Low temperature and anhydrous electron microscopy techniques to observe the infection process of the bacterial pathogen Xanthomonas fragariae on strawberry leaves.

Preserving the structural arrangement of the components of a bacterial infection process within a plant for microscopy study is a technical challenge because of the different requirements of each component for optimal preservation and visualization. We used low temperature scanning electron microscopy (cryo-SEM), anhydrous fixation at ambient temperature and freeze-substitution for transmission electron microscopy to examine fractured and sectioned strawberry leaves infected with Xanthomonas fragariae. Cryo-SEM images of fractured samples showed the bacterial colonization of mesophyll air spaces in the leaf, limited by the vascular bundles and the orientation and packing of bacteria in extracellular polysaccharide. Transmission electron microscopy of samples fixed using osmium tetroxide dissolved in FC-72 solvent at ambient temperature showed that the entire plant/bacteria/extracellular polysaccharide system was preserved in situ, and showed plasmolysis of mesophyll cells and disruption of organelles. In freeze-substitution samples, osmium tetroxide in FC-72 solvent gave superior preservation of the extracellular polysaccharide as compared to a conventional cocktail. In addition, strands believed to be xanthan were preferentially contrasted to show their density and orientation around the bacterial cells. We conclude that anhydrous fixation using osmium tetroxide in FC-72 at ambient temperature gave the best preservation of the entire system, and freeze-substitution using this same fixative enhanced the visualization of strands in the biofilm.

Interaction of ozone and negative air ions to control micro-organisms.

AIMS: The aims of this study were to investigate the effect of ozone and/or negative air ions (NAI) on the viability of bacteria. METHODS AND RESULTS: Dilute cell suspensions of Pseudomonas fluorescens, Erwinia carotovora pv. carotovora and Escherichia coli were inoculated onto agar and subsequently exposed to ozone and/or NAI. Ozone concentration was maintained at 100 +/- 5 nl l-1 and NAI at 106 ml-1. When exposed to a combination of ozone and NAI, viability among all three bacterial species decreased more rapidly when they were inoculated onto potato dextrose agar (PDA) than onto nutrient agar (NA). A subsequent test examined the effect of ozone and NAI alone or in combination on the bacteria inoculated onto PDA only. Treatment with NAI alone had no killing effect on any of the bacterial species. However, a strong interaction between ozone and NAI was observed. Pseudomonas fluorescens was most susceptible to the combined treatment. Cell viability was reduced to 0.7% after 6 h, while 76% of the cells remained viable when exposed to ozone alone. Viability of Erwinia carotovora pv. carotovora was reduced to 4% after 6 h in the combined treatment compared with 69% when exposed to ozone alone. Escherichia coli was relatively more resistant to the combined treatment; viability was reduced to 40% after 11 h compared with 70% in the ozone alone treatment. CONCLUSIONS: A strong synergism between ozone and NAI on bacterial cell death was found, but the degree of this effect varied depending on bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergism of ozone with NAI may provide an effective method of reducing food-borne disease and decay of fresh produce.

Evidence and characterization of a gene cluster required for the production of viscosin, a lipopeptide biosurfactant, by a strain of Pseudomonas fluorescens.

The genetic control of viscosin production was examined in a strain of Pseudomonas fluorescens (PfA7B) that causes broccoli head rot. Viscosin is a potent lipopeptide biosurfactant that enables the bacteria to come into intimate contact with the difficult-to-wet waxy heads of broccoli. Tn5 mutagenesis completely disrupted viscosin production as shown by HPLC analysis of the mutagenized cell lysates. The Vis- mutants retained their pectolytic capability and were able to decay potato slices. On broccoli, however, the Vis- mutants caused decay of wounded florets, but the decay failed to spread to adjacent nonwounded florets as had occurred with the wild-type PfA7B. Triparental matings of the Vis- mutants with their corresponding wild-type clones and the helper Escherichia coli HB101 carrying the mobilization plasmid pPK2013 resulted in three stable viscosin-producing transconjugants that caused typical decay of broccoli tissue. Linkage maps of clones and protein profiles showed that a 25-kb chromosomal DNA region of PfA7B affected the production of three high molecular mass proteins required for viscosin synthesis. These proteins, approximately 218, 215, and 137 kDa in size, likely compose a synthetase complex that assembles the nine amino acid peptide of viscosin and subsequently attaches this to the hydrophobic fatty acid component of the molecule. A probe made from this DNA region hybridized with DNA fragments of other phytopathogenic pseudomonads to varying degrees.