A novel feature for monitoring the enzymatic harvesting process of adherent cell cultures based on lens-free imaging.

Adherent cell cultures are often dissociated from their culture vessel (and each other) through enzymatic harvesting, where the detachment response is monitored by an operator. However, this approach is lacking standardisation and reproducibility, and prolonged exposure or too high concentrations can affect the cell's viability and differentiation potential. Quantitative monitoring systems are required to characterise the cell detachment response and objectively determine the optimal time-point to inhibit the enzymatic reaction. State-of-the-art methodologies rely on bulky imaging systems and/or features (e.g. circularity) that lack robustness. In this study, lens-free imaging (LFI) technology was used to develop a novel cell detachment feature. Seven different donors were cultured and subsequently harvested with a (diluted) enzymatic harvesting solution after 3, 5 and 7 days of culture. Cell detachment was captured with the LFI set-up over a period of 20 min (every 20 s) and by optimising the reconstruction of the LFI intensity images, a new feature could be identified. Bright regions in the intensity image were identified as detaching cells and using image analysis, a method was developed to automatically extract this feature, defined as the percentage of detached cell regions. Next, the method was quantitatively and qualitatively validated on a diverse set of images. Average absolute error values of 1.49%, 1.34% and 1.97% were obtained for medium to high density and overconfluent cultures, respectively. The detachment response was quantified for all conditions and the optimal time for enzyme inhibition was reached when approximately 92.5% of the cells were detached. On average, inhibition times of 9.6-11.1 and 16.2-17.2 min were obtained for medium to high density and overconfluent cultures, respectively. In general, overconfluent cultures detached much slower, while their detachment rate was also decreased by the diluted harvesting solution. Moreover, several donors exhibited similar trends in cell detachment behaviour, with two clear outliers. Using the novel feature, measurements can be performed with an increased robustness, while the compact LFI design could pave the way for in situ monitoring in a variety of culture vessels, including bioreactors.

The in planta proteome of wild type strains of the fire blight pathogen, Erwinia amylovora.

UNLABELLED: Erwinia amylovora is a Gram-negative plant pathogen that causes fire blight. This disease affects most members of the Rosaceae family including apple and pear. Here, an infection model is introduced to study proteomic changes in a highly virulent E. amylovora strain upon interaction with its host as compared to a lower virulent strain. For this purpose separate shoots of apple rootstocks were wound-infected and when infection became systemic, bacterial cells were isolated and processed for analysis in a proteomics platform combining 2-D fluorescence difference gel electrophoresis and mass spectrometry. Comparing the proteome of the isolates, significant abundance changes were observed in proteins involved in sorbitol metabolism, amylovoran production as well as in protection against plant defense mechanisms. Furthermore several proteins associated with virulence were more abundant in the higher virulent strain. Changes at the proteome level showed good accordance at the transcript level, as was verified by RT-qPCR. In conclusion, this infection model may be a valuable tool to unravel the complexity of plant-pathogen interactions and to gain insight in the molecular mechanisms associated with virulence of E. amylovora, paving the way for the development of plant-protective interventions against this detrimental disease. SIGNIFICANCE: During this research a first time investigation was performed on the proteome of E. amylovora, grown inside a susceptible host plant. This bacterium is the causal agent of fire blight, which can affect most members of the Rosaceae family including apple and pear. To do so, an artificial infection model on shoots of apple rootstocks was optimized and employed. When infection was systemic, bacterial cells were extracted from the plant tissue followed by extraction of the proteins from the bacteria. Further processing of the proteins was done by using a 2-D fluorescence difference gel electrophoresis analysis followed by mass spectrometry. By the use of two strains differing in their virulent ability, we were able to draw conclusions concerning virulence and behavior of different strains inside the host. This research provides a model to investigate plant-pathogen interactions and more importantly, we identified possible new targets for the development of novel control methods against this devastating disease.

A comparative proteome analysis reveals flagellin, chemotaxis regulated proteins and amylovoran to be involved in virulence differences between Erwinia amylovora strains.

Erwinia amylovora is a Gram-negative bacterium that causes the destructive disease fire blight affecting most members of the Rosaceae family, of which apple and pear are economically the most important hosts. E. amylovora has been considered as a homogeneous species in whole, although significant differences in virulence patterns have been observed. However, the underlying causes of the differences in virulence remain to be discovered. In a first-time comparative proteomic approach using E. amylovora, 2D differential in-gel electrophoresis (DIGE) was used to identify proteins that could explain the gradual difference in virulence between four different strains. Two important proteins were identified, FliC and CheY, both involved in flagella structure, motility and chemotaxis, which were more abundant in the least virulent strain. In the highly virulent strains the protein GalF, involved in amylovoran production, was more abundant, which was consistent with the higher expression of the gene and the higher amylovoran content in this strain in vitro. Together, these results confirm the involvement of amylovoran in virulence, but also imply an indirect role of flagellin in virulence as elicitor of plant defence. BIOLOGICAL SIGNIFICANCE: This research provides new insights into our current understanding of the virulence of Erwinia amylovora. This plant-pathogen is considered a homogeneous species although different strains show differences in virulence. Despite the efforts made on the genomic level which resulted in the discovery of virulence factors, the reason for the different virulence patterns between strains has not yet been identified. In our lab we used a comparative proteomic approach, which has never been published before, to identify proteins involved in these differences between strains and hereby possibly involved in virulence. Our results provide interesting insights in virulence and present us with the opportunity to glance into the proteome of E. amylovora.

Erwinia amylovora affects the phenylpropanoid-flavonoid pathway in mature leaves of Pyrus communis cv. Conférence.

Flavonoids, which are synthesized by the phenylpropanoid-flavonoid pathway, not only contribute to fruit colour and photoprotection, they also may provide antimicrobial and structural components during interaction with micro-organisms. A possible response of this pathway was assessed in both mature and immature leaves of shoots of 2-year-old pear trees cv. Conférence, which were inoculated with the gram-negative bacterium Erwinia amylovora strain SGB 225/12, were mock-inoculated or were left untreated. The phenylpropanoid-flavonoid pathway was analysed by histological studies, by gene expression using RT-qPCR and by HPLC analyses of the metabolites at different time intervals after infection. Transcription patterns of two key genes anthocyanidin reductase (ANR) and chalcone synthase (CHS) related to the phenylpropanoid-flavonoid pathway showed differences between control, mock-inoculated and E. amylovora-inoculated mature leaves, with the strongest reaction 48 h after inoculation. The impact of E. amylovora was also visualised in histological sections, and confirmed by HPLC, as epicatechin -which is produced via ANR- augmented 72 h after inoculation in infected leaf tissue. Besides the effect of treatments, ontogenesis-related differences were found as well. The increase of certain key genes, the rise in epicatechin and the visualisation in several histological sections in this study suggest a non-negligible impact on the phenylpropanoid-flavonoid pathway in Pyrus communis due to inoculation with E. amylovora. In this study, we propose a potential role of this pathway in defence mechanisms, providing a detailed analysis of the response of this system attributable to inoculation with E. amylovora.

Pathogenicity and infection strategies of the fire blight pathogen Erwinia amylovora in Rosaceae: state of the art.

Plants are host to a large amount of pathogenic bacteria. Fire blight, caused by the bacterium Erwinia amylovora, is an important disease in Rosaceae. Pathogenicity of E. amylovora is greatly influenced by the production of exopolysaccharides, such as amylovoran, and the use of the type III secretion system, which enables bacteria to penetrate host tissue and cause disease. When infection takes place, plants have to rely on the ability of each cell to recognize the pathogen and the signals emanating from the infection site in order to generate several defence mechanisms. These mechanisms consist of physical barriers and the production of antimicrobial components, both in a preformed and an inducible manner. Inducible defence responses are activated upon the recognition of elicitor molecules by plant cell receptors, either derived from invading micro-organisms or from pathogen-induced degradation of plant tissue. This recognition event triggers a signal transduction cascade, leading to a range of defence responses [reactive oxygen species (ROS), plant hormones, secondary metabolites, …] and redeployment of cellular energy in a fast, efficient and multiresponsive manner, which prevents further pathogen ingress. This review highlights the research that has been performed during recent years regarding this specific plant-pathogen interaction between Erwinia amylovora and Rosaceae, with a special emphasis on the pathogenicity and the infection strategy of E. amylovora and the possible defence mechanisms of the plant against this disease.

Preliminary results on the ability of pentatomidae to transfer fire blight Erwinia amylovora under controlled conditions.

With their piercing-sucking mouthparts stink bugs (Heteroptera: Pentatomidae), a major pest in especially organic orchards, create wounds in fruit of pear trees. As Erwinia amylovora (Burrill, Winslow et al.), a wide spread bacterial disease affecting many rosaceous plants including pome fruit trees and hawthorn, enters through openings in flowers, leaves, shoots and fruit, feeding punctures caused by these bugs might be inoculated with Erwinia bacteria. In order to investigate the ability of the bugs Pentotoma rufipes L. and Polomena prasina L. to transmit fire blight, insects were caught in an organically managed orchard without fire blight, brought into contact with artificially inoculated immature pear fruit/slices and transferred to healthy, mechanically wounded pear fruit/slices. After an incubation period potential transmission of bacteria was examined by evaluation of symptom expression (necrosis, ooze production). To assess the presence of bacteria on the exoskeleton of the tested bugs, all bugs were forced to walk on a semiselective nutrient agar medium. In another experiment the viability of Ea on the exoskeleton was tested -after previous contact with ooze- through washing and plating of the wash water. All experiments were conducted under optimal climatological conditions and according to quarantine standards. Results demonstrated the ability of stink bugs to transfer E. amylovora to fruit and the viability of bacteria on stink bugs externally - both under lab conditions.

Reduction in bacterial ooze formation on immature fruitlets after preventive treatments of Fosethyl-Al against fire blight Erwinia amylovora.

Fire blight, caused by the bacterium Erwinia amylovora (Burill Winslow et al.), is a very important bacterial disease on apple and pear orchards with devastating effects in some production area and in some years. Fire blight control consists in a whole strategy of measures that should start with control measures in and around the fruit tree nurseries. Only the use of Vacciplant (Laminarin), an inducer of the self-defence mechanism, is registered in Belgium since 2009. In other European countries Fosethyl-Al has been registered for fire blight control. Recently, research trials have been done at Pcfruit research station for several years on the activity of ALiette (fosethyl-Al) against fire blight. Fosethyl-Al, also a plant defence enhancing molecule, applied preventively 3 times at a dose of 3.75 kg/ha standard orchard (3 x 3000 g a.i./ha standard orchard), showed a reduction in the host susceptibility and decreased the disease development on artificial inoculated flower clusters and shoots. Also a clear reduction in the ooze droplet formation on artificially inoculated immature fruitlets has been observed with this molecule. This reduction in the bacterial ooze formation is considered as a very important factor in the spread of the disease in the orchard.

Bacteriocin Serratine-P as a biological tool in the control of fire blight Erwinia amylovora.

Fire blight, caused by the bacterium Erwinia amylovora (Burill Winslow et al.), is the most important bacterial disease in European pear growing. It can cause a lot of damage in some countries on apple and on pear trees in orchards and also in the fruit tree nurseries. In Belgium, the disease is present since 1972. Control of fire blight in Belgian fruit orchards is made on a broad basis of measurements in and around the fruit trees. The use of an antibiotic is allowed for application only during the primary blossom period under strict controlled regulations. The use of antobiotics in agriculture is strongly discussed on the European level today and will probably disappear in the near future. Therefore, the research on fire blight control concentrates on the possibilities of biological control with antagonistic bacteria such as Pantoea agglomerans (Erwinia herbicola), Bacillus subtilis or Pseudomonas syringae strain A 506. The use of Serratine-P, a phage tail-like bacteriocin, produced by Serratia plymiticum, shows an interesting antibacterial activity against Erwinia amylovora. Its mode of action consists in the perforation of the cytoplasmic membrane of the target cell, inducing perturbations in cellular exchanges and a final lysis of the bacterial cell. In this paper some trials are discussed on the use of Serratine-P at different doses and on different infection types on pear trees. The results indicate interesting protection possibilities on blossom- and fruit infections.