Influence of Culture Medium pH on Growth, Aggregation, and Biofilm Formation of Xylella fastidiosa

The major feature of Xylella fastidiosa growing in its hosts, as well as in culture media, is its cellular aggregation and biofilm formation, leading to partial obstruction of the xylem causing water stress in the plant. We report that growth, aggregation, and biofilm formation of X. fastidiosa are influenced by the medium pH. We have verified that X. fastidiosa cell aggregation is reversibly inhibited by decreasing the medium pH from 6.6 to 6.4. Biofilm formation on glass walls was affected as well, and a concomitant decrease in cell multiplication was observed below pH 6.4. The manipulation of culture medium pH can be used as a tool for the cloning of X. fastidiosa strains isolated from plant hosts, because different strains can inhabit the same plant. Also, X. fastidiosa mutants produced by gene manipulation can be isolated from cell aggregates containing transformed and untransformed cells.     

Grapevine xylem sap enhances biofilm development by Xylella fastidiosa

Xylella fastidiosa is able to form biofilms within xylem vessels of many economically important crops. Vessel blockage is believed to be a major contributor to disease development caused by this bacterium. This report shows that Vitis riparia xylem sap increases growth rate and induces a characteristic biofilm architecture as compared with biofilms formed in PD2 and PW media. In addition, stable cultures could be maintained, frozen and reestablished in xylem sap. These findings are important as xylem sap provides a natural medium that facilitates the identification of virulence determinants of Pierce's disease.     

Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media.

Development of in vitro-produced bovine embryos was studied in 3 two-step culture media: synthetic oviduct fluid (SOF), Gardner's G1/G2, and control (hamster embryo culture medium with 11 amino acids [HECM-6] followed by tissue culture medium 199 + 10% bovine calf serum). Modifications were made to reduce or eliminate protein. Glycolysis and Krebs cycle activity of morulae and blastocysts developed from selected immature oocytes were measured. There were no differences in development to the morula and blastocyst stages between SOF, G1/G2, or control (41%, 36%, and 46%, respectively), although more blastocysts developed in control medium than in G1/G2 (46%, 30%, respectively). Reducing or removing BSA during the initial culture period did not significantly reduce development to blastocyst (31%, 33%, respectively), although development was reduced in SOF with BSA removed from the final culture period (19%). There were no differences in development to the blastocyst stage between SOF, SOF with BSA removed during the initial culture period, and control (44%, 32%, 49%, respectively), but development was reduced in chemically defined protein-free medium throughout the culture period (21%). Krebs cycle activity did not differ between treatments; however, glycolysis was highest in the control embryos and lowest in embryos cultured in protein-free medium. Embryos that developed in the presence of serum appeared dark and granular and had elevated glycolytic rates compared to embryos developed in completely defined medium. This study shows that both metabolism and blastocyst development of embryos are altered by different culture media, implying a functional linkage between these two indicators of successful embryogenesis.     

Formulation and production of a blood-free and chemically defined virus production media for VERO cells

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.     

Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus

A chemically defined medium has been developed for anaerobic cultivation of hyaluronic-acid(HA)-producing Streptococcus zooepidemicus, which contains 11 amino acids essential to growth, and glutamine as a principal nitrogen source. The final HA concentration, the specific rate of HA production and HA-to-glucose yields were similar for growth in the chemically defined medium relative to growth in complex medium. Consequently cells cultivated on chemically defined medium can be expected to have similar activity regarding HA synthesis as compared to cells grown on complex media. However, the specific growth rate and volumetric HA production rate were found to be less favourable in the chemically defined media. Received: 4 October 1996 / Accepted: 25 October 1996     

<Emphasis Type="Italic">Xylella fastidiosa</Emphasis> Cultivation on a Minimal Solid Defined Medium

A simple defined solid medium containing citrate and succinate, three amino acids (L-glutamine, L-asparagine, and L-cysteine), hemin chloride, potato starch, gellan gum (GelRite), and mineral salts supported the growth of grape strains of Xylella fastidiosa, the bacterial pathogen that causes Pierces disease of grape. Isolation efficiency from infected grape plant samples, determined by the number of colony forming units recovered, on the defined medium was slightly less (10-fold) or indistinguishable from two standard rich media used for culturing X. fastidiosa, PWG and PD3, respectively. The bacterium also grew on media with citrate and L-glutamine as the only carbon and nitrogen sources. Potato starch was not essential for bacterial growth, but no growth was observed on media without hemin chloride. Agar inhibited bacterial growth when used as the gelling agent.     

Salivary enzymes are injected into xylem by the glassy-winged sharpshooter, a vector of Xylella fastidiosa

A few phytophagous hemipteran species such as the glassy-winged sharpshooter, Homalodisca vitripennis, (Germar), subsist entirely on xylem fluid. Although poorly understood, aspects of the insect's salivary physiology may facilitate both xylem-feeding and transmission of plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease of grape and other scorch diseases in many important crops. X. fastidiosa colonizes the anterior foregut (precibarium and cibarium) of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored in part by an exopolysaccharide (EPS) matrix that is reported to have a β-1,4-glucan backbone. Recently published evidence supports the following, salivation-egestion hypothesis for the inoculation of X. fastidiosa during vector feeding. The insect secretes saliva into the plant and then rapidly takes up a mixture of saliva and plant constituents. During turbulent fluid movements in the precibarium, the bacteria may become mechanically and enzymatically dislodged; the mixture is then egested back out through the stylets into plant cells, possibly including xylem vessels. The present study found that proteins extracted from dissected H. vitripennis salivary glands contain several enzyme activities capable of hydrolyzing glycosidic linkages in polysaccharides such as those found in EPS and plant cell walls, based on current information about the structures of those polysaccharides. One of these enzymes, a β-1,4-endoglucanase (EGase) was enriched in the salivary gland protein extract by subjecting the extract to a few, simple purification steps. The EGase-enriched extract was then used to generate a polyclonal antiserum that was used for immunohistochemical imaging of enzymes in sharpshooter salivary sheaths in grape. Results showed that enzyme-containing gelling saliva is injected into xylem vessels during sharpshooter feeding, in one case being carried by the transpiration stream away from the injection site. Thus, the present study provides support for the salivation-egestion hypothesis.     

Characterization of different cell culture media for expression of recombinant antibodies in mammalian cells: Presence of contaminating bovine antibodies

Several new cell culture media designed specifically for the expression of recombinant antibodies in Chinese hamster ovary (CHO) cells were investigated for the presence of bovine IgG. Three serum-free media, three protein-free (animal component free) media, as well as one chemically defined medium were included in the study. Employing a combination of affinity chromatography (Protein G or A columns), SDS-PAGE analysis, and peptide mass fingerprinting, two of the serum-free media were found to contain bovine IgG in the range of approximately 0.5 mg/L. The other five media did not contain detectable levels of contaminating Protein A or G-binding proteins such as bovine IgG.     

Application of competitive enzyme-linked immunosorbent assay for the quantification of imidacloprid titers in xylem fluid extracted from grapevines

A competitive enzyme-linked immunosorbent assay (ELISA) technique was evaluated for quantifying titers of imidacloprid in xylem fluid extracted from Vitis vinifera L. grapevines that were treated with systemic applications of the neonicotinoid insecticide Admire. Evidence of matrix effects, factors that compromise the precision and accuracy of the ELISA, was present in assays with undiluted xylem fluid. These effects could be eliminated by dilution of extracts in water, resulting in a lower sensitivity of the assay of 4 microg liter(-1). In a field trial conducted in a commercial vineyard, there was an excellent correlation between Admire application rates and xylem fluid concentrations of imidacloprid. At an Admire application rate of 1.17 liter ha(-1) (16 fl oz per acre), uptake of imidacloprid into vines was rapid. Imidacloprid was consistently detected in the xylem for up to 3 mo after application at concentrations known to be effective at managing populations of the sharpshooter Homalodisca coagulata Say, an important vector of Xylella fastidiosa Wells in California vineyards. The ELISA is a sensitive technique that can be used to study the behavior of systemic insecticides within crop systems and their impact on pest populations.     

Interactive effects of water stress and xylem-limited bacterial infection on the water relations of a host vine

Xylella fastidiosa, a xylem-limited bacterial pathogen that causes bacterial leaf scorch in its hosts, has a diverse and extensive host range among plant species worldwide. Previous work has shown that water stress enhances leaf scorch symptom severity and progression along the stem in Parthenocissus quinquefolia infected by X. fastidiosa. The objective here was to investigate the mechanisms underlying the interaction of water stress and infection by X. fastidiosa. Using the eastern deciduous forest vine, P. quinquefolia, infection and water availability were manipulated while measuring leaf water potentials (psi(L)), stomatal conductance (g(s)), whole shoot hydraulic conductance (K(h)), per cent xylem embolism, and xylem vessel dimensions. No significant differences in any of the physiological measurements were found between control and infected plants prior to drought. Drought treatment significantly reduced psi(L) and g(s) at all leaf positions throughout the day in late summer in both years of the study. In addition, infection significantly reduced psi(L) and g(s) in the most basal leaf positions in late summer in both years. Whole shoot hydraulic conductance was reduced by both low water and infection treatments. However, per cent embolized vessels and mean vessel diameter were affected by drought treatment only. These results imply that the major effect of infection by X. fastidiosa occurs due to reduced hydraulic conductance caused by clogging of the vessels, and not increased cavitation and embolism of xylem elements. The reduced K(h) caused by X. fastidiosa infection acts additively with the water limitation imposed by Drought stress.     

Effect of high molecular weight factors present in bovine oviduct-conditioned medium on in vitro bovine embryo development

To investigate the presence of embryotrophic factors in bovine oviduct-conditioned medium (BOCM), the high molecular weight fraction (> 10 KDa) from BOCM was added to 3 chemically defined embryo culture media (TCM199, DMEM/F12 and modified synthetic oviduct fluid [mSOF]). Zygotes were obtained by in vitro maturation and fertilization of oocytes. Conditioning of TCM199 with oviduct cells increased both cleavage to the 5- to 8-cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). The low molecular weight fraction (< 10 KDa) of BOCM maintained development to the 5- to 8-cell stage but did not allow development to the blastocyst stage. Adding the high molecular weight fraction to the inactive low molecular weight fraction restored bovine embryo development up to the blastocyst stage. This embryotrophic effect of the high molecular weight fraction was not observed when this fraction was added to TCM199 or DMEM/F12 medium. Whereas adding this fraction to mSOF medium significantly (P<0.05) increased embryo development up to the blastocyst stage (36%) in comparison with that of mSOF (15%) or BOCM (14%). These results show that BOCM contains high molecular weight factors promoting embryo development up to the blastocyst stage. Some chemically defined media mask the effect of these embryotrophic factors.     

Isolation of Ureolytic Peptostreptococcus productus from Feces Using Defined Medium; Failure of Common Urease Tests

Colony counts of fecal samples from three persons, obtained by using a chemically defined anaerobic roll-tube medium (containing glucose, maltose, glycerol, minerals, hemin, B-vitamins, methionine, volatile fatty acids, sulfide, bicarbonate, agar, carbon dioxide (gas phase), and 1 mM NH(4) (+) as main nitrogen source), averaged 60% of the 8.8 x 10(10) bacteria per g obtained when 0.2% Trypticase and 0.05% yeast extract were added to the otherwise identical medium. When 0.2% vitamin-free Casitone replaced Trypticase and yeast extract, counts were 94% those of the more complex medium. When urea-nitrogen was added to the defined medium as the main nitrogen source in place of NH(4) (+), counts of relatively large colonies averaged 1.0 x 10(9) per g of feces from five persons-1.1% of counts on the medium containing Trypticase and yeast extract. All of the organisms from the large colonies in the urea roll tubes were morphologically similar, and all six representative strains isolated were identified as urease-forming Peptostreptococcus productus, a species not previously known to produce urease. Ureolytic strains of Selenomonas ruminantium and P. productus were negative for urease activity in three assay media when inocula were from media containing complex nitrogen sources. The study documents that P. productus is the most numerous ureolytic species so far found in human feces and suggests that NH(4) (+) and more complex organic nitrogen sources strongly repress its production of urease. The study also indicates the efficacy of chemically defined media for direct selective isolation of nutritional groups of bacteria from feces.     

Two chemically defined media for the growth of Acholeplasma laidlawii strain A

A total of 11 tissue culture media and one synthetic medium were screened to determine whether they could support the growth of two strains of Acholeplasma. Two similar chemically defined media were found in which Acholeplasma laidlawii Strain A (Buchanan & Gibbons 1974) could grow. This is the first report of a fully defined medium for the growth of this organism (Greenaway 1981).     

Culture of endocrine pancreatic cells in protein-free,chemically defined media

Summary Cell suspensions prepared by collagenase digestion of pancreata obtained from 21.5-d-old rat fetuses were preincubated in RPMI medium containing 10% fetal bovine serum (FBS), to ensure cell adhesion. Twenty hours later, this medium was replaced by a chemically defined medium. Dulbecco's modified Eagle's (DME)-F12 was used alone or supplemented with various combinations of transferrin, sodium selenite, or Ultroser G. The evolution of the culture and the islet ultrastructure were similar in defined and serum-containing media. However, in the defined medium, the neoformed islets seemed less numerous, and the fibroblast layer less dense, when compared to the RPMI+10% FBS control medium. At Day 7, in defined media, the total insulin content per dish was half that of control cultures. None of the tested additives improved the yield of the cultures. The fractional insulin release per day was elevated in defined media. In subsequent incubations, glucose and leucine stimulated insulin release in a way characteristic of these cells of fetal origin. The labeling index of islet cells cultured in DME-F12 reached 10.7%, which is not far from that observed in RPMI+10% FBS. Such a defined medium is useful to study B cell physiology, avoiding the possible interaction of serum components with substances to be tested. The support of the Fonds National de la Recherche Scientifique and the ‘Loterie Nationale’ of Belgium is also acknowledged.     

Nutritional requirements of Xylella fastidiosa, which causes Pierce's disease in grapes

A defined medium (XF-26) containing 3 inorganic salts, 2 tricarboxylic acids, 17 amino acids, potato starch, phenol red, and agar was used as the starting point for the study. Deletions of one or more ingredients were performed to prepare various media. A medium was considered able to support growth of Xylella fastidiosa strains responsible for Pierce's disease in grapes, only after 10 serial passages had been completed. Of 3 inorganic salts, K2HPO4 and MgSO4 x 7H2O were essential, and (NH4)2HPO4 was nonessential for growth. Of the Krebs cycle intermediates, all (citrate, alpha-ketoglutarate, succinate, fumarate, malate, and oxaloacetate) but isocitrate supported growth of cultivated strains, whereas only citrate alone or citrate plus succinate supported the primary isolation of PD bacterium. Of 17 amino acids, 6 uncharged polar R groups (asparagine, cysteine, glutamine, glycine, serine, and threonine) supported growth, whereas 8 nonpolar R groups (alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine) or 3 positively charged polar groups (arginine, histidine, and lysine) did not. Starch proved to be nonessential.     

Birth of common marmoset (Callithrix jacchus) offspring derived from in vitro-matured oocytes in chemically defined medium

Optimization of oocyte culture conditions is a crucial aspect of reproductive biology and technology. In the present study, maturation of germinal vesicle-stage marmoset oocytes were evaluated in the following media: Waymouth medium, Waymouth medium containing porcine follicular fluid (pFF) (Waymouth-pFF medium), and porcine oocyte medium (POM). Oocytes cultured in Waymouth-pFF medium had higher maturation rates to the metaphase II stage than those cultured in Waymouth medium (36.1% vs. 24.8%, respectively, P < 0.05), indicating the suitability of this medium for culturing marmoset oocytes. Hence, maturation of marmoset oocytes cultured in POM was subsequently evaluated. The rate of maturation to the metaphase I stage was significantly higher and degradation rates were significantly lower in oocytes cultured in POM than those cultured in Waymouth medium. In addition, three offspring were successfully obtained after transfer of embryos matured in chemically defined medium. Therefore, we concluded that POM was suitable for marmoset oocyte culture. Furthermore, this was apparently the first report of marmoset offspring derived from oocytes cultured in chemically defined medium.