Environmental monitoring of the biocontrol agent Pantoea agglomerans CPA‐2 applied to citrus fruit at preharvest

The aim of this study was to evaluate the environmental fate and behaviour of the biocontrol product based on Pantoea agglomerans CPA‐2 cells after its release into the field. Three different methods, dilution plating, quantitative real‐time PCR (qPCR) and propidium monoazide combined with qPCR (PMA‐qPCR) were used to track the CPA‐2 population. Populations quantified by qPCR were statistically different compared with those by the PMA‐qPCR and dilution plating methods. In addition, the spray dispersion of the CPA‐2 treatment was evaluated using water‐sensitive papers. A lack of dispersion of the CPA‐2 treatment was observed at distances of 2.5 ± 0.5 m. The presence and persistence of CPA‐2 in the environment were monitored in different areas, by dilution plating and confirmed by conventional PCR. The results showed that CPA‐2 can survive but not proliferate on weed and leaves. The persistence on inert surfaces, such as motorised backpack sprayer, gloves and working clothes, was less than 7 days. In conclusion, PMA‐qPCR was a potential tool for fast and specific monitoring of viable populations of CPA‐2 and it gave valuable information on population behaviour. Moreover, the results demonstrate that CPA‐2 would not present a risk for the environment because of its low establishment, survival and dispersion.     

Biodegradation of naphthalene by enriched marine denitrifying bacteria

Numerous studies have been investigated on the PAHs biodegradation in aerobic and anaerobic environments; however, the biodegradation of PAHs under anoxic conditions, especially denitrifying conditions, has drawn less attention. In this study, four series of batch experiments were conducted to investigate the effect of temperature, pH, naphthalene concentration and nitrate concentration on the naphthalene degradation under denitrification condition. Our results showed that the degradation of naphthalene was most favorable at pH 7 and 25 °C. Results also indicated that 30 mg/l naphthalene inhibited the biodegradation and the removal efficiency was only 20.2%. Significant degradation (91.7% and 96.3%) of naphthalene occurred when nitrate concentrations were 1.0 and 5.0 mM. Moreover, the maximum degradation rates were 0.13 and 0.18 mg-NAP/(l h) depending on the concentration of nitrate. Based on 16S rDNA analysis, the denitrifying enriched culture was mainly composed of ??-Proteobacteria (19 clones out of a total of 23 clones) and Actinobacteria (4 clones). Using a primer set specific for naphthalene degrading functional gene nahAc, two operational taxonomy units were obtained in the clone library of nahAc. Both of them were closely related to nahAc genes of known species of Pseudomonas. Quantitative polymerase chain reaction (qPCR) was employed to quantify the change of naphthalene-degrading population during the degradation of naphthalene using nahAc gene as the biomarker. The maximum degradation rate and removal efficiency were strongly correlated with nahAc gene copy number, with R² of 0.69 and 0.79, respectively.     

The presence of atrazine and atrazine-degrading bacteria in the residential, cattle farming, forested and golf course regions of Lake Oconee

Aims: To assess the concentration of atrazine in Lake Oconee and develop a qPCR assay as a potential marker for the presence of atrazine‐degrading bacteria indicating atrazine contamination. Methods and Results: Water and sediment samples were collected from the Oconee Lake at four golf course sites, two residential sites, one cattle farming site and a forested site. Atrazine concentration at the study sites was determined using an ELISA kit and indicated the presence of atrazine from 0·72 ppb at the forested sites to 1·84 ppb at the golf course sites. QPCR results indicate the presence of atzA gene (atrazine chlorohydrolase) from 1·51 × 10² gene copies at the residential sites to 3·31 × 10⁵ gene copies per 100 ml of water at the golf course regions of the lake and correlated (r = 0·64) with atrazine concentration. Sediment samples had higher atzA gene copies compared with the water samples (P < 0·05). Conclusions: Atrazine concentration and the highest quantity of atzA gene were detected in the golf course regions of the lake. Overall, atrazine concentration monitored in Lake Oconee was below the Environment Protection Agency (EPA) regulatory standards. Significance and Impact of the Study: Quantitative PCR is an efficient technique for assessing the presence of atrazine catabolism gene as a functional marker for atrazine‐degrading bacteria and the presence of atrazine contamination.     

An international trial of quantitative PCR for monitoring Legionella in artificial water systems

Aims: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. Methods and Results: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l^?1) and culture (CFU l^?1) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l^?1 always exceeded CFU l^?1, and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. Conclusions: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. Significance and Impact of the Study: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.     

Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.