with the Small-Colony-Variant Phenotype

While coagulase-negative staphylococci (CoNS), with their ability to form a thick, multilayered biofilm on foreign bodies, have been identified as the major cause of implant-associated infections, no data are available about biofilm formation by staphylococcal small-colony variants (SCVs). In the past years, a number of device-associated infections due to staphylococcal SCVs were described, among them, several pacemaker infections due to SCVs of CoNS auxotrophic to hemin. To test the characteristics of SCVs of CoNS, in particular, to study the ability of SCVs to form a biofilm on foreign bodies, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in Staphylococcus epidermidis. In fact, this mutant displayed a stable SCV phenotype with tiny colonies showing strong adhesion to the agar surface. When the incubation time was extended to 48 h or a higher inoculum concentration was used, the mutant produced biofilm amounts on polystyrene similar to those produced by the parent strain. When grown under planktonic conditions, the mutant formed markedly larger cell clusters than the parental strain which were completely disintegrated by the specific beta-1,6-hexosaminidase dispersin B but were resistant to trypsin treatment. In a dot blot assay, the mutant expressed larger amounts of polysaccharide intercellular adhesin (PIA) than the parent strain. In conclusion, interrupting a hemin biosynthetic gene in S. epidermidis resulted in an SCV phenotype. Markedly larger cell clusters and the ability of the hemB mutant to form a biofilm are related to the augmented expression of PIA.

Time-Kill and Synergism Studies of Ceftobiprole against Enterococcus faecalis , Including β-Lactamase-Producing and Vancomycin-Resistant Isolates

Ceftobiprole (BAL9141) is an investigational cephalosporin with broad in vitro activity against gram-positive cocci, including enterococci. Ceftobiprole MICs were determined for 93 isolates of Enterococcus faecalis (including 16 beta-lactamase [Bla] producers and 17 vancomycin-resistant isolates) by an agar dilution method following the Clinical and Laboratory Standards Institute recommendations. Ceftobiprole MICs were also determined with a high inoculum concentration (10(7) CFU/ml) for a subset of five Bla producers belonging to different previously characterized clones by a broth dilution method. Time-kill and synergism studies (with either streptomycin or gentamicin) were performed with two beta-lactamase-producing isolates (TX0630 and TX5070) and two vancomycin-resistant isolates (TX2484 [VanB] and TX2784 [VanA]). The MICs of ceftobiprole for 50 and 90% of the isolates tested were 0.25 and 1 microg/ml, respectively. All Bla producers and vancomycin-resistant isolates were inhibited by concentrations of </=1 and </=4 microg/ml, respectively, at the standard inoculum concentration. Ceftobiprole MICs at a high inoculum concentration for a subset of five Bla(+) E. faecalis isolates were </=1 microg/ml. Bactericidal activity was observed against four isolates tested at concentrations as low as 1 microg/ml regardless of the production of beta-lactamase or vancomycin resistance. A combination of ceftobiprole (0.5 microg/ml) and streptomycin (25 microg/ml) was synergistic against Bla(+) TX0630 and TX5070. Ceftobiprole (0.5 microg/ml) plus gentamicin (10 microg/ml) was synergistic against VanB isolate TX2484 and showed enhanced killing, but not synergism, against TX2784 (VanA), despite the absence of high-level resistance to gentamicin. In conclusion, ceftobiprole exhibited good in vitro activity against E. faecalis, including Bla(+) and vancomycin-resistant strains, and exhibited synergism with aminoglycosides against selected isolates.

Amikacin-loaded vascular prosthesis as an effective drug carrier

Strong covalent immobilization of amikacin on Uni-Graft ® DV straight vascular prostheses made of gelatine-sealed poly(ethylene terephthalate) fibres was performed according to procedure described in the Polish Patent No. P-358934. The concentrations of amikacin in sample solutions were estimated either by HPLC or by UV spectroscopy method previously optimized for amikacin measurements. A high correlation was found between these two methods. It was found that the antibiotic was bound in mixed-type way via three types of interactions: strong covalent bonds (dominating amount: 81.84%) and weak interactions: physical adsorption and ionic bonds (18.19%). Even when total amount of physically and ionically attached drug has been released, the remaining covalently bound amount still locally protected the prostheses in vitro against bacteria. The release test was conducted in PBS at pH 7.4 at 37 °C and showed that about 15% of total drug amount was eluted from the matrix during the first 7 days of shaking, then no more antibiotic was released. It suggested that about 85% of amikacin attached to prosthesis modified in mixed-type mode was bound via covalent interactions. A bacterial inhibition test on Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 showed inhibition of growth for all strains at low inoculum concentrations up to 30 days as well as high inoculum concentration for E. coli. At high concentrations of S. aureus and P. aeruginosa, the modified prostheses showed slight bacteriostatic effect since 10th day of experiment. Amikacin-modified vascular prostheses might therefore be protected against bacterial infection locally, without long-lasting drug release to human system.

Arbuscular mycorrhiza induces gene expression of the apoplastic invertase LIN6 in tomato (Lycopersicon esculentum) roots

Extracellular invertases are suggested to play a crucial role in the arbuscular mycorrhiza (AM) symbiosis to fulfil the increased sink function of the mycorrhizal root and the supply of the obligate biotrophic AM fungus with hexoses. In tomato (Lycopersicon esculentum), LIN6 represents an apoplastic invertase which is described as a key enzyme in establishing and maintaining sink metabolism. In this study, transcript levels of LIN6 were analysed in tomato roots colonized with the AM fungus Glomus intraradices. Using real-time RT-PCR, a nearly 3-fold increase in LIN6 mRNA levels was detected at late stages of mycorrhization (11 weeks after inoculation). A 1.8-fold induction could already be achieved at earlier stages (5 weeks after inoculation) using higher inoculum concentrations, whereas wounding of non-mycorrhizal roots resulted in up to 12-fold enhanced LIN6 transcripts. As revealed by in situ hybridization, the expression of LIN6 upon mycorrhization was specifically restricted to colonized cells and to the central cylinder. Such a strongly localized pattern due to mycorrhizal cells and to the central core could also be shown for promoter activity using transgenic Nicotiana tabacum plants expressing the gene coding for beta-glucuronidase under the control of the LIN6 promoter. The moderate induction of LIN6 expression in mycorrhizal tomato roots compared with stress-stimulated induction suggested a fine-tuning in the activation of sink metabolism in the mutualistic interaction, avoiding stress-induced defence reactions.

Controle químico do cancro cítrico em plantas jovens sob manejo convencional e orgânico

O objetivo deste trabalho foi avaliar o desenvolvimento de porta-enxertos de Poncirus trifoliata Raf. e de enxertos de laranjeira "Valência" (Citrus sinensis Osb.), os índices de ataque de cancro cítrico, causado pela bactéria Xanthomonas citri pv. citri, artificialmente inoculada, e o controle dessa moléstia, com pulverizações cúpricas em sistemas de cultivo, convencional e orgânico, no Centro de Formação da EMATER, situado no município de Montenegro, Estado do Rio Grande do Sul. Foram testadas pulverizações cúpricas a cada 7, 14 e 21 dias, com concentrações de 0,15 e 0,30% de cobre metálico, utilizando calda bordalesa no sistema orgânico e oxicloreto de cobre no sistema convencional. Foram avaliados o crescimento do diâmetro do caule das plantas, a produção de matéria seca da parte aérea dos porta-enxertos, o número de folhas com lesões de cancro cítrico e o número de lesões presentes por folha atacada. Verificou-se que em ambos os sistemas de cultivo, no convencional e no orgânico, o desenvolvimento dos porta-enxertos foi semelhante, mas os enxertos cresceram mais no sistema convencional. Com elevada presença de fontes de inóculo, os tratamentos cúpricos não controlaram o cancro cítrico nos porta-enxertos. Já nos enxertos, com baixa presença de fontes de inóculo, tanto a calda bordalesa como o oxicloreto de cobre controlaram a doença, com melhor resposta na concentração de 0,3% de cobre metálico, aplicada em intervalos de 14 dias. Para melhorar o controle do cancro cítrico, com calda bordalesa, em sistemas orgânicos de cultivo, devem ser desenvolvidas medidas eficazes de controle da larva-minadora.

Efeito de fontes de silício na incidência e na severidade da antracnose do feijoeiro

Foram realizados três experimentos para estudar a influência do silício na redução da antracnose, causada por Colletotrichum lindemuthianum, em feijoeiro (Phaseolum vulgaris). No primeiro experimento plantas tratadas com silicato de cálcio foram inoculadas com 0, 10³, 10(4), 5x10(4), 10(5), 5x10(5) e 10(6) conídios/ml de C. lindemuthianum. Na concentração de 10(6) conídios/ml avaliou-se a diferença entre o silicato de cálcio e uma fonte de cálcio (óxido de cálcio). No segundo experimento foi avaliado o silicato de cálcio e de sódio na redução da antracnose. No terceiro experimento, por meio da microscopia eletrônica de varredura (MEV) e da microanálise de raios-X (MAX), estudou-se o efeito do silício nos mecanismos de resistência. Foram realizadas cinco avaliações da doença, utilizadas para calcular a área abaixo da curva de progresso da incidência (AACPI), da severidade (AACPS) e a duração da área foliar sadia (HAD), nos dois primeiros experimentos. Com o aumento da concentração do inóculo houve aumento na AACPI e AACPS e redução na HAD, porém a testemunha inoculada com a maior concentração de inóculo, e tratada apenas com cálcio (CaO), sem silicato, teve maiores AACPI e AACPS do que as plantas tratadas com silicato de cálcio. No segundo experimento, a menor AACPS e maior HAD foram obtidas no tratamento com silicato de sódio via foliar, que proporcionou redução de 62,4% na AACPS. Na MEV e na microanálise de raios X não se observaram a formação de barreira física e o acúmulo de silício externamente com a aplicação de silicato de cálcio, embora o elemento tenha contribuído para reduzir a antracnose.