Effect of oxygen on the growth and biofilm formation of Xylella fastidiosa in liquid media.

Xylella fastidiosa is a xylem-limited bacterial pathogen, and is the causative agent of Pierce's disease of grapevines and scorch diseases of many other plant species. The disease symptoms are putatively due to blocking of the transpiration stream by bacterial-induced biofilm formation and/or by the formation of plant-generated tylosis. Xylella fastidiosa has been classified as an obligate aerobe, which appears unusual given that dissolved O2 levels in the xylem during the growing season are often hypoxic (20-60 µmol L(-1)). We examined the growth and biofilm formation of three strains of X. fastidiosa under variable O2 conditions (21, 2.1, 0.21 and 0 % O2), in comparison to that of Pseudomonas syringae (obligate aerobe) and Erwinia carotovora (facultative anaerobe) under similar conditions. The growth of X. fastidiosa more closely resembled that of the facultative anaerobe, and not the obligate aerobe. Xanthomonas campestris, the closest genetic relative of X. fastidiosa, exhibited no growth in an N2 environment, whereas X. fastidiosa was capable of growing in an N2 environment in PW(+), CHARDS, and XDM2-PR media. The magnitude of growth and biofilm formation in the N2 (0 % O2) treatment was dependent on the specific medium. Additional studies involving the metabolism of X. fastidiosa in response to low O2 are warranted. Whether X. fastidiosa is classified as an obligate aerobe or a facultative anaerobe should be confirmed by gene activation and/or the quantification of the metabolic profiles under hypoxic conditions.

Nitrification and denitrification gene abundances in swine wastewater anaerobic lagoons.

Although anaerobic lagoons are used globally for livestock waste treatment, their detailed microbial cycling ofN is only beginning to become understood. Within this cycling, nitrification can be performed by organisms that produce the enzyme ammonia monooxygenase. For denitrification, the reduction of nitrite to nitric oxide can be catalyzed by two forms of nitrite reductases, and N,O can be reduced by nitrous oxide reductase encoded by the gene nosZ The objectives of this investigation were to (i) quantify the abundance of the amoA, nirK, nirS, and nosZ genes; (ii) evaluate the influence of environmental conditions on their abundances; and (iii) evaluate their abundance relative to denitrification enzyme activity (DEA). Samples were analyzed via real-time quantitative polymerase chain reaction and collected from eight typical, commercial anaerobic, swine wastewater lagoons located in the Carolinas. The four genes assayed in this study were present in all eight lagoons. Their abundances relative to total bacterial populations were 0.04% (amoA), 1.33% (nirS), 5.29% (nirK), and 0.27% (nosZ). When compared with lagoon chemical characteristics, amoA and nirK correlated with several measured variables. Neither nirS nor nosZ correlated with any measured environmental variables. Although no gene measured in this study correlated with actual or potential DEA, nosZ copy numbers did correlate with the disparity between actual and potential DEA. Phylogenetic analysis ofnosZdid not reveal any correlations to DEA rates. As with other investigations, analyses of these genes provide useful insight while revealing the underlying greater complexity of N cycling within swine waste lagoons.

Characterization of a microbial community capable of nitrification at cold temperature.

While the oxidation of ammonia is an integral component of advanced aerobic livestock wastewater treatment, the rate of nitrification by ammonia-oxidizing bacteria is drastically reduced at colder temperatures. In this study we report an acclimated lagoon nitrifying sludge that is capable of high rates of nitrification at temperatures from 5 degrees C (11.2mg N/g MLVSS/h) to 20 degrees C (40.4 mg N/g MLVSS/h). The composition of the microbial community present in the nitrifying sludge was investigated by partial 16S rRNA gene sequencing. After DNA extraction and the creation of a plasmid library, 153 partial length 16S rRNA gene clones were sequenced and analyzed phylogenetically. Over 80% of these clones were affiliated with the Proteobacteria, and grouped with the beta- (114 clones), gamma- (7 clones), and alpha-classes (2 clones). The remaining clones were affiliated with the Acidobacteria (1 clone), Actinobacteria (8 clones), Bacteroidetes (16 clones), and Verrucomicrobia (5 clones). The majority of the clones belonged to the genus Nitrosomonas, while other clones affiliated with microorganisms previously identified as having floc forming or psychrotolerance characteristics.

Influence of xylem fluid chemistry on planktonic growth, biofilm formation and aggregation of Xylella fastidiosa.

Xylella fastidiosa is the causal agent of Pierce's disease in grapevines. The mechanisms of pathogenicity are largely due to occlusion of xylem vessels by aggregation of X. fastidiosa and biofilm formation. Xylella fastidiosa was subjected to xylem fluids with varying chemistries to examine the effects of nutritional components on bacterial growth in vitro. The exposure of X. fastidiosa to xylem fluids collected from different Vitis genotypes resulted in highly significant differences in both planktonic growth and biofilm formation. Planktonic growth of X. fastidiosa in Vitis xylem fluid was correlated to the concentration of citric acid, amino acids (glutamic acid, glutamine, histidine, valine, methionine, isoleucine and phenylalanine) and inorganic ions (copper, magnesium, phosphorus and zinc). Biofilm formation was correlated to many amino acids at 1 h of incubation. Xylem fluid from Vitis rotundifolia cv. Noble (fluid that supported low planktonic growth) was supplemented with the compounds that were correlated above to levels found in Vitis champinii cv. Ramsey (fluid that supported high planktonic growth) to determine the direct impact of xylem constituents on the growth characteristics of X. fastidiosa. Augmentation of fluid from Noble with the amino acids listed above, citric acid, calcium and magnesium resulted in increased planktonic growth and aggregation.