text:
Wolfgang Gerhardt (* 31. Dezember 1943 in Helpershain) ist ein deutscher Politiker (FDP).

Er war von 1987 bis 1991 Hessischer Staatsminister für Wissenschaft und Kunst, von 1995 bis 2001 FDP-Bundesvorsitzender sowie von 1998 bis 2006 Vorsitzender der FDP-Bundestagsfraktion und damit von November 2005 bis April 2006 auch Oppositionsführer.
Von 2006 bis 2018 war er Vorstandsvorsitzender der Friedrich-Naumann-Stiftung für die Freiheit, seitdem ist er Ehrenvorsitzender ihres Vorstandes.

Ausbildung und Beruf
Nach dem Abitur im Jahr 1963 an der Albert-Schweitzer-Schule Alsfeld absolvierte Gerhardt von 1963 bis 1969 ein Studium der Erziehungswissenschaften, Germanistik und Politik an der Universität Marburg. Im Jahr 1970 erfolgte dort seine Promotion über die Bildungspolitik der FDP nach 1945 bei Leonhard Froese zum Dr. phil. Ab 1969 arbeitete er bei der Friedrich-Naumann-Stiftung, zunächst als Leiter des Regionalbüros in Hannover, dann als Referent in der Inlandsabteilung. 1971 wechselte er als persönlicher Referent des Ministers Hanns-Heinz Bielefeld ins Hessische Ministerium des Innern, wo er unter Bielefelds Amtsnachfolger Ekkehard Gries bis zu seinem Einzug in den Landtag das Ministerbüro leitete.

Partei
Gerhardt ist seit 1965 Mitglied der FDP. Zunächst engagierte er sich hochschul- und jugendpolitisch, unter anderem war er 1967/68 Landesvorsitzender des Liberalen Studentenbundes Deutschlands und stellvertretender Landesvorsitzender der Deutschen Jungdemokraten in Hessen. Von 1982 bis 1995 war er Landesvorsitzender der FDP Hessen. Ab 1982 war er Mitglied im FDP-Bundesvorstand. Im Jahr 1985 wurde er zunächst Stellvertretender Vorsitzender, im Jahr 1995 dann als Nachfolger des damaligen Außenministers Klaus Kinkel Bundesvorsitzender der FDP. Dieses Amt behielt er bis zum Mai 2001, als er es nach innerparteilicher Kritik an den bisherigen Generalsekretär Guido Westerwelle abgab.

Anschließend war Gerhardt von 2002 bis 2012 Stellvertretender Präsident der Liberalen Internationale.

Für den Fall einer Regierungsbeteiligung nach der Bundestagswahl 2005 war Wolfgang Gerhardt für das Amt des Bundesaußenministers vorgesehen.

Im Juni 2010 wurde Gerhardt zum Mitglied der Programmkommission gewählt, die bis zum Jahr 2013 ein neues FDP-Programm erarbeiten sollte.

Abgeordneter

Gerhardt war von 1978 bis 1982, von 1983 bis 1987 sowie von 1991 bis 1994 Mitglied des Hessischen Landtages. Hier war er von 1983 bis 1987 und von 1991 bis 1994 Vorsitzender der Landtagsfraktion.

Von 1994 bis 2013 war Gerhardt Mitglied des Deutschen Bundestages. Nach der Bundestagswahl 1998 wurde er am 5. Oktober 1998 als Nachfolger von Hermann Otto Solms zum Vorsitzenden der FDP-Bundestagsfraktion gewählt. Als solcher übernahm Gerhardt mit Antritt der Regierung Merkel am 22. November 2005 den Posten des Oppositionsführers. Am 1. Mai 2006 gab er den Fraktionsvorsitz an den Parteivorsitzenden Guido Westerwelle ab.

Gerhardt zog stets über die Landesliste Hessen in den Deutschen Bundestag ein. Bei der Bundestagswahl 2013 kandidierte er nicht mehr.

Öffentliche Ämter
Am 24. April 1987 wurde Gerhardt Hessischer Minister für Wissenschaft und Kunst, Bevollmächtigter des Landes Hessen beim Bund und zugleich Stellvertreter des Ministerpräsidenten Walter Wallmann. Nachdem die CDU-FDP-Koalition bei der Landtagswahl 1991 ihre Mehrheit eingebüßt hatte, schied er am 5. April 1991 aus der Regierung aus.

Familie
Gerhardt wurde im Jahr 1943 als Sohn eines Berufssoldaten geboren. Sein Vater fiel im Jahr 1944. Er wuchs bei seiner Mutter auf einem Bauernhof auf. Er ist verheiratet und hat zwei Kinder.

Ehrungen
 2006: Thomas-Dehler-Preis
 2008: Reinhold-Maier-Medaille
 2011: Wilhelm-Leuschner-Medaille

Zitate
"Wir müssen aus dem Schlafwagen heraus!" (Gerhardt über die rot-grüne Reformpolitik auf dem FDP-Bundesparteitag 2005)

"Nicht der Staat gewährt den Bürgern Freiheit, sondern die Bürger dem Staat Einschränkungen ihrer Rechte." (am 29. März 2006 im Bundestag/ Anmerkung: Dieses Zitat stammt ursprünglich aus den "Wiesbadener Grundsätzen" aus dem Jahre 1997)

Veröffentlichungen
 (Hrsg.) Die Kraft der Freiheit. Geschichte, Gegenwart und Zukunft des Liberalismus, Hohenheim-Verlag, Stuttgart [u. a.] 2008, ISBN 978-3-89850-165-1.
 Thomas Dehler im Bundestag, in: Haus der Geschichte der Bundesrepublik Deutschland (Hrsg.): Thomas Dehler und seine Politik, Nicolaische Verlagsbuchhandlung, Berlin 1998, ISBN 3-87584-721-0, S. 83–90.
 Es geht. Wir haben alle Chancen, Edition Ferenczy bei Bruckmann, München 1997, ISBN 3-7654-2883-3.
 Der Euro-DM-Markt. Marktteilnehmer, Zinsbildung und geldpolitische Bedeutung. Eine Untersuchung aus der Sicht der Bundesrepublik Deutschland (= Veröffentlichungen des HWWA-Institut für Wirtschaftsforschung Hamburg), Verlag Weltarchiv, Hamburg 1984, ISBN 3-87895-255-4.
 Die bildungspolitische Diskussion in der FDP von 1945–1951, Universität Marburg 1971 (Dissertation).

Literatur

 Jochen Lengemann: MdL Hessen. 1808–1996. Biographischer Index (= Politische und parlamentarische Geschichte des Landes Hessen. Bd. 14 = Veröffentlichungen der Historischen Kommission für Hessen. Bd. 48, 7). Elwert, Marburg 1996, ISBN 3-7708-1071-6, S. 142–143.

Weblinks

 Lebenslauf bei der Friedrich-Naumann-Stiftung für die Freiheit
 Biographie beim Deutschen Bundestag

Einzelnachweise

Bundestagsabgeordneter (Hessen)
Mitglied des Auswärtigen Ausschusses (Deutscher Bundestag)
Fraktionsvorsitzender (FDP Hessen)
Wissenschaftsminister (Hessen)
Kunstminister (Hessen)
Vorsitzender der FDP-Bundestagsfraktion
Bundesvorsitzender der FDP
Vorsitzender der FDP Hessen
Mitglied der Jungdemokraten
Vorsitzender der Friedrich-Naumann-Stiftung
Träger des Thomas-Dehler-Preises der Thomas-Dehler-Stiftung
Träger der Reinhold-Maier-Medaille
Träger der Wilhelm-Leuschner-Medaille
Deutscher
Geboren 1943
Mann

redpajama_set_name: RedPajamaWikipedia


text:
The Correctional Reception Center is a state prison for men located in Orient, Pickaway County, Ohio, opened in 1987, owned and operated by the Ohio Department of Rehabilitation and Correction.

The facility holds a maximum of 1500 inmates at various security levels.  The prison is immediately adjacent to the state's Pickaway Correctional Institution.

Ariel Castro committed suicide while incarcerated at the facility in September 2013.

References

Prisons in Ohio
Buildings and structures in Pickaway County, Ohio
1987 establishments in Ohio


Israeli airstrikes had pounded the area in recent days, targeting tunnels used by Palestinian militants to smuggle weapons in from Egypt. A few streetlamps glowed dimly and stray dogs roamed around trash piles, but otherwise taxis were the only things moving.
Driving north road from Rafah, traffic thinned almost to nothing. In Gaza City, the streets were pitch dark except for a couple of small grocery stores, a mobile phone shop, a used-television dealer. Powered by generators, the shops glowed like hospital operating rooms even though few customers milled about.
Overhead, however, there was an unmistakable reminder of the war: the persistent buzz of Israeli drones, which circled the city for much of the night and broke the eerie quiet.


text:
MAIN TRIAL SITE 2023
MAIN TRIAL SITE HISTORY
R&D BOOKS
BACK TO CURRENT PROJECTS
Redefining dryland salinity management for new generation land custodians
Commencement date
This project aims to address the significant knowledge gap of regenerative dryland salinity management.
Funding Provider
Project lead organisation
Liebe Group
Dryland salinity is a major cause of land degradation in the southwest of WA, with more than one million hectares being classified as severely salt-affected and a further 2.8 to 4.5 million hectares at risk. Saline soils are inhospitable for most common flora and fauna species, decreasing the area's biodiversity and making them an unsuitable environment both for agricultural purposes and native habitat.
Additionally, soils with higher levels of salinity often become bare with minimal ground cover, increasing erosion and further degradation. Throughout the Northern Zone of Rejuvenated Drainage hydrozone (i.e. Liebe Group Region), the past 20 years of drier seasons have seen saline areas spread at a variable rate.
More recently concerns have arisen due to the increasing variability of the climate that is affecting rainfall patterns with wetter summers and later seasonal breaks becoming more prevalent. The most recent major analysis of groundwater trends for the region was conducted in 2014, which resulted in highly complicated and technical outputs that were not easily interpreted at a landholder level.
The broadacre industry is also seeing a new generation of landholders taking on the primary decision making roles in their enterprises. It has been identified that although these landholders have strong values as custodians of the land for future generations, there is perceived conflict between production efficiencies of modern farming systems and the salinity management practices implemented by previous generations (ie contour banks, deep drainage).
Results and Reports
Redefining Dryland Salinity - Catchment Review
© 2020 by Liebe Group Inc
E: admin@liebegroup.org.au
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Information, recommendations, suggestions or opinions on this website do not necessarily represent the policy or views of the Liebe Group. This website is not a scientific journal and all information has been prepared solely as a means of disseminating information to the members of the Liebe Group and the community. Content on this website has been prepared in good faith on the basis of information available at the date of publication. Although all reasonable care has been taken in producing the reports within, no person should act on the basis of the contents of this publication without first obtaining specific independent professional advice. Products may be identified by propriety or trade names to help readers identify them, however, this is not an endorsement or recommendation of any product or manufacturer referred to. The Liebe Group will not be liable for any loss, damage, cost or expense incurred or arising by reason of any person using or relying on the information provided on this website.

redpajama_set_name: RedPajamaCommonCrawl


Capricorn the mountain goat is on a mission. It's one focus is to climb the mountain of its desires and reach the pinnacle; no matter what the cost in terms of time, effort or endurance. The power of goals matter to Capricorn energy. Achievement is the ultimate reward.
Where you find Capricorn and it's planetary ruler, Saturn, is where you need to achieve and be recognized and rewarded for your efforts. This quest is not limited to a job or avocation. Perhaps your deepest desire is to be successful within your friendship circle, or in your spiritual practice. As with any chart the possibilities are endless.
Capricorn key words include the three `r's': responsibility, respect and reliability. We tend to rise to the heights of success when we put forth the effort reflected by these qualities. Saturn's key words include; structure, discipline and reality. We get `real' where Capricorn and Saturn resonate within our charts and build what we want whether that be a physical structure, a career, a relationship, or anything else of our imagination.
Manifestation happens with Capricorn and Saturn. Behind every dream is hard work. Capricorn and Saturn strategize to perform the 99% sweat equity behind the 1% of inspiration.
And herein lies the negative side of Capricorn. It's said that `power corrupts, and absolute power corrupts absolutely.' There is an isolation within a strong Capricorn infused person; it's lonely on top of the mountain. Furthermore, an attitude can develop in which people become commodities.
A balanced use of Capricorn energy is to `use' tools to garner success, but only if the `tools' are of the material type. When an individual uses another person a misuse of the potent Capricorn energy occurs resulting in dehumanization.


text:
Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions
H.C. Ramirez-Saad
Research output: Thesis › internal PhD, WU

Microbial Ecology studies aim to describe and assess the behavior and activity of microorganisms in their natural environments (Brock 1966). Nowadays it is clear that the large number of existing microorganisms has surpassed our capabilities to rapidly characterise them by traditional culturing methods. This has resulted in a poor understanding of the structure and composition of microbial communities. As an alternative, microbial communities can be described on the basis of 16S rRNA sequence diversity, without the bias-introducing step of cultivation.

In the present thesis a molecular analysis is given of two ecosystems that harbour several uncultured bacteria. The first part of the thesis is focused on the detection and characterisation of Frankia in actinorhizal nodules and soil. Elucidation of the actual diversity within the family Frankiaceae was hampered by the inability to obtain isolates from all known actinorhizal plants. So far, the Nod +/Fix +Frankia symbionts in root nodules of plants from the families Coriariaceae, Datiscaceae, Rosaceae and Rhamnaceae (with exceptions reported by Carú 1993, Carú et al . 1990, and Carrasco et al . 1995) have resisted isolation. Best opportunities to characterise those uncultured endophytes require molecular methods that relay heavily on an easy and efficient technique to extract DNA from the respective actinorhizal nodules. Chapter 2 describes the techniques to isolate DNA from root nodules of different actinorhizal plants such as Casuarina sp, Alnus sp and Ceanothus sp. The procedure has also been successfully applied by Wolters et al . (1997b) in the minuscule ineffective nodules on Alnus glutinosa .

Several attempts to characterise the uncultured endophytes from Coriaria sp. and Datisca sp. plants pointed on the one hand, to the presence in those actinorhizae of Frankia -related actinomycetes. This assumption was based mainly on the repeated isolation from those nodules of Nod -/Fix -Frankia -like strains (Hafeez 1983, Mirza et. al . 1994b, c). On the other hand, the effective (Fix +), non-isolated symbionts showed to be phylogenetically closely related (Mirza et al. 1994a), forming a separate lineage within the genus, in spite of the distant geographical distribution of the plants (Nick et al. 1992).

The work described in Chapter 3 is focussed on the localisation and phylogenetic position of the nitrogen-fixing Frankia and Nod -/Fix -actinomycetes, both present in root nodules of the Mexican actinorhizal plant Ceanothus caeruleus . Application of the TGGE technique allowed localising the Nod -/Fix -actinomycete in the outer layers of the C. caeruleus nodules. Similar bacteria were also detected in Hippophaë rhamnoides nodules induced with soil inoculum that was collected in the vicinity of the former plant. The fact that a second nodule inhabitant was commonly present in these nodules containing recalcitrant endophytes may allow some speculations about their possible role in the symbiosis. However, it seems worthwhile to apply the same TGGE methodology to other actinorhizal nodules, even to those containing Frankia strains that are easy-to-isolate (i.e. Elaeagnus,Casuarina, Alnus spp.), since the detection of Frankia -related actinomycetes, in addition to the Fix +endophyte, would provide further evidence about the need for their presence. Coincidentally, the Nod -/Fix -isolates from Coriaria, Datisca and Ceanothus are phylogenetically related, pointing again to a certain specificity for their presence in the nodules. This relatedness has also been supported by analysis of low molecular weight RNA (i.e. 5S rRNA and tRNA's) using staircase electrophoresis (Velázquez et al. 1998).

The 16S rDNA sequence from the non-isolated Fix +endophyte in C. caeruleus root nodules (Chapter 3), was the first full sequence obtained from a field-collected Ceanothus symbiont. Parsimony and phylogenetic distance analyses grouped it within the Dryas cluster that originally contained only the uncultured endophytes from Dryas , Coriaria and Datisca as proposed by Normand et al. (1996). Benson et al . (1996) redefined this cluster by adding other uncultured endophytes present in Ceanothus griseus (Rhamnaceae), Purshia tridentata and Dryas drummondii (Rosaceae) root nodules. Since the determined partial 16S rDNA sequences were almost identical, they suggested that the Frankia diversity from these actinorhizal plant families might be low. However, Clawson et al. (1998) demonstrated that Frankia isolates obtained from several genera within the Rhamnaceae (i.e. Talguenea , Colletia , Discaria, Retanilla and Trevoa ) were phylogenetically different than those in Ceanothus, grouping in the Elaeagnus cluster. These findings were consistent with morphological differences of the endophytes in planta , since the vesicles found in the Ceanothus symbionts resemble more to those in the Rosaceae, while all the latter host plants in the Rhamnaceae family have endophytes like those in Elaeagnus .

The results reported in the first part of the thesis have demonstrated that TGGE and sequence analysis of 16S rDNA provide an accurate picture for the identification of recalcitrant endophytes in root nodules of actinorhizal plants. It has also been demonstrated that besides the N 2 -fixing endophyte, root nodules of C. caeruleus also harbour Frankia -related actinomycetes. Since these have also been observed in other actinorhizae, a further study is needed to understand the possible function of these co-symbionts.

The work described in the second part of the thesis was addressing the changes occurring under chlorobenzoate stress in the soil bacterial community and other selected groups of bacteria present in peat soil collected from a natural Alnus glutinosa stand. A combination of culturing and non-culture based approaches was used for the assessment. Among the latter approaches, the possibilities offered by TGGE were exploited in several ways. Profiling of complex communities and subsequent analysis of specific bacterial groups has been one of the major applications of TGGE (Felske et al . 1996). With this approach, major population shifts induced by either 3CBA or 2,5DCB were detected in the uncultured bacterial community (Chapter 4). Although only the former compound was readily metabolised in soil, both xenobiotics promoted similar changes. Several bacterial populations were reduced or suppressed, while few others were enriched in time, as assessed by shifts in the TGGE banding patterns of the total bacterial community.

To characterise the soil-enriched bacterial populations, 3CBA-degrading enrichment cultures were obtained and their composition was addressed by TGGE. Further isolation attempts were directed by this means to prove that the isolated strains were indeed the same enriched organisms as detected in soil. One of the enrichment cultures contained two of the soil-enriched bacteria as predominant components. Although isolation was not achieved, both bacteria were identified as belonging to the genus Burkholderia . The bacterial group detected as predominantly enriched in both spiked soils was not present in any of the enrichment cultures, suggesting that the microorganisms belonging to this groups are either unable to degrade 3CBA or not growing under the used culturing conditions. In any case their fitness to the soil conditions imposed by the addition of chlorobenzoates was high, but the mechanisms involved were not elucidated. These bacteria were also identified as Burkholderia by partial 16S rDNA sequence analysis (Chapter 4).

The diversity (H) and the equitability (J) indices are important parameters used by ecologists to assess the species richness and the species evenness, respectively, within a community. As the estimation of such indices relies heavily on species definition and individuals enumeration, their application in microbial ecology studies is seldomly possible. Furthermore, assessment of H and J in uncultured bacterial communities must rely on the interpretation of community fingerprints, which should provide means to distinguish between species or operational taxonomic units (OTU), and to estimate their abundance. TGGE community profiling offers both possibilities, and the community changes occurring in the model soil system were evaluated with this original approach (Chapter 5).

In addition, H and J indices were also estimated for the fluorescent pseudomonads group, a selected culturable fraction of the bacterial community. OTU recognition was addressed by using TGGE as a ribotype-fingerprinting technique for the isolated fluorescent pseudomonads. Estimation of H and J at the community level without culturing by TGGE profiling, and at the group level by a combination of culturing and TGGE ribotyping should allow to address and compare the population changes occurring, since the target molecule used in both TGGE was the same. Such comparison was only partially possible since most of the bands corresponding to the fluorescent pseudomonads could not be assessed in the community profiles. However, estimation of H and J indices indicated a clear reduction of species richness and individuals abundance in the uncultured community, which was related to the presence of chlorobenzoates in soil. Evaluation of population shifts by indexed values as H and J proved to be a useful means for analysing the community structure in time, and may be used to assess short and long-term responses of a bacterial community to environmental perturbations.

Chapter 6 describes the changes in the total frankiae  in soil and in the fraction of the population that is able to produce root nodules in Alnus glutinosa seedlings . Culture-independent approaches based on the most probable number concept were used, one in combination with a Frankia -specific PCR detection and another in combination with a plant-nodulation bioassay. After 15 days of incubation in the presence of chlorobenzoates both fractions of the soil Frankia populations were reduced in more than one order of magnitude, while the populations in the unspiked control soil were not affected. The results indicated that 3CBA and 2,5DCB both had a negative effect on the size of the native Frankia population from the used peat forest soil. This negative effect was also evident during in vitro experiments using Frankia strains isolated from Alnus sp. The presence of 1 mM 3CBA in the culture medium, in addition to the normal carbon source, resulted in reduction or suppression of biomass yield.

The influence of Alnus glutinosa on the dechlorination of 3CBA by Pseudomonas sp. strain B13 was assessed in hydroponic cultures. It was expected that root exudates could enhance the dechlorination activity of Pseudomonas B13. When the bacteria were incubated in the presence of an alder plant, only a slight increase in the dechlorination rate of 3CBA was registered in comparison to the control without plant. The main observed effect in the alder plants appears to be a protection against 3CBA toxicity, as the alders inoculated with Pseudomonas B13 showed a better survival rate and grew more vigorously than the non-inoculated plants.

The toolbox for microbial ecology studies is increasing constantly by means of developing new techniques or by adapting foreign tools into the field, such as indices to evaluate species diversity and eveness. Although the information provided by these community parameters facilitates comparisons and assessment of changes, their suitability to evaluate bacterial communities is still uncomplete. Estimation of diversity indices requires the recognition of bacterial species as discrete units, and this condition is far from real in natural environments. Species-independent approaches to evaluate diversity must be developed, that consider the bacterial diversity as a continuous range of phylogenetically related taxonomic units.

In conclusion, the work described in the first part of the thesis strengthen the current phylogenetic division of the Frankiaceae, by adding new evidence supporting two of the already described clusters ( Dryas cluster, Nod -/Fix -cluster). Although the current taxonomic status of the latter cluster must be better evaluated in order to assess its pertinence to the genus Frankia . In addition, the common occurrence of Nod -/Fix -Frankia -like actinomycetes in nodules containing recalcitrant endophytes, mainly from the Dryas cluster, was also demonstrated for Ceanothus actinorhiza. In the second part, the suitability and versatility of molecular tools such as TGGE were demonstrated by their application in community profiling and estimation of bacterial diversity. Community changes occurring in stressed and unstressed soil systems were easily detected and assessed by this means.

In addition, specific populations such as the Burkholderia -like bacteria that strongly reacted to the addition of chlorobenzoates to soil were further characterised. Moreover, TGGE was shown to be a fast ribotyping technique that may enable its use in combination with the community profiles to address shifts of specific groups within bacterial communities. It is tempting to suggest that this general approach will be of importance to direct the isolation of hitherto uncultured bacteria from soil.

de Vos, W.M., Promotor
Akkermans, A.D.L., Promotor
Ramirez Saad
soil bacteria
frankia
benzoates
https://edepot.wur.nl/195239
Dive into the research topics of 'Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions'. Together they form a unique fingerprint.
Frankia Agriculture & Biology 100%
soil bacteria Agriculture & Biology 81%
Ceanothus Agriculture & Biology 77%
ecology Agriculture & Biology 54%
actinorhizae Agriculture & Biology 47%
Datisca Agriculture & Biology 38%
Rhamnaceae Agriculture & Biology 38%
Ramirez-Saad, H. C. (1999). Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions. Ramirez Saad. https://edepot.wur.nl/195239
Ramirez-Saad, H.C.. / Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions. S.l. : Ramirez Saad, 1999. 119 p.
@phdthesis{da03c6c3582f4788a94173e2eb318b88,
title = "Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions",
abstract = "Microbial Ecology studies aim to describe and assess the behavior and activity of microorganisms in their natural environments (Brock 1966). Nowadays it is clear that the large number of existing microorganisms has surpassed our capabilities to rapidly characterise them by traditional culturing methods. This has resulted in a poor understanding of the structure and composition of microbial communities. As an alternative, microbial communities can be described on the basis of 16S rRNA sequence diversity, without the bias-introducing step of cultivation.In the present thesis a molecular analysis is given of two ecosystems that harbour several uncultured bacteria. The first part of the thesis is focused on the detection and characterisation of Frankia in actinorhizal nodules and soil. Elucidation of the actual diversity within the family Frankiaceae was hampered by the inability to obtain isolates from all known actinorhizal plants. So far, the Nod +/Fix +Frankia symbionts in root nodules of plants from the families Coriariaceae, Datiscaceae, Rosaceae and Rhamnaceae (with exceptions reported by Car{\'u} 1993, Car{\'u} et al . 1990, and Carrasco et al . 1995) have resisted isolation. Best opportunities to characterise those uncultured endophytes require molecular methods that relay heavily on an easy and efficient technique to extract DNA from the respective actinorhizal nodules. Chapter 2 describes the techniques to isolate DNA from root nodules of different actinorhizal plants such as Casuarina sp, Alnus sp and Ceanothus sp. The procedure has also been successfully applied by Wolters et al . (1997b) in the minuscule ineffective nodules on Alnus glutinosa .Several attempts to characterise the uncultured endophytes from Coriaria sp. and Datisca sp. plants pointed on the one hand, to the presence in those actinorhizae of Frankia -related actinomycetes. This assumption was based mainly on the repeated isolation from those nodules of Nod -/Fix -Frankia -like strains (Hafeez 1983, Mirza et. al . 1994b, c). On the other hand, the effective (Fix +), non-isolated symbionts showed to be phylogenetically closely related (Mirza et al. 1994a), forming a separate lineage within the genus, in spite of the distant geographical distribution of the plants (Nick et al. 1992).The work described in Chapter 3 is focussed on the localisation and phylogenetic position of the nitrogen-fixing Frankia and Nod -/Fix -actinomycetes, both present in root nodules of the Mexican actinorhizal plant Ceanothus caeruleus . Application of the TGGE technique allowed localising the Nod -/Fix -actinomycete in the outer layers of the C. caeruleus nodules. Similar bacteria were also detected in Hippopha{\"e} rhamnoides nodules induced with soil inoculum that was collected in the vicinity of the former plant. The fact that a second nodule inhabitant was commonly present in these nodules containing recalcitrant endophytes may allow some speculations about their possible role in the symbiosis. However, it seems worthwhile to apply the same TGGE methodology to other actinorhizal nodules, even to those containing Frankia strains that are easy-to-isolate (i.e. Elaeagnus,Casuarina, Alnus spp.), since the detection of Frankia -related actinomycetes, in addition to the Fix +endophyte, would provide further evidence about the need for their presence. Coincidentally, the Nod -/Fix -isolates from Coriaria, Datisca and Ceanothus are phylogenetically related, pointing again to a certain specificity for their presence in the nodules. This relatedness has also been supported by analysis of low molecular weight RNA (i.e. 5S rRNA and tRNA's) using staircase electrophoresis (Vel{\'a}zquez et al. 1998).The 16S rDNA sequence from the non-isolated Fix +endophyte in C. caeruleus root nodules (Chapter 3), was the first full sequence obtained from a field-collected Ceanothus symbiont. Parsimony and phylogenetic distance analyses grouped it within the Dryas cluster that originally contained only the uncultured endophytes from Dryas , Coriaria and Datisca as proposed by Normand et al. (1996). Benson et al . (1996) redefined this cluster by adding other uncultured endophytes present in Ceanothus griseus (Rhamnaceae), Purshia tridentata and Dryas drummondii (Rosaceae) root nodules. Since the determined partial 16S rDNA sequences were almost identical, they suggested that the Frankia diversity from these actinorhizal plant families might be low. However, Clawson et al. (1998) demonstrated that Frankia isolates obtained from several genera within the Rhamnaceae (i.e. Talguenea , Colletia , Discaria, Retanilla and Trevoa ) were phylogenetically different than those in Ceanothus, grouping in the Elaeagnus cluster. These findings were consistent with morphological differences of the endophytes in planta , since the vesicles found in the Ceanothus symbionts resemble more to those in the Rosaceae, while all the latter host plants in the Rhamnaceae family have endophytes like those in Elaeagnus .The results reported in the first part of the thesis have demonstrated that TGGE and sequence analysis of 16S rDNA provide an accurate picture for the identification of recalcitrant endophytes in root nodules of actinorhizal plants. It has also been demonstrated that besides the N 2 -fixing endophyte, root nodules of C. caeruleus also harbour Frankia -related actinomycetes. Since these have also been observed in other actinorhizae, a further study is needed to understand the possible function of these co-symbionts.The work described in the second part of the thesis was addressing the changes occurring under chlorobenzoate stress in the soil bacterial community and other selected groups of bacteria present in peat soil collected from a natural Alnus glutinosa stand. A combination of culturing and non-culture based approaches was used for the assessment. Among the latter approaches, the possibilities offered by TGGE were exploited in several ways. Profiling of complex communities and subsequent analysis of specific bacterial groups has been one of the major applications of TGGE (Felske et al . 1996). With this approach, major population shifts induced by either 3CBA or 2,5DCB were detected in the uncultured bacterial community (Chapter 4). Although only the former compound was readily metabolised in soil, both xenobiotics promoted similar changes. Several bacterial populations were reduced or suppressed, while few others were enriched in time, as assessed by shifts in the TGGE banding patterns of the total bacterial community.To characterise the soil-enriched bacterial populations, 3CBA-degrading enrichment cultures were obtained and their composition was addressed by TGGE. Further isolation attempts were directed by this means to prove that the isolated strains were indeed the same enriched organisms as detected in soil. One of the enrichment cultures contained two of the soil-enriched bacteria as predominant components. Although isolation was not achieved, both bacteria were identified as belonging to the genus Burkholderia . The bacterial group detected as predominantly enriched in both spiked soils was not present in any of the enrichment cultures, suggesting that the microorganisms belonging to this groups are either unable to degrade 3CBA or not growing under the used culturing conditions. In any case their fitness to the soil conditions imposed by the addition of chlorobenzoates was high, but the mechanisms involved were not elucidated. These bacteria were also identified as Burkholderia by partial 16S rDNA sequence analysis (Chapter 4).The diversity (H) and the equitability (J) indices are important parameters used by ecologists to assess the species richness and the species evenness, respectively, within a community. As the estimation of such indices relies heavily on species definition and individuals enumeration, their application in microbial ecology studies is seldomly possible. Furthermore, assessment of H and J in uncultured bacterial communities must rely on the interpretation of community fingerprints, which should provide means to distinguish between species or operational taxonomic units (OTU), and to estimate their abundance. TGGE community profiling offers both possibilities, and the community changes occurring in the model soil system were evaluated with this original approach (Chapter 5).In addition, H and J indices were also estimated for the fluorescent pseudomonads group, a selected culturable fraction of the bacterial community. OTU recognition was addressed by using TGGE as a ribotype-fingerprinting technique for the isolated fluorescent pseudomonads. Estimation of H and J at the community level without culturing by TGGE profiling, and at the group level by a combination of culturing and TGGE ribotyping should allow to address and compare the population changes occurring, since the target molecule used in both TGGE was the same. Such comparison was only partially possible since most of the bands corresponding to the fluorescent pseudomonads could not be assessed in the community profiles. However, estimation of H and J indices indicated a clear reduction of species richness and individuals abundance in the uncultured community, which was related to the presence of chlorobenzoates in soil. Evaluation of population shifts by indexed values as H and J proved to be a useful means for analysing the community structure in time, and may be used to assess short and long-term responses of a bacterial community to environmental perturbations.Chapter 6 describes the changes in the total frankiae in soil and in the fraction of the population that is able to produce root nodules in Alnus glutinosa seedlings . Culture-independent approaches based on the most probable number concept were used, one in combination with a Frankia -specific PCR detection and another in combination with a plant-nodulation bioassay. After 15 days of incubation in the presence of chlorobenzoates both fractions of the soil Frankia populations were reduced in more than one order of magnitude, while the populations in the unspiked control soil were not affected. The results indicated that 3CBA and 2,5DCB both had a negative effect on the size of the native Frankia population from the used peat forest soil. This negative effect was also evident during in vitro experiments using Frankia strains isolated from Alnus sp. The presence of 1 mM 3CBA in the culture medium, in addition to the normal carbon source, resulted in reduction or suppression of biomass yield.The influence of Alnus glutinosa on the dechlorination of 3CBA by Pseudomonas sp. strain B13 was assessed in hydroponic cultures. It was expected that root exudates could enhance the dechlorination activity of Pseudomonas B13. When the bacteria were incubated in the presence of an alder plant, only a slight increase in the dechlorination rate of 3CBA was registered in comparison to the control without plant. The main observed effect in the alder plants appears to be a protection against 3CBA toxicity, as the alders inoculated with Pseudomonas B13 showed a better survival rate and grew more vigorously than the non-inoculated plants.The toolbox for microbial ecology studies is increasing constantly by means of developing new techniques or by adapting foreign tools into the field, such as indices to evaluate species diversity and eveness. Although the information provided by these community parameters facilitates comparisons and assessment of changes, their suitability to evaluate bacterial communities is still uncomplete. Estimation of diversity indices requires the recognition of bacterial species as discrete units, and this condition is far from real in natural environments. Species-independent approaches to evaluate diversity must be developed, that consider the bacterial diversity as a continuous range of phylogenetically related taxonomic units.In conclusion, the work described in the first part of the thesis strengthen the current phylogenetic division of the Frankiaceae, by adding new evidence supporting two of the already described clusters ( Dryas cluster, Nod -/Fix -cluster). Although the current taxonomic status of the latter cluster must be better evaluated in order to assess its pertinence to the genus Frankia . In addition, the common occurrence of Nod -/Fix -Frankia -like actinomycetes in nodules containing recalcitrant endophytes, mainly from the Dryas cluster, was also demonstrated for Ceanothus actinorhiza. In the second part, the suitability and versatility of molecular tools such as TGGE were demonstrated by their application in community profiling and estimation of bacterial diversity. Community changes occurring in stressed and unstressed soil systems were easily detected and assessed by this means.In addition, specific populations such as the Burkholderia -like bacteria that strongly reacted to the addition of chlorobenzoates to soil were further characterised. Moreover, TGGE was shown to be a fast ribotyping technique that may enable its use in combination with the community profiles to address shifts of specific groups within bacterial communities. It is tempting to suggest that this general approach will be of importance to direct the isolation of hitherto uncultured bacteria from soil.",
keywords = "bodembacteri{\"e}n, frankia, microbi{\"e}le ecologie, benzoaten, soil bacteria, frankia, microbial ecology, benzoates",
author = "H.C. Ramirez-Saad",
note = "WU thesis 2631 Proefschrift Wageningen",
publisher = "Ramirez Saad",
Ramirez-Saad, HC 1999, 'Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions', Doctor of Philosophy, S.l.. <https://edepot.wur.nl/195239>
Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions. / Ramirez-Saad, H.C.
S.l. : Ramirez Saad, 1999. 119 p.
T1 - Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions
AU - Ramirez-Saad, H.C.
N1 - WU thesis 2631 Proefschrift Wageningen
N2 - Microbial Ecology studies aim to describe and assess the behavior and activity of microorganisms in their natural environments (Brock 1966). Nowadays it is clear that the large number of existing microorganisms has surpassed our capabilities to rapidly characterise them by traditional culturing methods. This has resulted in a poor understanding of the structure and composition of microbial communities. As an alternative, microbial communities can be described on the basis of 16S rRNA sequence diversity, without the bias-introducing step of cultivation.In the present thesis a molecular analysis is given of two ecosystems that harbour several uncultured bacteria. The first part of the thesis is focused on the detection and characterisation of Frankia in actinorhizal nodules and soil. Elucidation of the actual diversity within the family Frankiaceae was hampered by the inability to obtain isolates from all known actinorhizal plants. So far, the Nod +/Fix +Frankia symbionts in root nodules of plants from the families Coriariaceae, Datiscaceae, Rosaceae and Rhamnaceae (with exceptions reported by Carú 1993, Carú et al . 1990, and Carrasco et al . 1995) have resisted isolation. Best opportunities to characterise those uncultured endophytes require molecular methods that relay heavily on an easy and efficient technique to extract DNA from the respective actinorhizal nodules. Chapter 2 describes the techniques to isolate DNA from root nodules of different actinorhizal plants such as Casuarina sp, Alnus sp and Ceanothus sp. The procedure has also been successfully applied by Wolters et al . (1997b) in the minuscule ineffective nodules on Alnus glutinosa .Several attempts to characterise the uncultured endophytes from Coriaria sp. and Datisca sp. plants pointed on the one hand, to the presence in those actinorhizae of Frankia -related actinomycetes. This assumption was based mainly on the repeated isolation from those nodules of Nod -/Fix -Frankia -like strains (Hafeez 1983, Mirza et. al . 1994b, c). On the other hand, the effective (Fix +), non-isolated symbionts showed to be phylogenetically closely related (Mirza et al. 1994a), forming a separate lineage within the genus, in spite of the distant geographical distribution of the plants (Nick et al. 1992).The work described in Chapter 3 is focussed on the localisation and phylogenetic position of the nitrogen-fixing Frankia and Nod -/Fix -actinomycetes, both present in root nodules of the Mexican actinorhizal plant Ceanothus caeruleus . Application of the TGGE technique allowed localising the Nod -/Fix -actinomycete in the outer layers of the C. caeruleus nodules. Similar bacteria were also detected in Hippophaë rhamnoides nodules induced with soil inoculum that was collected in the vicinity of the former plant. The fact that a second nodule inhabitant was commonly present in these nodules containing recalcitrant endophytes may allow some speculations about their possible role in the symbiosis. However, it seems worthwhile to apply the same TGGE methodology to other actinorhizal nodules, even to those containing Frankia strains that are easy-to-isolate (i.e. Elaeagnus,Casuarina, Alnus spp.), since the detection of Frankia -related actinomycetes, in addition to the Fix +endophyte, would provide further evidence about the need for their presence. Coincidentally, the Nod -/Fix -isolates from Coriaria, Datisca and Ceanothus are phylogenetically related, pointing again to a certain specificity for their presence in the nodules. This relatedness has also been supported by analysis of low molecular weight RNA (i.e. 5S rRNA and tRNA's) using staircase electrophoresis (Velázquez et al. 1998).The 16S rDNA sequence from the non-isolated Fix +endophyte in C. caeruleus root nodules (Chapter 3), was the first full sequence obtained from a field-collected Ceanothus symbiont. Parsimony and phylogenetic distance analyses grouped it within the Dryas cluster that originally contained only the uncultured endophytes from Dryas , Coriaria and Datisca as proposed by Normand et al. (1996). Benson et al . (1996) redefined this cluster by adding other uncultured endophytes present in Ceanothus griseus (Rhamnaceae), Purshia tridentata and Dryas drummondii (Rosaceae) root nodules. Since the determined partial 16S rDNA sequences were almost identical, they suggested that the Frankia diversity from these actinorhizal plant families might be low. However, Clawson et al. (1998) demonstrated that Frankia isolates obtained from several genera within the Rhamnaceae (i.e. Talguenea , Colletia , Discaria, Retanilla and Trevoa ) were phylogenetically different than those in Ceanothus, grouping in the Elaeagnus cluster. These findings were consistent with morphological differences of the endophytes in planta , since the vesicles found in the Ceanothus symbionts resemble more to those in the Rosaceae, while all the latter host plants in the Rhamnaceae family have endophytes like those in Elaeagnus .The results reported in the first part of the thesis have demonstrated that TGGE and sequence analysis of 16S rDNA provide an accurate picture for the identification of recalcitrant endophytes in root nodules of actinorhizal plants. It has also been demonstrated that besides the N 2 -fixing endophyte, root nodules of C. caeruleus also harbour Frankia -related actinomycetes. Since these have also been observed in other actinorhizae, a further study is needed to understand the possible function of these co-symbionts.The work described in the second part of the thesis was addressing the changes occurring under chlorobenzoate stress in the soil bacterial community and other selected groups of bacteria present in peat soil collected from a natural Alnus glutinosa stand. A combination of culturing and non-culture based approaches was used for the assessment. Among the latter approaches, the possibilities offered by TGGE were exploited in several ways. Profiling of complex communities and subsequent analysis of specific bacterial groups has been one of the major applications of TGGE (Felske et al . 1996). With this approach, major population shifts induced by either 3CBA or 2,5DCB were detected in the uncultured bacterial community (Chapter 4). Although only the former compound was readily metabolised in soil, both xenobiotics promoted similar changes. Several bacterial populations were reduced or suppressed, while few others were enriched in time, as assessed by shifts in the TGGE banding patterns of the total bacterial community.To characterise the soil-enriched bacterial populations, 3CBA-degrading enrichment cultures were obtained and their composition was addressed by TGGE. Further isolation attempts were directed by this means to prove that the isolated strains were indeed the same enriched organisms as detected in soil. One of the enrichment cultures contained two of the soil-enriched bacteria as predominant components. Although isolation was not achieved, both bacteria were identified as belonging to the genus Burkholderia . The bacterial group detected as predominantly enriched in both spiked soils was not present in any of the enrichment cultures, suggesting that the microorganisms belonging to this groups are either unable to degrade 3CBA or not growing under the used culturing conditions. In any case their fitness to the soil conditions imposed by the addition of chlorobenzoates was high, but the mechanisms involved were not elucidated. These bacteria were also identified as Burkholderia by partial 16S rDNA sequence analysis (Chapter 4).The diversity (H) and the equitability (J) indices are important parameters used by ecologists to assess the species richness and the species evenness, respectively, within a community. As the estimation of such indices relies heavily on species definition and individuals enumeration, their application in microbial ecology studies is seldomly possible. Furthermore, assessment of H and J in uncultured bacterial communities must rely on the interpretation of community fingerprints, which should provide means to distinguish between species or operational taxonomic units (OTU), and to estimate their abundance. TGGE community profiling offers both possibilities, and the community changes occurring in the model soil system were evaluated with this original approach (Chapter 5).In addition, H and J indices were also estimated for the fluorescent pseudomonads group, a selected culturable fraction of the bacterial community. OTU recognition was addressed by using TGGE as a ribotype-fingerprinting technique for the isolated fluorescent pseudomonads. Estimation of H and J at the community level without culturing by TGGE profiling, and at the group level by a combination of culturing and TGGE ribotyping should allow to address and compare the population changes occurring, since the target molecule used in both TGGE was the same. Such comparison was only partially possible since most of the bands corresponding to the fluorescent pseudomonads could not be assessed in the community profiles. However, estimation of H and J indices indicated a clear reduction of species richness and individuals abundance in the uncultured community, which was related to the presence of chlorobenzoates in soil. Evaluation of population shifts by indexed values as H and J proved to be a useful means for analysing the community structure in time, and may be used to assess short and long-term responses of a bacterial community to environmental perturbations.Chapter 6 describes the changes in the total frankiae in soil and in the fraction of the population that is able to produce root nodules in Alnus glutinosa seedlings . Culture-independent approaches based on the most probable number concept were used, one in combination with a Frankia -specific PCR detection and another in combination with a plant-nodulation bioassay. After 15 days of incubation in the presence of chlorobenzoates both fractions of the soil Frankia populations were reduced in more than one order of magnitude, while the populations in the unspiked control soil were not affected. The results indicated that 3CBA and 2,5DCB both had a negative effect on the size of the native Frankia population from the used peat forest soil. This negative effect was also evident during in vitro experiments using Frankia strains isolated from Alnus sp. The presence of 1 mM 3CBA in the culture medium, in addition to the normal carbon source, resulted in reduction or suppression of biomass yield.The influence of Alnus glutinosa on the dechlorination of 3CBA by Pseudomonas sp. strain B13 was assessed in hydroponic cultures. It was expected that root exudates could enhance the dechlorination activity of Pseudomonas B13. When the bacteria were incubated in the presence of an alder plant, only a slight increase in the dechlorination rate of 3CBA was registered in comparison to the control without plant. The main observed effect in the alder plants appears to be a protection against 3CBA toxicity, as the alders inoculated with Pseudomonas B13 showed a better survival rate and grew more vigorously than the non-inoculated plants.The toolbox for microbial ecology studies is increasing constantly by means of developing new techniques or by adapting foreign tools into the field, such as indices to evaluate species diversity and eveness. Although the information provided by these community parameters facilitates comparisons and assessment of changes, their suitability to evaluate bacterial communities is still uncomplete. Estimation of diversity indices requires the recognition of bacterial species as discrete units, and this condition is far from real in natural environments. Species-independent approaches to evaluate diversity must be developed, that consider the bacterial diversity as a continuous range of phylogenetically related taxonomic units.In conclusion, the work described in the first part of the thesis strengthen the current phylogenetic division of the Frankiaceae, by adding new evidence supporting two of the already described clusters ( Dryas cluster, Nod -/Fix -cluster). Although the current taxonomic status of the latter cluster must be better evaluated in order to assess its pertinence to the genus Frankia . In addition, the common occurrence of Nod -/Fix -Frankia -like actinomycetes in nodules containing recalcitrant endophytes, mainly from the Dryas cluster, was also demonstrated for Ceanothus actinorhiza. In the second part, the suitability and versatility of molecular tools such as TGGE were demonstrated by their application in community profiling and estimation of bacterial diversity. Community changes occurring in stressed and unstressed soil systems were easily detected and assessed by this means.In addition, specific populations such as the Burkholderia -like bacteria that strongly reacted to the addition of chlorobenzoates to soil were further characterised. Moreover, TGGE was shown to be a fast ribotyping technique that may enable its use in combination with the community profiles to address shifts of specific groups within bacterial communities. It is tempting to suggest that this general approach will be of importance to direct the isolation of hitherto uncultured bacteria from soil.
AB - Microbial Ecology studies aim to describe and assess the behavior and activity of microorganisms in their natural environments (Brock 1966). Nowadays it is clear that the large number of existing microorganisms has surpassed our capabilities to rapidly characterise them by traditional culturing methods. This has resulted in a poor understanding of the structure and composition of microbial communities. As an alternative, microbial communities can be described on the basis of 16S rRNA sequence diversity, without the bias-introducing step of cultivation.In the present thesis a molecular analysis is given of two ecosystems that harbour several uncultured bacteria. The first part of the thesis is focused on the detection and characterisation of Frankia in actinorhizal nodules and soil. Elucidation of the actual diversity within the family Frankiaceae was hampered by the inability to obtain isolates from all known actinorhizal plants. So far, the Nod +/Fix +Frankia symbionts in root nodules of plants from the families Coriariaceae, Datiscaceae, Rosaceae and Rhamnaceae (with exceptions reported by Carú 1993, Carú et al . 1990, and Carrasco et al . 1995) have resisted isolation. Best opportunities to characterise those uncultured endophytes require molecular methods that relay heavily on an easy and efficient technique to extract DNA from the respective actinorhizal nodules. Chapter 2 describes the techniques to isolate DNA from root nodules of different actinorhizal plants such as Casuarina sp, Alnus sp and Ceanothus sp. The procedure has also been successfully applied by Wolters et al . (1997b) in the minuscule ineffective nodules on Alnus glutinosa .Several attempts to characterise the uncultured endophytes from Coriaria sp. and Datisca sp. plants pointed on the one hand, to the presence in those actinorhizae of Frankia -related actinomycetes. This assumption was based mainly on the repeated isolation from those nodules of Nod -/Fix -Frankia -like strains (Hafeez 1983, Mirza et. al . 1994b, c). On the other hand, the effective (Fix +), non-isolated symbionts showed to be phylogenetically closely related (Mirza et al. 1994a), forming a separate lineage within the genus, in spite of the distant geographical distribution of the plants (Nick et al. 1992).The work described in Chapter 3 is focussed on the localisation and phylogenetic position of the nitrogen-fixing Frankia and Nod -/Fix -actinomycetes, both present in root nodules of the Mexican actinorhizal plant Ceanothus caeruleus . Application of the TGGE technique allowed localising the Nod -/Fix -actinomycete in the outer layers of the C. caeruleus nodules. Similar bacteria were also detected in Hippophaë rhamnoides nodules induced with soil inoculum that was collected in the vicinity of the former plant. The fact that a second nodule inhabitant was commonly present in these nodules containing recalcitrant endophytes may allow some speculations about their possible role in the symbiosis. However, it seems worthwhile to apply the same TGGE methodology to other actinorhizal nodules, even to those containing Frankia strains that are easy-to-isolate (i.e. Elaeagnus,Casuarina, Alnus spp.), since the detection of Frankia -related actinomycetes, in addition to the Fix +endophyte, would provide further evidence about the need for their presence. Coincidentally, the Nod -/Fix -isolates from Coriaria, Datisca and Ceanothus are phylogenetically related, pointing again to a certain specificity for their presence in the nodules. This relatedness has also been supported by analysis of low molecular weight RNA (i.e. 5S rRNA and tRNA's) using staircase electrophoresis (Velázquez et al. 1998).The 16S rDNA sequence from the non-isolated Fix +endophyte in C. caeruleus root nodules (Chapter 3), was the first full sequence obtained from a field-collected Ceanothus symbiont. Parsimony and phylogenetic distance analyses grouped it within the Dryas cluster that originally contained only the uncultured endophytes from Dryas , Coriaria and Datisca as proposed by Normand et al. (1996). Benson et al . (1996) redefined this cluster by adding other uncultured endophytes present in Ceanothus griseus (Rhamnaceae), Purshia tridentata and Dryas drummondii (Rosaceae) root nodules. Since the determined partial 16S rDNA sequences were almost identical, they suggested that the Frankia diversity from these actinorhizal plant families might be low. However, Clawson et al. (1998) demonstrated that Frankia isolates obtained from several genera within the Rhamnaceae (i.e. Talguenea , Colletia , Discaria, Retanilla and Trevoa ) were phylogenetically different than those in Ceanothus, grouping in the Elaeagnus cluster. These findings were consistent with morphological differences of the endophytes in planta , since the vesicles found in the Ceanothus symbionts resemble more to those in the Rosaceae, while all the latter host plants in the Rhamnaceae family have endophytes like those in Elaeagnus .The results reported in the first part of the thesis have demonstrated that TGGE and sequence analysis of 16S rDNA provide an accurate picture for the identification of recalcitrant endophytes in root nodules of actinorhizal plants. It has also been demonstrated that besides the N 2 -fixing endophyte, root nodules of C. caeruleus also harbour Frankia -related actinomycetes. Since these have also been observed in other actinorhizae, a further study is needed to understand the possible function of these co-symbionts.The work described in the second part of the thesis was addressing the changes occurring under chlorobenzoate stress in the soil bacterial community and other selected groups of bacteria present in peat soil collected from a natural Alnus glutinosa stand. A combination of culturing and non-culture based approaches was used for the assessment. Among the latter approaches, the possibilities offered by TGGE were exploited in several ways. Profiling of complex communities and subsequent analysis of specific bacterial groups has been one of the major applications of TGGE (Felske et al . 1996). With this approach, major population shifts induced by either 3CBA or 2,5DCB were detected in the uncultured bacterial community (Chapter 4). Although only the former compound was readily metabolised in soil, both xenobiotics promoted similar changes. Several bacterial populations were reduced or suppressed, while few others were enriched in time, as assessed by shifts in the TGGE banding patterns of the total bacterial community.To characterise the soil-enriched bacterial populations, 3CBA-degrading enrichment cultures were obtained and their composition was addressed by TGGE. Further isolation attempts were directed by this means to prove that the isolated strains were indeed the same enriched organisms as detected in soil. One of the enrichment cultures contained two of the soil-enriched bacteria as predominant components. Although isolation was not achieved, both bacteria were identified as belonging to the genus Burkholderia . The bacterial group detected as predominantly enriched in both spiked soils was not present in any of the enrichment cultures, suggesting that the microorganisms belonging to this groups are either unable to degrade 3CBA or not growing under the used culturing conditions. In any case their fitness to the soil conditions imposed by the addition of chlorobenzoates was high, but the mechanisms involved were not elucidated. These bacteria were also identified as Burkholderia by partial 16S rDNA sequence analysis (Chapter 4).The diversity (H) and the equitability (J) indices are important parameters used by ecologists to assess the species richness and the species evenness, respectively, within a community. As the estimation of such indices relies heavily on species definition and individuals enumeration, their application in microbial ecology studies is seldomly possible. Furthermore, assessment of H and J in uncultured bacterial communities must rely on the interpretation of community fingerprints, which should provide means to distinguish between species or operational taxonomic units (OTU), and to estimate their abundance. TGGE community profiling offers both possibilities, and the community changes occurring in the model soil system were evaluated with this original approach (Chapter 5).In addition, H and J indices were also estimated for the fluorescent pseudomonads group, a selected culturable fraction of the bacterial community. OTU recognition was addressed by using TGGE as a ribotype-fingerprinting technique for the isolated fluorescent pseudomonads. Estimation of H and J at the community level without culturing by TGGE profiling, and at the group level by a combination of culturing and TGGE ribotyping should allow to address and compare the population changes occurring, since the target molecule used in both TGGE was the same. Such comparison was only partially possible since most of the bands corresponding to the fluorescent pseudomonads could not be assessed in the community profiles. However, estimation of H and J indices indicated a clear reduction of species richness and individuals abundance in the uncultured community, which was related to the presence of chlorobenzoates in soil. Evaluation of population shifts by indexed values as H and J proved to be a useful means for analysing the community structure in time, and may be used to assess short and long-term responses of a bacterial community to environmental perturbations.Chapter 6 describes the changes in the total frankiae in soil and in the fraction of the population that is able to produce root nodules in Alnus glutinosa seedlings . Culture-independent approaches based on the most probable number concept were used, one in combination with a Frankia -specific PCR detection and another in combination with a plant-nodulation bioassay. After 15 days of incubation in the presence of chlorobenzoates both fractions of the soil Frankia populations were reduced in more than one order of magnitude, while the populations in the unspiked control soil were not affected. The results indicated that 3CBA and 2,5DCB both had a negative effect on the size of the native Frankia population from the used peat forest soil. This negative effect was also evident during in vitro experiments using Frankia strains isolated from Alnus sp. The presence of 1 mM 3CBA in the culture medium, in addition to the normal carbon source, resulted in reduction or suppression of biomass yield.The influence of Alnus glutinosa on the dechlorination of 3CBA by Pseudomonas sp. strain B13 was assessed in hydroponic cultures. It was expected that root exudates could enhance the dechlorination activity of Pseudomonas B13. When the bacteria were incubated in the presence of an alder plant, only a slight increase in the dechlorination rate of 3CBA was registered in comparison to the control without plant. The main observed effect in the alder plants appears to be a protection against 3CBA toxicity, as the alders inoculated with Pseudomonas B13 showed a better survival rate and grew more vigorously than the non-inoculated plants.The toolbox for microbial ecology studies is increasing constantly by means of developing new techniques or by adapting foreign tools into the field, such as indices to evaluate species diversity and eveness. Although the information provided by these community parameters facilitates comparisons and assessment of changes, their suitability to evaluate bacterial communities is still uncomplete. Estimation of diversity indices requires the recognition of bacterial species as discrete units, and this condition is far from real in natural environments. Species-independent approaches to evaluate diversity must be developed, that consider the bacterial diversity as a continuous range of phylogenetically related taxonomic units.In conclusion, the work described in the first part of the thesis strengthen the current phylogenetic division of the Frankiaceae, by adding new evidence supporting two of the already described clusters ( Dryas cluster, Nod -/Fix -cluster). Although the current taxonomic status of the latter cluster must be better evaluated in order to assess its pertinence to the genus Frankia . In addition, the common occurrence of Nod -/Fix -Frankia -like actinomycetes in nodules containing recalcitrant endophytes, mainly from the Dryas cluster, was also demonstrated for Ceanothus actinorhiza. In the second part, the suitability and versatility of molecular tools such as TGGE were demonstrated by their application in community profiling and estimation of bacterial diversity. Community changes occurring in stressed and unstressed soil systems were easily detected and assessed by this means.In addition, specific populations such as the Burkholderia -like bacteria that strongly reacted to the addition of chlorobenzoates to soil were further characterised. Moreover, TGGE was shown to be a fast ribotyping technique that may enable its use in combination with the community profiles to address shifts of specific groups within bacterial communities. It is tempting to suggest that this general approach will be of importance to direct the isolation of hitherto uncultured bacteria from soil.
KW - bodembacteriën
KW - frankia
KW - microbiële ecologie
KW - benzoaten
KW - soil bacteria
KW - microbial ecology
KW - benzoates
M3 - internal PhD, WU
PB - Ramirez Saad
Ramirez-Saad HC. Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions. S.l.: Ramirez Saad, 1999. 119 p.


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text:
<?php

namespace Oro\Bundle\PlatformBundle\Tests\Unit\DependencyInjection;

use Symfony\Component\DependencyInjection\ContainerBuilder;
use Symfony\Component\DependencyInjection\Definition;

use Oro\Bundle\PlatformBundle\DependencyInjection\Compiler\UpdateDoctrineEventHandlersPass;

class UpdateDoctrineEventHandlersPassTest extends \PHPUnit_Framework_TestCase
{
    /** @var UpdateDoctrineEventHandlersPass */
    protected $compiler;

    /** @var \PHPUnit_Framework_MockObject_MockObject|ContainerBuilder */
    protected $container;

    protected function setUp()
    {
        $this->compiler = new UpdateDoctrineEventHandlersPass();
        $this->container = $this->getMockBuilder('Symfony\Component\DependencyInjection\ContainerBuilder')
            ->disableOriginalConstructor()->getMock();
    }

    /**
     * @param bool $hasConnectionParameter
     * @param bool $hasExcludedParameter
     * @param mixed $connectionParameter
     * @param mixed $excludedParameter
     * @param array $tags
     *
     * @dataProvider dataProvider
     */
    public function testProcess(
        $hasConnectionParameter,
        $hasExcludedParameter,
        $connectionParameter,
        $excludedParameter,
        array $tags = []
    ) {
        $this->container->expects($this->any())->method('hasParameter')
            ->willReturnOnConsecutiveCalls($hasConnectionParameter, $hasExcludedParameter);
        $this->container->expects($this->any())->method('getParameter')
            ->willReturnOnConsecutiveCalls($connectionParameter, $excludedParameter);

        $this->container->expects($this->any())->method('findTaggedServiceIds')
            ->willReturn(['id' => ['event' => ['tag1' => []]]]);

        $definition = new Definition();
        $this->container->expects($this->any())->method('getDefinition')->willReturn($definition);

        $this->compiler->process($this->container);

        $this->assertEquals($tags, $definition->getTags());
    }

    /** @return array */
    public function dataProvider()
    {
        return [
            'missing connections' => [false, false, [], [], []],
            'missing excluded connections' => [true, false, [], [], []],
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redpajama_set_name: RedPajamaGithub


Kubodera, T. (2000)Family Chiroteuthidae.In: Okutani, T. (ed.), Marine Mollusks in Japan. Tokai University Press, Tokyo, 1071 (in Japanese).
Okutani, T. (2005)Cuttlefishes and Squids of the World.National Cooperative Association of Squid Processors, Tokyo. 253 pp. (in Japanese).


text:
Michigan Department of Civil Rights: Discrimination against LGBT community hurts state economy
Updated Apr 03, 2019; Posted Jan 30, 2013
About 150 people marched through downtown Holland in 2011, urging city officials to add sexual orientation and gender identity to Holland's anti-discrimination ordinance.The city voted down the measure.
By Melissa Anders | manders@mlive.com
LANSING, MI — A report from the Michigan Department of Civil Rights found that Michigan's economy would benefit from an anti-discrimination law that includes protections for lesbian, gay, bisexual and transgender residents.
The study found that the "current state of discrimination" compels some professionals and college graduates to leave Michigan in favor of more welcoming states. It also impacts businesses' competitive advantage in recruiting and retaining a skilled workforce, ability to attract a diverse customer base and employee productivity and morale, according to the report.
"The report conclusively establishes that the economic implications of LGBT inclusion/exclusion are real, they are substantial, they are predictable and they must be a part of any informed policy discussion," MDCR Executive Director Daniel Krichbaum wrote in a letter given to Michigan civil rights commissioners on Monday.
Related: Is Michigan gay friendly? Advocates encouraged by changing attitudes, but seek fairness first
The one-year project was funded with a $100,000 grant from the Tides Foundation, an LGBT advocacy group.
Leslee Fritz, the MDCR's director of public affairs, defended the department's use of the grant from the advocacy group.
"There were no pre-determined outcomes and that shows in the report — it is thorough and thoughtful, using all existing data and research available," she said. "In addition, our outreach efforts included as many people as we could reach on both sides of this oft-debated issue."
The project included public forums, surveys and archival research. It largely focused on the Elliott Larsen Civil Rights Act of 1976, which prohibits discrimination in employment, housing and public accommodations based on religion, race, color, national origin, age, sex, height, weight, familial status, or marital status.
Democratic Sen. Rebekah Warren of Ann Arbor plans to reintroduce legislation that stalled in committee last session that would ban discrimination based on sexual orientation, gender identity or expression.
Republican Rep. Tom McMillin of Rochester introduced a bill that also stalled in committee last session that would have prohibited municipalities from adding to their local ordinances any protected class not already included in the state anti-discrimination law.
McMillin and Warren did not return phone calls requesting comment.
The MDCR has long supported adding sexual orientation and gender identity/expression to the state's anti-discrimination law, Fritz said. She said she was most surprised by the study's finding that many people don't realize Michigan doesn't already outlaw discrimination against the LGBT community.
"We need to do a better job of educating the public and doing public outreach to make sure that people understand that what they want in their community … is not the way Michigan law is today," she said.
The study did not put a dollar figure on the policy's economic impact, but it outlined anecdotal and survey evidence that suggests the law's exclusion of the LGBT community negatively affects everything from household incomes and public assistance programs to consumer spending and tourism.
About one-fifth of the 180 testimonials given to the MDCR alluded to people leaving the state for reasons related to discrimination, the report said.
"Anywhere I decide to call home will need to provide protections from discrimination and civil unions, at a minimum. I won't even consider staying here after graduation, no matter how good the job offer," according to one person's testimony. The report did not release full names for most people who testified to protect their privacy.
Holland City Councilman Brian Burch, who voted with the majority against adding sexual orientation to the city's anti-discrimination ordinance, countered that there are larger issues the state should address when it comes to young people leaving the state.
"Is it a factor for some people? I imagine it is," he said. "If we're talking about brain drain, we need to talk about larger issues, creating opportunities for students, jobs people want … valuing education."
He also said that Holland is a leader in certain job growth measures.
Social issues also can't be ignored, said James Muffett, president of Citizens for Traditional Values, a conservative organization based in Lansing.
"I think that this report leaves a lot to be discussed about the social impact, the impact on families, the impact on children," he said.
Muffett said he's against "creating a laundry list of protected classes in the law." Civil rights protections should include immutable characteristics that can't be changed, he said. Adding sexual preference means that other characteristics, such as someone's weight, also would have to be included.
Krichbaum said the MDCR report did not set out to address the moral and religious issues related to homosexuality.
The report outlined several recommendations for the Michigan Civil Rights Commission. It urged the commission to pursue non-discrimination policy changes and to educate citizens, business owners and local officials about the issue.
Equality Michigan applauded the department for its work.
"The Legislature and Governor Snyder, who were elected on promises of focusing on economic barriers, now have a roadmap to cultivating the diverse and inclusive Michigan that has proven to result in economic growth," Emily Dievendorf, Equality Michigan's policy director, said in a statement.
Email Melissa Anders at manders@mlive.com. Follow her on Twitter: @MelissaDAnders. Download the MLive app for iPhone and Android.