Whole cell fatty acid analysis as a tool for classification of phytopathogenic pseudomonas bacteria

In this thesis some members of the plant pathogenic bacterial genus Pseudomonas have been studied. Conventional morphological, biochemical, physiological and pathogenitcity tests as well as a 'finger-print' technique, viz. automated whole cell fatty acid analysis, were used. The taxonomy of the plant pathogenic Pseudomonas bacteria is in many cases unsettled. Not in the last place this is because plant pathogenicity is difficult to assess and its value in taxonomy is differently estimated. The genus Pseudomonas has been subdivided on the basis of DNA-r(ibosomal) RNA homology studies into four rRNA groups. Fluorescent plant pathogens are found in group I, non-fluorescent species in group II (Chapter 1.2.).Whole cell fatty acid analysis has been found very useful in the classification of phytopathogenic bacteria. Especially the development of the Microbial Identification System (MIS) of Microbial ID. Inc. Newark, USA enables rapid and reliable determination of fatty acid patterns and has enhanced its use in bacterial taxonomy (Chapter 1.3.). The MIS has been used in all my studies on fatty acids of plant pathogens.At first a bacterium was studied which was well known in our laboratory, viz. the fluorescent P.syringae subsp. savastanoi, causing excrescences on Oleaceae and Nerium oleander. Based on pathogenicity, host range and plant hormone production three pathogenic varieties of this bacterium could be distinguished (Chapter II.1.). With fatty acid analysis (FAA), the three pathovars could also be distinguished, leading to the notion that pathogenicity of primary plant pathogens is not just one phenotypic factor, but it is also reflected in the bacterial membranes, where most fatty acids are present (Chapter II.2.).Subsequently the important non-fluorescent plant pathogen P.solanacearum was investigated. From this bacterium, which causes a devastating vascular disease on many different food crop plants, both biochemical and pathogenic varieties have been described. The occurrence of biochemical varieties could not be confirmed by FAA. Apparently differences in the ability to metabolize a few carbon compounds have no effect on fatty acid composition. As was the case with P.s. subsp. savastanoi, pathogenic varieties of P. solanacearum could be discriminated by FAA. Fatty acid patters of P. solanacearum were also studied in relation to those of other members of rRNA-group II, such as P.cepacia , P. gladioli , P. caryophylli and P. pickettii. The taxonomic patterns found were in good congruence with those determined in DNA-DNA homology studies by other authors. This once more confirms that FAA is a powerful additional tool in the classification of bacteria (Chapter II.3.).Finally the very complex group of the fluorescent, oxidase positive soft rot Pseudomonas bacteria was studied. These bacteria are opportunistic plant pathogens, especially important in post harvest situations. They have been found to be biochemically indistinguishable from saprophytic pseudomonads such as P.fluorescens biovars, P.putida and P.chlororaphis (incl. P.aureofaciens). On the basis of their ability to hydrolyze pectin and to cause soft rot, they have been named P. marginalis. In this study soft rot strains were biochemically similar to biovars of P. fluorescens or intermediates of these biovars, unknown forms of P. fluorescens, or similar to P. putida and P.chlororaphis. With FAA, oxidase positive sof rot pseudomonads were all found in a heterogeneous super cluster with saprophytic strains biochemically identified as P. fluorescens biovars or intermediates, P.chlororaphis and P.putida.Therefore it is suggested to abandon the use of ' P.marginalis ' and to name oxidase positive fluorescent soft rot bacteria ' P. fluorescens ', with some additional information between brackets, e.g. P.fluorescens (pectolytic, soft rot strain).P. aeruginosa strains from plants, animals and men were found in a very homogeneous cluster, well separated from the P.fluorescens supercluster. A supposed plant pathogenicity of P.aeruginosa could not be confirmed. This bacterium can multiply and cause some necrotic action only occasionally on plant material under special (unknown) circumstances. Certain non-pectolytic, non-soft rot strains of P. fluorescens are described which cause bacterial stripe symptoms on Iris sp. The pathogenicity factors of the Iris strains have not been substantiated (Chapter II.4.).Fatty acid analysis has been shown to be a welcome and useful tool in elucidation of natural relations between plant pathogenic Pseudomonas bacteria. Fatty acid analysis in combination with other methods such as conventional phenotypic tests and DNA-and protein fingerprinting may lead to a better understanding of this interesting group of bacteria, not in the last place to achieve a better disease control (Chapter III)

Occurence of Pseudomonas Syrengae pathovars in stone fruits in the Netherlands and availability of strains from different hosts of this pathogen

SFT Meeting in Skierniewice (Polen 27-28 maart 2009) over de fruitteelt in Nederland en de aanwezigheid van pseudomonas syringae in fruitgewasse

Bruinrot bij aardappel

De quarantaineziekte bruinrot van aardappel, veroorzaakt door de bacterie Ralstonia (voorheen : Pseudomonas solanacearum) werd in Nederland voor het eerst met zekerheid aangetroffen in 1992. Het betrof een geïsoleerd geval in Zuid-Limburg, dat later verbonden bleek te zijn met een eerder opgetreden besmetting in België. Sindsdien is door een grote inspanning van de Plantenziektenkundige Dienst (PD), de Nederlandse Algemene Keuringsdienst (NAK), het bedrijfsleven en het onderzoek (Plant Research International (PRI - voorheen IPO) en Wageningen Universiteit veel bereikt. Dit artikel geeft een overzicht van de aanpak en het onderzoek sinds de uitbraak van bruinrot in 1995 en werpt een blik vooruit naar toekomstige ontwikkelinge

Effectiviteit van middelen tegen Rhizoctonia in sla en radijs

Rhizoctonia is een veelvoorkomend probleem in sla en radijs en kan veel opbrengstverlies geven. Uit eerder onderzoek van Wageningen UR Glastuinbouw in radijs werd gevonden dat het enige toegelaten fungicide tegen Rhizoctonia in de praktijk niet voldoende werkt. Door het kleine middelenpakket in de bladgewassen was het daarom noodzakelijk om op zoek te gaan naar nieuwe alternatieven. Het doel van dit onderzoek was om onder gecontroleerde kascondities na te gaan welke middelen Rhizoctonia effectief kunnen bestrijden in sla en radijs en het middel Rizolex kunnen vervangen of aanvullen. De proeven zijn in de wintermaanden (december tot februari) uitgevoerd. Voor beide gewassen zijn een aantal veelbelovende alternatieven naar voren gekomen. Nieuwe effectieve middelen tegen Rhizoctonia in sla De slaplanten werd in diepe containers met zand geteeld en het zand was van tevoren besmet met Rhizoctonia. Twee middelen werden toegediend als grondbehandeling en de andere vier als bladbespuiting. Twee weken na planten werd de eerste bladaantasting door Rhizoctonia zichtbaar bij de controlebehandeling. Vanuit het blad groeide deze aantasting door naar de plantvoet. Planten die behandeld waren met Rizolex vertoonden in de laatste week vlak voor de oogst uitval doordat de aangetaste plantvoet afbrak. Twee middelen (oa. Signum die nog geen toelating heeft voor dit doel) die als bladbehandeling waren toegediend gaven een goede bescherming tegen aantasting van Rhizoctonia en presteerden beter dan Rizolex. De andere drie geteste middelen gaven geen betere bescherming ten opzichte van Rizolex. Nieuwe effectieve middelen tegen Rhizoctonia in radijs De radijszaden werden in diepe containers geteeld en het zand in alle containers was van tevoren besmet met Rhizoctonia. De middelen werden in overleg met de fabrikant toegediend als zaadbehandeling, bodem_ of bladbespuiting. Door de extra belichting kon na acht weken de proef worden geoogst. In de controlebehandelingen was gemiddeld 20% knoluitval door Rhizoctonia. De behandeling met Trianum (met de schimmel Trichoderma harzianum) reduceerde de aantasting met 35% ten opzichte van de controleplanten, maar dit gaf helaas nog steeds te veel uitval van knollen. Drie nieuwe middelen gaven net als Rizolex slechts 1% knolaantasting. Eén daarvan die als bladbehandeling was toegediend gaf groeiremming, maar reduceerde wel de aantasting evengoed als Rizolex. De behandeling waarbij Rizolex gecombineerd werd met een zaadbehandeling en een ander nieuw middel gaf geen enkele knolaantasting. Doordat de planten behandeld met de nieuwe middelen effectief Rhizoctonia aantasting bestreden, was de productie ook hoger dan van de planten in de controlebehandeling. Dit betekende vooral een hogere fractie van grove knollen (> 25 mm)

Bladrandjes en Ca bij tomaat: Fysiologische achtergronden van cel- en weefselstevigheid in relatie tot het ontstaan van bladrandjes en infectie met Botrytis cinerea L.

Tip burn of tomato leaves is often seen by growers as indication for maximum crop performance, however grey mould (botrytis), can easily infect through the necrotic leaf edges. In this desk study factors that are influencing the occurrence of tip burn and calcium (Ca) deficiency were studied. Cells formed during periods of Ca shortage have weaker membranes and walls and ‘burst’ after a climatic shock resulting in plasmolysis and disintegration of the membranes. Possibly the disruption of the Ca homeostasis in the cytoplasm. Botrytis uses dead tissue as an entrance to infect the plant. The weak cell walls and solute leaking caused by disruption of the membranes facilitates the infection process of the fungus. Ca uptake and transport are affected by high fruit load, EC and K/Ca in the root environment and transpiration and root pressure. These factors sometimes interact and sometimes are independently effective, resulting in a complex situation. Thus preventing heavy fruit loads in susceptible periods and stimulating Ca uptake and distribution will alleviate the problem. This, in combination with the prevention of climate shocks will help to reduce the occurrence of tip burn in tomato

A review of Monte Carlo simulations of polymers with PERM

In this review, we describe applications of the pruned-enriched Rosenbluth method (PERM), a sequential Monte Carlo algorithm with resampling, to various problems in polymer physics. PERM produces samples according to any given prescribed weight distribution, by growing configurations step by step with controlled bias, and correcting "bad" configurations by "population control". The latter is implemented, in contrast to other population based algorithms like e.g. genetic algorithms, by depth-first recursion which avoids storing all members of the population at the same time in computer memory. The problems we discuss all concern single polymers (with one exception), but under various conditions: Homopolymers in good solvents and at the $\Theta$ point, semi-stiff polymers, polymers in confining geometries, stretched polymers undergoing a forced globule-linear transition, star polymers, bottle brushes, lattice animals as a model for randomly branched polymers, DNA melting, and finally -- as the only system at low temperatures, lattice heteropolymers as simple models for protein folding. PERM is for some of these problems the method of choice, but it can also fail. We discuss how to recognize when a result is reliable, and we discuss also some types of bias that can be crucial in guiding the growth into the right directions.Comment: 29 pages, 26 figures, to be published in J. Stat. Phys. (2011

Arrhythmogenic mechanisms in the isolated perfused hypokalaemic murine heart

AIM: Hypokalaemia is associated with a lethal form of ventricular tachycardia (VT), torsade de pointes, through pathophysiological mechanisms requiring clarification. METHODS: Left ventricular endocardial and epicardial monophasic action potentials were compared in isolated mouse hearts paced from the right ventricular epicardium perfused with hypokalaemic (3 and 4 mm [K(+)](o)) solutions. Corresponding K(+) currents were compared in whole-cell patch-clamped epicardial and endocardial myocytes. RESULTS: Hypokalaemia prolonged epicardial action potential durations (APD) from mean APD(90)s of 37.2 ± 1.7 ms (n = 7) to 58.4 ± 4.1 ms (n =7) and 66.7 ± 2.1 ms (n = 11) at 5.2, 4 and 3 mm [K(+)](o) respectively. Endocardial APD(90)s correspondingly increased from 51.6 ± 1.9 ms (n = 7) to 62.8 ± 2.8 ms (n = 7) and 62.9 ± 5.9 ms (n = 11) giving reductions in endocardial–epicardial differences, ΔAPD(90), from 14.4 ± 2.6 to 4.4 ± 5.0 and −3.4 ± 6.0 ms respectively. Early afterdepolarizations (EADs) occurred in epicardia in three of seven spontaneously beating hearts at 4 mm [K(+)](o) with triggered beats followed by episodes of non-sustained VT in nine of 11 preparations at 3 mm. Programmed electrical stimulation never induced arrhythmic events in preparations perfused with normokalemic solutions yet induced VT in two of seven and nine of 11 preparations at 4 and 3 mm [K(+)](o) respectively. Early outward K(+) current correspondingly fell from 73.46 ± 8.45 to 61.16±6.14 pA/pF in isolated epicardial but not endocardial myocytes (n = 9) (3 mm [K(+)](o)). CONCLUSIONS: Hypokalaemic mouse hearts recapitulate the clinical arrhythmogenic phenotype, demonstrating EADs and triggered beats that might initiate VT on the one hand and reduced transmural dispersion of repolarization reflected in ΔAPD(90) suggesting arrhythmogenic substrate on the other