Drop Of Suspension Research Articles

Preparation, characterization and evaluation of novel elastic nano-sized niosomes (ethoniosomes) for ocular delivery of prednisolone

Niosomes embodying ethanol and minimum amount of cholesterol (ethoniosomes) could be promising ocular delivery systems for water soluble and insoluble drugs. This manuscript reports on novel nano-sized elastic niosomes (ethoniosomes) composed of Span 60: cholesterol (7:3 mol/mol) and ethanol, for ocular delivery of prednisolone acetate (Pred A) and prednisolone sodium phosphate (Pred P). These ethoniosomes were prepared with the thin film hydration (TFH) and ethanol injection (EI) methods, characterized for percentage entrapment efficiency (% EE), size, zeta potential, morphology, elasticity, in vitro release and physical stability. Ocular irritation, bioavailability and anti-inflammatory effects were evaluated and compared with the conventional suspension and solution eye drops. The prepared ethoniosomal vesicles (EV) had a Z-average diameter of 267 nm, zeta potential of approximately −40 mV and % change in size after extrusion of 4%. They were physically stable for at least 2 months at 4 °C. The prepared EV showed good ocular tolerability using the modified Draize’s test and the estimated relative ocular bioavailability for Pred A EV and Pred P EV was 1.54 and 1.75 times greater than that for the suspension and solution eye drops, respectively. The time required for complete healing from the clove oil-induced severe ocular inflammation was reduced to half with Pred A and Pred P EV. More interestingly, the intraocular pressure (IOP) elevation side effect recorded for Pred A and Pred P EV was significantly less than that for the conventional suspension and solution eye drops.

Frequency distribution of virulence factors in uropathogenic Escherichia coli isolated from Kermanshah in 2011-2012.

Background:Uropathogenic Escherichia coli (UPEC) can cause urinary tract infection (UTI). To prevent urine flow lavage, UPEC has acquired several virulence factors called adhesins. These adhesins are expressed and controlled by different genes.Aim:This study was aimed to determine some of the most important genes that control virulence factors of UPEC (pyelonephritis associated pili [pap], S fimbrial adhesion [sfa] and A fimbrial adhesion [afa] genes), which code for adhesins and phenotypic factors.Materials and Methods:In total, 205 UPEC isolates from in- and out-patients with UTI were obtained. Polymerase chain reaction was used for gene amplification. One drop of bacterial suspension, one of red blood cells and one of peripheral blood smear were mixed for hemagglutination (HA). Formation of a clump was considered to be positive. Bacteria were grown on blood agar to determine hemolysis. Surface hydrophobicity was determined using the SAT test.Result:Frequencies of pap, afa and sfa were 42 (20.5%), 17 (8.3%) and 44 (21.5%), respectively. Frequencies of HA, hemolysis and hydrophobicity were 138 (67.3%), 56 (27.3%) and 39 (19%), respectively. Among HA-positive bacteria, 103 (74.6%) were mannose resistant. Our results highlight higher frequency of HA than that of other virulence factors, indicating a crucial role of this virulence factor in UPEC.Discussion:We concluded that major differences exist in the prevalence of virulence factors among different UPEC isolated from different countries. The association observed between pathogenicity and virulence factors may promote UPEC survival and growth within the urinary tract. Detecting these genes as the primary controllers of UPEC virulence factors may aid in better management of related infections.

Liquid–air interface self-assembly: A facile method to fabricate long-range nanoparticle monolayers

A liquid–air interfacial assembly approach is a technique to fabricate long-range well-ordered nanostructures in either two or three dimensions. In this work, magnetic nanoparticles were synthesized by co-reduction of iron acetylacetonate (Fe(acac)3) and platinum acetylacetonate (Pt(acac)2) in benzyl ether. A few drops of the as-made nanoparticle suspension with varying concentrations were spread on the surface of diethylene glycol, ethylene glycol, and deionized water. After solvent evaporation, a substrate was placed under the liquid surface and then gently lifted up to collect nanoparticle layers. Although the nanoparticles are irregular in shape, they could well self-assemble on the diethylene glycol surface, forming an extended monolayer with small defects. By contrast, multilayer domains were frequently obtained when deionized water and ethylene glycol were used as the liquid subphases. An appropriate nanoparticle concentration was also a crucial factor in this approach. The lower concentration could not form an extended monolayer while the higher concentration led to multilayer assemblies. The order of the nanoparticle layers also strongly depended on the uniformity of the nanoparticles.

First Report of Corynespora cassiicola Causing Leaf Spot on Akebia trifoliate.

Akebia species have been used for centuries in medicinal practices in a few Asian countries such as China and Japan. The dried stems of Akebia trifoliata are known as mutong in the Chinese pharmacopoeia (4) and mokutsu in Kampo, the traditional Chinese medicine developed in Japan (2). In China, the plant is grown in the provinces of Shandong, Hebei, Shanxi, Henan, Gansu, and some provinces in the south of the Yangtze River basin. During the summer of 2012, a leaf spot disease was detected on A. trifoliata grown in Nanning, Guangxi, China. The disease occurred and spread rapidly in July under conditions of high temperature and high humidity. The symptoms appeared on three sites that we inspected; disease incidences were higher than 80%. Initial symptoms consisted of small (less than 5 mm in diameter), circular, purple-brown leaf spots. Spots later enlarged and became elliptical, circular, or irregular with gray-white centers and dark brown rims. The centers were slightly concave. The spots could coalesce with each other, resulting in leaf desiccation and wilting. A fungal isolate was obtained from symptomatic leaf tissue that taken from a field (22°50'N, 108°22'E) in Nanning, Guangxi, China. Single-spore culture of the isolate was incubated on potato dextrose agar (PDA) for 7 days in the dark at 28°C. Conidiophores were straight to slightly curved, unbranched, and pale brown. Conidia (19.0 to 140.5 μm long and 7.0 to 11.0 μm wide) were formed singly or in chains, obclavate to cylindrical, straight or curved, pale brown, with a rounded apex and truncate base, and 1 to 13 pseudosepta. Morphological characteristics of the isolate were similar to the descriptions of Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei (1). Genomic DNA of the isolate was extracted and used for PCR amplification of rDNA-ITS (internal transcribed spacer) sequence with primers ITS1 and ITS4. The PCR products were purified and sequenced. The sequence (GenBank Accession No. KC977496) was used in BLAST searches to interrogate GenBank for sequence similarity. High sequence similarity of 100% was obtained with several C. cassiicola strains. Pathogenicity of the isolate was investigated to demonstrate Koch's postulate. Young, healthy, fully expanded green leaves of A. trifoliata were surface sterilized. Fifteen leaves were inoculated with 10-μl drops of conidia suspension (105 conidia per ml) and 10 leaves were inoculated with the same volume of sterile water to serve as controls. All the leaves were placed in a humid chamber for 5 days. Spots with similar symptoms to those observed in the field developed on all inoculated leaves. The pathogen was reisolated and identified as C. cassiicola. The controls remained symptomless. According to previous reports, A. trifoliata was infected by Alternaria tenuissima in China and by Colletotrichum acutatum in Japan (3). To our knowledge, this is the first report of C. cassiicola found on Akebia species worldwide. Furthermore, this new disease primarily affects plantations and reduces the quality and yield of the medicine. Some effective measures should be taken to control this disease.

Polymer dots for quantifying the total hydrophobic pathogenic lysates in a single drop

A selective, rapid, small sample load (2–4μL), and sensitive quantification method for the hydrophobic cellular biomolecules of pathogenic bacteria and their biosensing application were reported. The present approach is based on using polythiophene polymer dots (2.5nm), which were prepared via the oxidation/polymerization reactions and then were characterized using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR), ultraviolet spectroscopy (UV), and matrix (surface) assisted laser desorption/ionization mass spectrometry (M(S)ALDI-MS). The present method requires only gentle agitation for a single drop of aqueous bacteria suspension (10μL, Pseudomonas aeruginosa (1×104cfu/mL) and Staphylococcus aureus (1×105cfu/mL)) with 1mL of polythiophene (0.5mg/mL) in chloroform, and the time required for quantifying the total hydrophobic was significantly reduced to less than 3min. The polythiophene polymer dots is also a quantitative assessment of bacteria for aqueous and blood samples if exposed to more than 4–5μL of pathogenic bacteria and thus, it is a new biosensor for quantitative hydrophobic portions. The fluorescence intensity of polythiophene was enhanced after adding different volumes of pathogenic bacteria with low colony units. The standard bacteria suspensions of P. aeruginosa and S. aureus have low LOD (limits of detection) for 2μL (1×104cfu/mL) and 4μL (1×105cfu/mL), respectively. Further, the pathogenic bacteria were spiked into mouse blood and the total hydrophobic biomolecules were quantified. This method is extremely rapid as it does not require any culture steps prior to analysis and also no need for any separation or post sample treatments.

Comment on “Effects of Particle Shape on Growth Dynamics at Edges of Evaporating Drops of Colloidal Suspensions”

A Comment on the Letter by P. J. Yunker et al., Phys. Rev. Lett. 110, 035501 (2013). The authors of the Letter offer a Reply.Received 5 June 2013DOI:https://doi.org/10.1103/PhysRevLett.111.209601© 2013 American Physical Society

Bahasa Indonesia

Bacterial blight disease on soybean caused by Pseudomonas syringae pv. glycinea is an important factor causing yield loss in Indonesia. Bacteriophage isolated from irrigation water around the soybean field can be used as indicator for the presence of phytopathogenic bacteria. The objectives of this research was to obtain suitable composition of detector materials to detect P. syringae pv. glycinea using bacteriophage. Composition of detector kit contains of nutrient broth medium with 0.1% of bromothymol blue, 10 g talk and 1 g CMC which will be rubbed on to the detector paper and caused green colour development (pH ± 7) when the paper was dipped on to bacteria suspension, added by a drop of bacteriophage suspension and incubated for 24 hour, the colour will be changed. Yellow color indicated growth activity of P. syringae pv. glycinea where as blue colour indicated suppression of P. syringae pv. glycinea.

Morphology of growth and first to fourth moments in a Monte Carlo particle-deposition model with a novel adherence-potential barrier

Two models of particle depositions with different adhesion dynamics have been developed and studied using Monte Carlo simulations. They have been compared for the complete range of Peclet numbers and they differ in the particle-adhesion dynamics, with an adherence-potential barrier in one of them. In general, the time course of the two models is different in ballistic cases, but similar in diffusive cases. Thus, they complement each other, enabling a description of the morphology for most cases. Depending on their deposits, these systems have been characterized morphologically, as compact, homogeneous, or open. They have also been described by the following parameters, which have been analyzed: from the first to the fourth moments, porosity, and the fractal dimension. Also, the time course of the parameters has been studied and described, especially the Anomalous and Family-Vicsek scaling. The third and fourth moments and the fractal dimension have been characterized, defining different stages in their time course, and studying their behavior for the complete range of Peclet numbers. Moreover, the inclusion of an adherence-potential barrier enables the description of new universality class intervals needed to explain recent experimental systems, e.g., at the edges of evaporated drops of colloidal suspensions, electro-convection on nematic liquid crystals, and polynuclear growth model. Finally, the characterization of the systems required not only a dynamic exponent (β) by universality classes but also a third (skewness) and fourth (kurtosis) moments for a complete description.

Colloidal particles in a drying suspension: A phase field crystal approach

Using a phase field crystal model we study the structure and dynamics of a drop of colloidal suspension during evaporation of the solvent. We model an experimental system where contact line pinning of the drop on the substrate is non-existent. Under such carefully controlled conditions, evaporation of the drop produces an ordered or disordered arrangement of the colloidal residue depending only on the initial average density of solute and the drying rate. We obtain a non-equilibrium phase boundary showing amorphous and crystalline phases of single component and binary mixtures of colloidal particles in the density-drying rate plane. While single-component colloids order in the two-dimensional triangular lattice, a symmetric binary mixture of mutually repulsive particles can be ordered into three triangular sub-lattices in two dimensions. Two of them are occupied by the two species of particles with the third sub-lattice vacant.

Spatial Resolution in Micrometric Periodic Assemblies of Magnetotactic Bacteria and Magnetic Nanoparticles

We developed a simple method for obtaining micro arrays of magnetic nanoparticles using audio tapes. We present spatial micro-arrangements of magnetotactic bacteria (Magnetospirillum gryphiswaldense), magnetite (Fe3O4) and functionalized cobalt ferrite (CoFe2O4) nanoparticles. Computer generated square audio waves of different frequencies (100 Hz-10 kHz) were recorded leading to magnetic patterns of different micrometer spatial wavelengths. Drops of aqueous suspensions were deposited on the tapes to control particle density, and bacteria and particles were trapped at locations where magnetic energy is minimized, as observed using conventional optical microscopy after the dispersium medium was evaporated. We discuss the spatial limits of the magnetic nanoparticles and the bacteria assemblies, concluding that cell walls of bacteria inhibit agglomeration and optimize spatial organization.

The cellular and flagella morphologies of ulcerogenic Helicobacter pylori paediatric strains.

Helicobacter pylori is a pathogenic spiral-shaped, microaerophilic, gram-negative bacterium, that inhabits the human stomach. Infection is usually acquired during childhood and always elicits an acute immune response that is, however, inefficient in bacteria clearance. Therefore, in the absence of effective treatment, infection and gastritis (non ulcer dyspepsia, NUD) persist throughout the patient’s life. Depending on its severity and pattern, in about 15% of infected adults, this silent destruction of the gastric mucosa may further progress to peptic ulcer disease (PUD) (gastric and duodenal ulcers, GU and DU respectively) and/or gastric cancer. Infection with H. pylori is also the major cause for the development of paediatric PUD, a rare event that may occur shortly after infection. In addition to the still undisclosed genetic susceptibility of these young patients, the virulence of the implicated H. pylori strain plays a crucial role in the paediatric PUD pathogenesis. Recently, we proved by in vitro infection assays that, compared with paediatric NUD-associated isolates, a group of paediatric ulcerogenic-strains present a greater ability to induce a marked decrease in the gastric cells viability and to cause them severe cytoskeleton damage and mucins’ production/secretion impairment. Moreover, we showed that their enhanced virulence result from a synergy between the ability to better adapt to the hostility of their niche and the expression of cagA, vacAs1, oipA ‘‘on’’ status, homB and jhp562 virulence factors. Accordingly, these ulcerogenic strains share a particular proteome profile, providing them with better antioxidant defences, a metabolism favouring the biosynthesis of aromatic amino acids and higher motility.We are now characterizing/comparing the cellular and flagella morphologies of H. pylori strains isolated from Portuguese children, associated with DU, GU or NUD, belonging to the vast and multiethnic collection of the Instituto Nacional de Saúde Dr. Ricardo Jorge (Portugal). For that, bacteria were grown in H. pylori selective medium (Biogerm, Maia, Portugal) at 37ºC in a microaerobic environment (Anoxomat®, MART Microbiology BV, Drachten, The Netherlands) for 24 h. For Leifson staining analysis, a drop of each bacterial suspension (in PBS) was spread in cleaned microscope slides, stained with the Leifson dye solution until a golden film developed on the dye surface and a precipitate appeared throughout the sample, and analysed by optical microscopy. For Transmission-Electronic-Microscopy (TEM) studies bacterial pellets were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.Corroborating the better swimming abilities of the PUD strains, as previously shown by motility assays, optical microscopy analysis of Leifson stained slides demonstrated marked differences in the morphology of the studied strains (Figure 1). The H. pylori strain associated with DU (Hp 1152/04) seem longer than all the others and, in contrast, that associated with GU (Hp 499/02) is the shortest one and presents a, more pronounced, spiral morphology. Moreover, our preliminary data on TEM analysis indicate the presence of more abundant and apparently more organized flagella in the GU-associated strain Hp 499/02, in contrast to the NUD control strain, Hp 655/99 (Figure 2).Work supported by Research Grant 2011 – Sociedade Portuguesa de Gastrenterologia.

An Outbreak of Leaf Spot Caused by Stemphylium solani on Eggplant in Malaysia

In 2011, a severe gray leaf spot was observed on eggplant (Solanum melongena) in major eggplant growing areas in Malaysia, including the Pahang, Johor, and Selangor states. Disease incidence was >70% in severely infected areas of about 150 ha of eggplant greenhouses and fields examined. Symptoms initially appeared as small (1 to 5 mm diameter), brownish-black specks with concentric circles on the lower leaves. The specks then coalesced and developed into greyish-brown, necrotic lesions, which also appeared on the upper leaves. Eventually, the leaves senesced and were shed. Tissue cut from the edges of leaf spots were surface-sterilized in 1% NaOCl for 2 min, rinsed in sterilized water, dried, and incubated on potato dextrose agar (PDA). Fungal colonies were greyish green to light brown, and produced a yellow pigment. Single, muriform, brown, oblong conidia formed at the terminal end of each conidiophore, were each 21.6 to 45.6 μm long and 11.5 to 21.6 μm wide, and contained 2 to 7 transverse and 1 to 4 longitudinal septa. The conidiophores were tan to light brown and ≤220 μm long. Based on these morphological criteria, 25 isolates of the fungus were identified as Stemphylium solani (1). To produce conidia in culture, 7-day-old single-conidial cultures were established on potato carrot agar (PCA) and V8 juice agar media under an 8-h/16-h light/dark photoperiod at 25°C (4). Further confirmation of the identification was obtained by molecular characterization in which fungal DNA was extracted and the internal transcribed spacer (ITS) region of ribosomal DNA amplified using primers ITS5 and ITS4 (2), followed by direct sequencing. A BLAST search in the NCBI database revealed that the sequence was 99% identical with published ITS sequences for two isolates of S. solani (Accession Nos. AF203451 and HQ840713). The amplified ITS region was deposited in GenBank (JQ736023). Pathogenicity testing of a representative isolate was performed on detached, 45-day-old eggplant leaves of the cv. 125066-X under laboratory conditions. Four fully expanded leaves (one wounded and two non-wounded leaflets/leaf) were placed on moist filter paper in petri dishes, and each leaflet inoculated with a 20-μl drop of a conidial suspension containing 1 × 105 conidia/ml in sterilized, distilled water (3). The leaves were wounded by applying pressure to leaf blades with the serrated edge of forceps. Four control leaves were inoculated similarly with sterilized, distilled water. Inoculated leaves were incubated in humid chambers at 25°C with 95% RH and a 12-h photoperiod. After 7 days, symptoms similar to those observed in the original fields developed on both wounded and non-wounded inoculated leaves, but not on control leaves, and S. solani was reisolated consistently from the symptoms using the same method as the original isolations. Control leaves remained asymptomatic and the fungus was not isolated from these leaves. The pathogenicity testing was repeated with similar results. To our knowledge, this is the first report of S. solani on eggplant in Malaysia.

Comparison of artificial inoculation methods for studying pathogenesis of Alternaria brassicae (Berk.) Sacc on Brassica juncea (L.) Czern. (Indian mustard)

Establishment of disease by artificial inoculation is essential for studies of various aspects of plant pathology. Keeping this in mind, five inoculation methods were compared namely spraying, infiltration, wounding, spore suspension drop and spore suspension drop along with agarose method. The findings of the present study suggest that out of five inoculation methods used, spore suspension drop along with agarose inoculation method was most ideal as this fixed the inoculum on the target site. The mean value of number of initial disease lesions in drop plus agarose method was highest in all of the time intervals of observation namely 312.2, 484.2, 664.2, 734.2 and 799.2 at 24, 48, 72, 96 and 120 h after pathogen inoculation compared to other methods respectively. Statistical analysis software (SAS) was used to find out the significant comparison among the different methods. The spore suspension drop along with agarose method has the advantage of being accurate and precise, and it was also easy to handle the inoculated plants. Keywords : Artificial inoculation, Brassica , Alternaria , pathogenicity, screening African Journal of Biotechnology Vol. 12(18), pp. 2422-2426

FACTORS AFFECTING SPORE GERMINATION OF COCHLIOBOLUS SATIVUS OVER HOST LEAF SURFACE

The effect of spore concentration, incubation period, culture age, buffer concentration, pH and incubation temperature on germination of Cochliobolus sativus over host leaves has been investigated. The data revealed that for optimum germination and germ tube growth of C. sativus over cleared host leaves, the drops of spore suspension (105 spores per ml), prepared in phosphate buffer (0.01 M, pH 6) using spores collected from 20-day old cultures, should be incubated at 28 ± 1°C for 16 h.

Ocular penetration of topical antibiotics: study on the penetration of chloramphenicol, tobramycin and netilmicin into the anterior chamber after topical administration

To compare penetration in the aqueous humour of topically applied antibiotics. Randomized prospective study, Department of Ophthalmology, University of Perugia, Italy Patients undergoing cataract surgery. One hundred twenty-two patients were included: 14 received one drop of chloramphenicol suspension; 12 one application of chloramphenicol gel; 11 one drop of netilmicin suspension; 13 one drop of tobramycin suspension; 37 repeated instillations of chloramphenicol suspension every 10 min for a total of four drops; and 35 repeated instillations of chloramphenicol gel every 10 min for a total of four drops. Samples were taken immediately before surgery from the anterior chamber in order to determine the antibiotic by means of high-performance liquid chromatography. Samples were taken 45-190 min after the eye drops were instilled. Intraocular penetration of chloramphenicol, netilmicin and tobramicyn. After a single administration, netilmicin and tobramycin were undetectable, whereas the chloramphenicol suspension reached a mean concentration of 0.23 ± 0.21 μg/mL, and the chloramphenicol gel a mean concentration of 0.13 ± 0.14 μg/mL. After repeated administrations, the mean concentrations of the chloramphenicol suspension and gel were 0.60 ± 0.26 μg/mL and 0.58 ± 0.18 μg/mL, respectively. Tobramycin and netilmicin do not reach detectable concentrations, whereas chloramphenicol, after multiple administrations, reaches concentrations that are effective against Haemophilus influenzae and Haemophilus parainfluenzae, Legionella pneumophila, Moraxella catarrhalis, Neisseria meningitidis, Pasteurella multocida and Streptococcus pneumoniae. This means that chloramphenicol can be rationally used in the prophylaxis and treatment of infections supported by sensitive germs.

Effects of Particle Shape on Growth Dynamics at Edges of Evaporating Drops of Colloidal Suspensions

We study the influence of particle shape on growth processes at the edges of evaporating drops. Aqueous suspensions of colloidal particles evaporate on glass slides, and convective flows during evaporation carry particles from drop center to drop edge, where they accumulate. The resulting particle deposits grow inhomogeneously from the edge in two dimensions, and the deposition front, or growth line, varies spatiotemporally. Measurements of the fluctuations of the deposition front during evaporation enable us to identify distinct growth processes that depend strongly on particle shape. Sphere deposition exhibits a classic Poisson-like growth process; deposition of slightly anisotropic particles, however, belongs to the Kardar-Parisi-Zhang universality class, and deposition of highly anisotropic ellipsoids appears to belong to a third universality class, characterized by Kardar-Parisi-Zhang fluctuations in the presence of quenched disorder.

Pseudomonas viridiflava in imported carrot seeds

In 2005, a sample of carrot seeds var. Kikuyo imported from Japan was submitted to the Instituto Biológico for bacteriological phytosanitary certification. Circular, convex and creamy colored colonies were obtained. These isolates produced a green fluorescent pigment on King’s B medium and incited a hypersensitivity reaction in tobacco leaves. They were oxidase and arginine dihydrolase negative, did not produce levan but caused soft rot in potato slices and were identified as Pseudomonas viridiflava. Artificial inoculations by infiltration of bacterial suspensions at the crown area or by placing a drop of bacterial suspension on this area and pricking with a sterile needle, produced light to dark brown areas around the injured points 5 days after inoculation. The bacterium was then re-isolated. Although this bacterium could be an opportunistic pathogen and was isolated from asymptomatic samples, eventual losses in the field and post-harvest storage is possible.

Interferência de ulvana no desenvolvimento e melanização de apressórios de Colletotrichum gloeosporioides

The algal polysaccharide ulvan induces resistance in apple to Glomerella leaf spot. However, changes in the pre-infectious process of Colletotrichum gloeosporioides have been reported in apple plants previously treated with ulvan. Thus, this study aimed to verify if the polysaccharide interferes directly in the development of pre-infective structures. For this, 10 µL drops of spore suspension (2x105 conidia.m L-1) were distributed on cellophane paper segments settled on glass slides. The conidial suspension was prepared with aqueous solution with (10 mg.mL-1) or without ulvan. Samples were incubated at 25oC and 100% relative humidity. At every two hours of incubation and over a period of 16 hours, 100 conidia per drop were microscopically (200x) examined. The rates of conidia germination, apressoria formation and melanization were quantified. Germination of conidia was not affected. Ulvan stimulated the elongation of the germ tube but retarded the differentiation of the first apressorium (sessile). The polysaccharide accelerated the formation of second apressorium, but inhibited its melanization.

First Report of Pseudomonas viridiflava Causing a Bacterial Blight of Artichoke Bract Leaves.

In 2006, a disease was observed on two artichoke (Cynara scolymus L. cv. Lardati) fields in Crete, Greece, covering ~2 ha. Symptoms developed after several days of rainy and windy weather and >70% of capitula were affected, resulting in unmarketable produce. Initial symptoms were water-soaked, dark green spots on bracts with many sunken, necrotic, often elongated lesions, each with a brown-black center and surrounded by a water-soaked halo with a dark red-brown margin. Symptoms were more severe on inner bracts. Isolations from symptomatic, surface-disinfected bracts onto King's B agar medium (KB) consistently yielded yellow bacterial colonies that produced a green-blue fluorescent pigment. Ten selected artichoke isolates, all gram-negative, presented the LOPAT profile (- - + - +) and were levan negative, oxidase negative, potato rot positive, arginine dihydrolase negative, and showed tobacco hypersensitive reaction. All isolates used L-arabinose, D(-)-tartrate, and L-lactate, but not sucrose, L(+)-tartrate, or trigonelline. Results were identical to those obtained with the reference strain of Pseudomonas viridiflava NCPPB 1249 (3), and strains PV3005 and PV3006 from eggplant (1). Based on these biochemical tests, 10 isolates were identified as P. viridiflava group II members of the LOPAT determinative scheme of Lelliott (1,2). Two artichoke isolates (PV608 and PV609) were selected for molecular characterization. The identity and phylogenetic analysis were determined by multilocus sequence typing with the gyrB, rpoD, and rpoB genes (PV608 Accession Nos. JN383375, JN383363, and JQ267546; PV609 Accession Nos. JN383376, JN383364, and JQ267547). BLAST searches showed highest nucleotide sequence identity (96%) with GenBank sequences of P. viridiflava reference strains NCPPB 963 and CFBP 2107. Pathogenicity of 10 artichoke isolates and reference strains was tested twice on detached capitulum bracts of artichoke cv. Lardati, as well as 4-week-old tomato plants of cv. ACE, and Chrysanthemum indicum cv. Reagan plants. Each isolate was inoculated onto 10 bracts by placing 15 μl of bacterial suspension (5 × 106 CFU/ml) of a 48-h culture in KB broth on the surface of the bract, and pricking the bract through the drop of bacterial suspension with a sterile needle. Each isolate was also inoculated onto five tomato and five chrysanthemum plants by dipping a sterile toothpick in the appropriate bacterial culture and pricking the surface of the stem. Ten control plants were inoculated similarly with sterile, distilled water. Inoculated bracts and plants were kept in boxes lined with moist filter paper at 25 to 30°C and 80 to 100% relative humidity. Lesions developed on detached bracts within 72 h and were similar to those observed on the naturally infected plants. On tomato and chrysanthemum plants, pith necrosis and wilting symptoms were induced within 1 week of inoculation. Symptoms were not observed on control bracts and plants. Bacterial colonies were reisolated from bract lesions and stems with pith necrosis, but not from control plants, and the reisolates had the same LOPAT profile as the original isolates of P. viridiflava, thus fulfilling Koch's postulates. To our knowledge, this is the first report in the world of P. viridiflava causing a disease of artichoke bracts.

First Report of Tomato Gray Leaf Spot Disease Caused by Stemphylium solani in Malaysia.

In June 2011, tomatoes (Solanum lycopersicum) in major growing areas of the Cameron Highlands and the Johor state in Malaysia were affected by a leaf spot disease. Disease incidence exceeded 80% in some severely infected regions. Symptoms on 50 observed plants initially appeared on leaves as small, brownish black specks, which later became grayish brown, angular lesions surrounded by a yellow border. As the lesions matured, the affected leaves dried up and became brittle and later developed cracks in the center of the lesions. A survey was performed in these growing areas and 27 isolates of the pathogen were isolated from the tomato leaves on potato carrot agar (PCA). The isolates were purified by the single spore technique and were transferred onto PCA and V8 agar media for conidiophore and conidia production under alternating light (8 hours per day) and darkness (16 hours per day) (4). Colonies on PCA and V8 agar exhibited grey mycelium and numerous conidia were formed at the terminal end of conidiophores. The conidiophores were up to 240 μm long. Conidia were oblong with 2 to 11 transverse and 1 to 6 longitudinal septa and were 24 to 69.6 μm long × 9.6 to 14.4 μm wide. The pathogen was identified as Stemphylium solani on the basis of morphological criteria (2). In addition, DNA was extracted and the internal transcribed spacer region (ITS) was amplified by universal primers ITS5 and ITS4 (1). The PCR product was purified by the commercial PCR purification kit and the purified PCR product sequenced. The resulting sequences were 100% identical to published S. solani sequences (GenBank Accestion Nos. AF203451 and HQ840713). The amplified ITS region was deposited with NCBI GenBank under Accession No. JQ657726. A representative isolate of the pathogen was inoculated on detached 45-day-old tomato leaves of Malaysian cultivar 152177-A for pathogenicity testing. One wounded and two nonwounded leaflets per leaf were used in this experiment. The leaves were wounded by applying pressure to leaf blades with the serrated edge of a forceps. A 20-μl drop of conidial suspension containing 105 conidia/ml was used to inoculate these leaves (3). The inoculated leaves were placed on moist filter paper in petri dishes and incubated for 48 h at 25°C. Control leaves were inoculated with sterilized distilled water. After 7 days, typical symptoms for S. solani similar to those observed in the farmers' fields developed on both wounded and nonwounded inoculated leaves, but not on noninoculated controls, and S. solani was consistently reisolated. To our knowledge, this is the first report of S. solani causing gray leaf spot of tomato in Malaysia.