A new method for the identification of phytoplasmas in strawberries (Fragaria fragaria)

Motivation:Phytoplasmas are plant pathogens that generally inhabit the phloem and are transmitted from plant to plant by vector insects that feed on phloem. Their genome is small, with a high content of genes (1). The presence of extrachromosomal DNA has also been detected (2). Serological and molecular studies have shown that phytoplasmas contain a gene encoding a membrane protein and this is unique for each specie and helps to identify what type of phytoplasma is, according to its taxonomic classification (3). Different phytoplasmas had been detected and identified in strawberries plants (Fragaria fragaria). Symptoms observed on strawberries were yellow coloration of leaves, plant stunting, and reduced leaf size and the virescence were observed in the inflorescence. PCR analyses as well as sequencing of 16S ribosomal gene enabled the identification of phytoplasmas belonging to two ribosomal groups, namely stolbur and aster yellows, but the eficiency for this detection is not reliable. Here we are working to develop a new method to easily detect and identify this pathogen in plant samples before symptoms appear.Methods: 70 samples of strawberries infected with phytoplasma were tested, by,analysing leaves, crown and root in the strawberry plant to determine the most optimal zone to be sampled. The identification of phytoplasmas required a double PCRs  using phytoplasma universal primer pair P1/P7, followed by nested PCR with Fu5/Ru3 primers. Further nested PCR reactions on R16mF2/R1 amplicons were also performed with R16F2N/R2 primer pair, specific for phytoplasma belonging to aster yellow to determine their class. Asymptomatic samples were also analysed as negative controls in each PCR reaction. The amplified DNA was sequenced and the sequence analysed by Blast.Results:With this PCR method, the presence of phytoplasma was mainly detected in strawberries roots but not in leaves and crowns. This technique allowed us to detect presence of high amount of phytoplasma because a positive result in the first PCR, low amount with a positive result only in the second one, or no presence because negative result in both. Finally,, the sequencing of the amplified DNA, allowed us to determine if they belong to aster yellow or stolbur because they DNA identity.Conclusions: This new phytoplasma identification method is effective. It would be interesting to obtain a specific primer for each specie of phytoplasma and thus have a more precise method

The role of Doppler ultrasonography in vascular access surveillance-controversies continue

Chronic hemodialysis therapy required regular entry into the patient's blood stream with adequate flow. The use of arteriovenous fistulas and grafts is linked with lower morbidity and mortality than the use of catheters. However, these types of accesses are frequently affected by stenoses, which decrease the flow and lead to both inadequate dialysis and access thrombosis. The idea of duplex Doppler ultrasound surveillance is based on the presumption that in-time diagnosis of an asymptomatic significant stenosis and its treatment prolongs access patency. Details of performed trials are conflicting, and current guidelines do not support ultrasound surveillance. This review article summarizes the trials performed and focuses on the reasons of conflicting results. We stress the need of precise standardized criteria of significant access stenosis and the weakness of the metaanalyses performed.Nephrolog

The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine

Aims: The objective of this study was to investigate the effects of free molecular and bound forms of sulphur dioxide and oxygen on the viability and culturability of a selected strain of Acetobacter pasteurianus and a selected strain of Brettanomyces bruxellensis in wine. Methods and Results: Acetic acid bacteria and Brettanomyces/Dekkera yeasts associated with wine spoilage were isolated from bottled commercial red wines. One bacterium, A. pasteurianus strain A8, and one yeast, B. bruxellensis strain B3a, were selected for further study. The resistance to sulphur dioxide and the effect of oxygen addition on these two selected strains were determined by using plating and epifluorescence techniques for monitoring cell viability in wine. Acetobacter pasteurianus A8 was more resistant to sulphur dioxide than B. bruxellensis B3a, with the latter being rapidly affected by a short exposure time to free molecular form of sulphur dioxide. As expected, neither of these microbial strains was affected by the bound form of sulphur dioxide. The addition of oxygen negated the difference observed between plate and epifluorescence counts for A. pasteurianus A8 during storage, while it stimulated growth of B. bruxellensis B3a. Conclusions: Acetobacter pasteurianus A8 can survive under anaerobic conditions in wine in the presence of sulphur dioxide. Brettanomyces bruxellensis B3a is more sensitive to sulphur dioxide than A. pasteurianus A8, but can grow in the presence of oxygen. Care should be taken to exclude oxygen from contact with wine when it is being transferred or moved. Significance and Impact of the Study: Wine spoilage can be avoided by preventing growth of undesirable acetic acid bacteria and Brettanomyces/Dekkera yeasts through the effective use of sulphur dioxide and the management of oxygen throughout the winemaking process. © 2005 The Society for Applied Microbiology.Articl