First Report of Stewart's Wilt of Maize in Argentina Caused by Pantoea stewartii.

Stewart's wilt is a serious disease of corn (Zea mays L.) caused by the bacterium Pantoea stewartii subsp. stewartii (Pss). Typical symptoms of infected fields and dent corn are longitudinal streaks with irregular or wavy margins, which are parallel to the veins and may extend the length of the leaf. These pale to green yellow lesions become dry and brown as the disease progresses producing a leaf blight (4). During the growing seasons 2010 to 2011 and 2011 to 2012, symptoms of bacterial leaf blight of corn were observed in central Argentina maize fields, with an incidence of 54% in Córdoba province. To identify the pathogen, leaves from 10 symptomatic maize plants per field were collected from 15 fields covering a representative geographical area. High populations of morphologically uniform bacteria were isolated from leaf tissues by conventional methods using King's medium B agar (2). Ten representative facultatively anaerobic gram-negative, non-fluorescing, non-motile, catalase positive and oxidase negative rod-shaped and yellow-pigmented bacterial isolates were evaluated further. The biochemical profile obtained was: fermentative metabolism, negative indol, acetoin and hydrogen sulfide production, negative gelatin hydrolysis (22°C), positive acid production from D-glucose and lactose, negative gas production from D-glucose, and negative nitrate reduction (1). All the isolates produced a 300-bp band with PCR using the species specific primer pair PST3581/PST3909c (3). The Pss ATCC 8199 and Pseudomonas fluorescens ATCC 13525 strains were used as positive and negative controls for the PCR assays, respectively. The pathogenicity test was performed by stem inoculation of five to ten P2069 YR maize plants (one to two leaf growth stage) grown in growth chamber. Plants were inoculated by syringe with a 107 to 108 cell/ml bacterial suspension and kept in a humid chamber at 25 to 27°C. Plants inoculated with Pss ATCC 8199 or with sterile water were used as positive and negative control treatments, respectively. The development of symptoms similar to those originally found in the field was observed on all the plants inoculated with the different isolates at 7 to 10 days post inoculation. In addition, symptoms on inoculated plants were similar to those observed for the positive control treatment. No symptoms were found on negative controls. Koch's postulates were fulfilled since bacteria isolated from symptomatic tissue had identical characteristics to isolates used to inoculate plants and to the reference Pss strain for biochemical tests and PCR reaction mentioned above. To our knowledge, this is the first report of P. stewartii subsp. stewartii isolated from diseased maize in Argentina. References: (1) J. G. Holt et al. Page 179 in: Bergey's manual of determinative bacteriology. Williams and Wilkins, Baltimore, MD, 1994. (2) OEPP/EPPO. Bulletin OEPP/EPPO Bulletin, 36: 111, 2006. Pantoea stewartii subsp. stewartii diagnostic. (3) A. Wensing et al. Appl. Environ. Microbiol. 76:6248, 2010. (4) D. G. White Page 4 in: Compendium of corn disease. The American Phytopathology Society, 1999.

Biodegradable bioartificial materials made with chitosan and poly(vinyl alcohol). Part I: Physicochemical characterization.

Polymers have widespread applications in therapeutics, and their use can play important structural and functional roles in different disease conditions. Bioartificial biodegradable materials, to be used as biomaterials and, in particular, as localized drug carriers, were prepared mixing chitosan (CHI) and poly(vinyl alcohol) (PVA), then manufactured as films, and finally cross-linked with glutaraldehyde (GTA), both in the absence and in the presence of the edible plasticizer sorbitol (SOR). The materials were characterized by Fourier-transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), ther-mogravimetric analysis (TGA), X-ray diffraction, scanning electron microscopy (SEM), and tensile test. The FTIR spectroscopy and the X-ray diffraction indicated that the presence of CHI lowers the crystallinity of PVA, and that the cross-linking with GTA does not modify the interactions between the two polymers, but only forms bridges between the different chains. In addition, the thermodynamic parameters for PVA, evaluated from the DSC measurements, confirmed that the PVA structure was less crystalline in the blends than in the pure state. The addition of SOR as a plasticizer to the CHI/PVA blends generally improved the characteristics of the films, making the cross-linked films less brittle, as confirmed by the SEM measurements and by the mechanical test. The TGA measurements confirmed the presence of chemical interactions between the polymers, as indicated by the DSC measurements. On the whole, the physicochemical properties of the blends showed the strong interactions existing between the component materials.

Composites between hydroxyapatite and poly(epsilon-caprolactone) synthesized in open system at room temperature.

The hydroxyls present on the surface of hydroxyapatite (HA) granules, annealed at 700 composite function, 900 composite function and 1,100 composite function C, are able to initiate the polymerization of epsilon-caprolactone (CL), not only at 185 composite function C under vacuum, but also at room temperature in open system. A polymer layer ionically linked to the substrate is formed on HA surface, enhancing the compatibility between the organic phase and the inorganic one in composite biomaterials. We studied the characteristics of the polymer, produced by the reaction carried out at room temperature in open system, as well as the percentages of the poly(epsilon-caprolactone) (PCL) ionically bonded to the HA structure and of the "free" one. Both percentages appear very dependent on the annealing temperature; in particular, HA annealed for 1 h at 1,100 composite function C is the most efficient initiator of the reaction leading to ionically bonded PCL. The percentages of "free" polymer are much higher than at 185 composite function C under vacuum. Its formation is attributed to the role of water in opening the CL rings, and to the presence of CO(3) (2-) and HPO(4) (2-) ions in the HA annealed at lower temperatures. The presence of water appears to be the limiting factor for the production of PCL not bonded to the HA structure.

Cross-linked ionomeric materials from poly(styrene-alt-maleic anhydride) and poly(ethylene glycol) for biomedical applications: a preliminary investigation.

Two ionomeric materials, cross-linked through the formation of polyoxyethylene bridges, were synthesized by the reaction of poly(styrene-alt-maleic anhydride) (PSMA) with poly(ethylene glycol) (PEG), carried out in the absence of external catalysts. The reaction was carried out at room temperature, both in bulk with excess glycol, and in acetone solution with a 20:1 ratio of anhydride rings to hydroxyl groups. The materials were characterized by scanning electron microscopy (SEM), total reflection Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The SEM showed a quite uniform porous structure for the material synthesized in bulk, and two distinct phases for that synthesized in acetone solution, a sponge-like structure and a denser one. The FTIR spectra showed that the first material underwent the cross-linking reaction to a greater extent than the second one. Both TGA and DSC confirmed the formation of cross-linked structures. Such tri-dimensional networks, owing to the presence of the carboxyl groups, could easily entrap either cationic drugs, in view of a possible controlled release, or poisonous metal cations, when they must be removed from blood. The second use can be made easier by the hemocompatibility, ascertained in preceding studies on other materials, synthesized by the reaction between maleic anhydride copolymers and hydroxyl-containing macromolecules. Another possible use is the production of ion exchanging gels, as fillers for both ion exchange and liquid chromatography columns, which could be easily regenerated.

Release of 5-fluorouracil by biodegradable poly(ester-ether-ester)s. Part I: release by fused thin sheets.

The 5-fluorouracil release by biodegradable epsilon-caprolactone and L-lactide copoly(ester-ether-ester)s was tested. The drug-copolymer mixture was formed by fusion in thin sheets, which were dipped in Dulbecco's PBS for time intervals ranging from one hour to two months. Each experiment shows a fast initial release, which subsequently slows down and stops at a limiting value, depending on the copolymer composition. This behavior was attributed to an extraction of the drug present on the sheet surface, due only to its shape, and to hydrogen bonds between the drug and the copolymers. The results obtained lead to a possibility of using such copolymers as "time-delayed" drug-releasing systems, when formed in specimens with smaller surface-to-volume ratio, which could minimize the fast initial extraction.

Fibers by bioresorbable poly(ester-ether-ester)s as potential suture threads: a preliminary investigation.

Fibers made by a bioresorbable poly(epsilon-caprolactone)-block-poly(oxyethylene)-block-poly(epsilon-caprolactone) copolymer, having a number average molecular mass of about 200,000 Da and an average molar composition of 66% oxycaproyl units and 34% oxyethylene units, were melt-spun, with the aim at using them as suture threads. Their properties were investigated by the stress-strain test and by differential scanning calorimetry (DSC). The results obtained show that the properties of this material depend very strongly on the alignment of its macromolecules. In particular, the only partial alignment, obtainable by a relatively moderate drawing just after the extrusion, leads to values of elongation at break too high for use of the fibers as suture threads. The DSC analysis reveals interesting properties of the material, but also confirms their strong dependence on the extrusion procedure and on the mechanical treatment. In conclusion, the results of this preliminary study show that the spinning technique must be improved, and that further investigations are necessary to ascertain the possibility of using these poly(ester-ether-ester)s for the fabrication of suture threads.

Periodontal membranes from composites of hydroxyapatite and bioresorbable block copolymers.

Biomembranes are frequently proposed as devices for "guided bone regeneration." Such membranes consist generally of a thin sheet of polymeric material, mostly textured from polymeric yarns or clots, which all have a diffuse very fine winding porosity. The cross-section size of the holes of such porosity is nanometric (diameter < 0.1 microm); thus these holes can be indicated as nanoholes. Whatever the method of production, the surface density of nanoholes (number per square centimeter) has to be as high as possible. It is important also that no variation of this density occurs. The fine dimension of these microholes allows the crossing of small molecules (O2, CO2, H2O, sugars, many nutritional organic compounds and even some simple proteins) but not other larger molecules and particulates, including cells of any kind. These biomembranes have, consequently, a semipermeable behavior, providing the functional role which is the interposition of a barrier for the cells, separating the bone from the surrounding soft tissues. The kinetic of proliferation of osteoblasts is lower than that of fibroblasts. Most membranes of this kind are not resorbable. The main problem for the resorbable ones is the speed of size increase of the holes during the time. Their diameter must not exceed a threshold value until the reconstruction of bone is complete, otherwise soft tissue cells will invade the growing bone tissue with formation of undesirable mixed tissue. The present paper deals with a resorbable membrane made with a composite polymer/ceramic. A poly(epsilon-caprolactone)-block-poly(oxyethylene)-block-poly(epsilon-caprolactone) copolymer is the polymeric matrix which contains dispersed ceramic hydroxyapatite microgranules, a stiff filling additive. The main possible use is that of periodontal membranes. The copolymer, obtained by thermal polymerization of epsilon-caprolactone onto poly(ethylene-glycol), presents good biological tolerance, is resorbable under physiological conditions and can promote cell growth. Histological tests, performed 6 months after implantation, showed that the polymeric matrix is almost totally resorbed. New-formed bone colonizes even the innermost parts of the membrane, with bone trabeculae closely surrounding the hydroxyapatite granules.

New composites of hydroxyapatite and bioresorbable macromolecular material.

Composite materials were prepared by mixing in different proportions of hydroxyapatite (HA) and poly(epsilon-caprolactone-oxyethylene-epsilon-caprolactone) block copolymer (PCL-POE-PCL) to produce a new resorbable material for biomedical applications. This material has proved to be very interesting for production of periodontal membranes. Mechanical properties are linearly proportional to the amount of HA introduced. Fourier transform infrared (FTIR) investigations have pointed out that HA is able to influence some close epsilon-caprolactone molecules to start its homopolymerization giving PCL with an end chain ionic bonding. HA grains are therefore surrounded by a film of PCL which grants close connection of HA grains within copolymeric matrix. This interface bond with PCL is, however, an interesting occurrence for preparations of HA/PCL composites.

In vitro cytotoxicity testing of chitosan-containing polyelectrolyte complexes.

Two chitosan-containing polyelectrolyte complexes, chitosan-poly(acrylic acid) and chitosan-poly(styrenesulphonate), were synthesized by polymerizing acrylic acid and sodium styrenesulphonate in the presence of chitosan and chitosan hydrochloride, respectively. The complexes were studied by optical microscopy and tested for cytotoxicity by the Neutral Red uptake, Kenacid Blue R-Binding and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assays. The optical microscopy confirmed the differences in crystallinity and structure already found for the two polycomplexes by other characterization techniques. The cytoxicity tests showed different influences on the cell activity by the extracts of the two polyelectrolyte complexes. Such results were discussed and correlated to the different structures of the two materials.

The activation of human plasma prekallikrein as a hemocompatibility test for biomaterials. II. Contact activation by EVAL and EVAL-SMA copolymers.

The activation of human plasma prekallikrein (PKK) to kallikrein (KK), induced by the contact of blood with foreign materials, is a useful in vitro hemocompatibility test. Kallikrein is easily detected by its reaction with the chromogenic substrate H-D-Pro-Phe-Arg-pNA, which releases p-nitroaniline, revealed by its absorption at 405 nm. This test, which was already carried out by evaluating PKK activation by the 'end-point' method, has been carried out in this work by the more accurate 'initial velocity' method, i.e. by evaluating the activation from the initial rates of the KK-substrate reaction. The tests were carried out on the following materials: borosilicate glass (as a high-activation reference material), silicone (as a low-activation reference material), the commercial biomaterial Cardiothane 51, three graft copolymers synthesized in our laboratory by reacting ethylene-vinyl alcohol copolymer (EVAL) with styrene-maleic anhydride copolymer (SMA), and EVAL itself. A mathematical treatment based on a simple kinetic model has been used for a first-approximation evaluation of the PKK-activating power of the materials tested. The quite low activating power of the EVAL-SMA copolymers, which are easily processable into water-permeable hollow fibers, suggests the possibility of their use in blood dialyzers.