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Het project N2Africa is onlangs de tweede fase ingegaan met als doel dat in 2020 een half miljoen kleine boeren in Afrika, ten zuiden van de Sahara, stikstofbinding hebben geïntegreerd in hun bedrijfsvoering. Op een manier die hen past, zegt Ken Giller. Stikstofbinding verbetert de bodemvruchtbaarheid, terwijl de teelt van bonen, die samen met bacteriën de stikstof vastleggen, een belangrijke aanvulling vormt op het menu en op het inkomen van de boer.

Hoogleraar Ken Giller propageert onder Afrikaanse boeren het gebruik van peulvruchten. Die hebben dankzij hulp van bacteriën geen stikstofmeststof nodig. In Wageningen onderzoekt hoogleraar Ton Bisseling de finesses van deze symbiose.

Het project N2Africa is onlangs de tweede fase ingegaan met als doel dat in 2020 een half miljoen kleine boeren in Afrika, ten zuiden van de Sahara, stikstofbinding hebben geïntegreerd in hun bedrijfsvoering. Op een manier die hen past, zegt Ken Giller. Stikstofbinding verbetert de bodemvruchtbaarheid, terwijl de teelt van bonen, die samen met bacteriën de stikstof vastleggen, een belangrijke aanvulling vormt op het menu en op het inkomen van de boer.

Onderdeel van het nieuwe Gemeenschappelijk Landbouw Beleid (GLB) is de vergroening. Deze vergroening houdt in dat 30% van de directe betalingen in Pijler 1 alleen uitbetaald worden aan boeren als zij voldoen aan verplichte beheersmaatregelen die gunstig zijn voor natuur en biodiversiteit en ook voor milieu en klimaat. Dit rapport bevat adviezen over de nationale invulling van de vergroening vanuit het perspectief van natuur en biodiversiteit. Er wordt verkend welke elementen wel of niet erkend zouden moeten worden als ecological focus area (EFA) en welke graslanden buiten Natura 2000 in aanmerkingen komen voor een ploeg- en omzetverbod. Tenslotte wordt advies uitgebracht over welke gebieden met natuurlijke handicaps in aanmerking zouden kunnen komen voor een ‘top-up’ en wat de hoogte van deze betaling kan zijn.

Onderdeel van het nieuwe Gemeenschappelijk Landbouw Beleid (GLB) is de vergroening. Deze vergroening houdt in dat 30% van de directe betalingen in Pijler 1 alleen uitbetaald worden aan boeren als zij voldoen aan verplichte beheersmaatregelen die gunstig zijn voor natuur en biodiversiteit en ook voor milieu en klimaat. Dit rapport bevat adviezen over de nationale invulling van de vergroening vanuit het perspectief van natuur en biodiversiteit. Er wordt verkend welke elementen wel of niet erkend zouden moeten worden als ecological focus area (EFA) en welke graslanden buiten Natura 2000 in aanmerkingen komen voor een ploeg- en omzetverbod. Tenslotte wordt advies uitgebracht over welke gebieden met natuurlijke handicaps in aanmerking zouden kunnen komen voor een ‘top-up’ en wat de hoogte van deze betaling kan zijn.

Hoogleraar Ken Giller propageert onder Afrikaanse boeren het gebruik van peulvruchten. Die hebben dankzij hulp van bacteriën geen stikstofmeststof nodig. In Wageningen onderzoekt hoogleraar Ton Bisseling de finesses van deze symbiose.

The effect of calcium on the nodulation of lucerne was studied using EGTA, a specific calcium-chelator. First, the effects of the chelator were tested on hydroponically grown plants at pH 7.0. Optimal numbers of nodules were obtained in nutrient solution containing 0.2 mM CaCl2. When 0.4 mM EGTA was given additionally, nodulation was completely inhibited. Nodulation was restored specifically with CaCl2, but not with MgCl2. For studies in an acid soil (pH-H2O 5.2), lucerne seedlings were grown in rhizotrons. 67% of the seedlings became nodulated when the soil around the seed was neutralized locally with 1.0 μmol of K2CO3 in drops of 12 μL volume. When native calcium was removed with 2 μmol of EGTA, nodulation was reduced to 12%. However, addition of EGTA to soil resulted in a drop of pH from 6.1 to 5.2. A phosphate buffer could also not keep soil-pH sufficiently stable. Such pH-decreases could be avoided by placing agar blocks containing 6 μmol of EGTA for three hours on freshly developed roots. This treatment reduced nodulation from 87% to 32%, with soil-pH lowering only from 6.2 to 6.0. Nodulation could be restored by adding 2 μmol of CaCl2. The depletion of soil-calcium could depress nodule formation only during the first day after inoculation.

Leguminous plants are able to establish a symbiotic interaction with nitrogen-fixing soil bacteria, generally called rhizobia. This host-specific interaction results in the formation of a completely new organ, the root nodule, where the bacteria are hosted intracellularly and are able to fix atmospheric nitrogen. A successful interaction requires the strict coordination of two processes: the formation of the nodule organ from root cortical cells and infection of this new organ by the bacteria through tubular structures, called infection threads, whose formation is started in the root hairs. To initiate these processes a molecular dialogue is required between the two partners. Upon perception of flavonoids secreted by the plant the rhizobia produce lipo-chitooligosaccharidic signalling molecules, the Nod factors, which are essential and in most cases sufficient for the induction of symbiotic responses in the host plant. These Nod factors have a common basic structure consisting of three to fiveb-1,4-linked N -acetyl-D-glucosamine subunits with an acyl chain linked to the non-reducing terminal sugar residue. Depending on the rhizobial species, the structure of the acyl chain can vary and specific decorations at the reducing and non-reducing terminal glucosamine residues can be present, which function as major determinants of the host-specificity of the interaction. Nod factors are biologically active at nano- to picomolar concentrations and their activity depends on their structure, which implies that they are perceived by specific receptors. The major goal of the research described in this thesis was to unravel the molecular basis of Nod factor perception. Therefore we focussed on the pea SYM2 gene, which was proposed to be involved in Nod factor perception. In the pea accession Afghanistana SYM2 A allele was identified, which controls rhizobial infection in a Nod factor structure dependent manner. Only Rhizobium leguminosarumbvviciae strains that contain the bacterial nodulation gene, nodX , which specifically acetylates the reducing terminal sugar residue of pentameric Nod factors, are allowed to successfully infect pea plants containing the SYM2 A allele fromAfghanistan. This suggests that SYM2 A is involved in recognizing these nodX modified Nod factors and it would be attractive to clone this gene.However, positional cloning in pea is severely hampered because of its large genome size (~5000 Mb) and it is difficult to transform this legume. Therefore we decided to use a synteny based positional cloning approach in the phylogenetically closely related model legume Medicago truncatula (Medicago).In Chapter 1 an overview is given of the different steps of nodulation with a focus on the molecular basis of Nod factor perception and transduction as it was known at the start of the research.In Chapter 2 the level of microsynteny between pea and Medicago was studied in the SYM2 region. This resulted in the delineation of a ~350 kb physical BAC contig representing the SYM2 orthologous region in Medicago.A conservedgene content was observed in the SYM2 orthologous regions, which supports the idea that Medicago can be used as intergenomic vehicle to clone pea genes.Because SYM2 represent a natural occurring variation in pea, sequence analysis could not be used to identify the SYM2 gene. Therefore we developed a reverse genetics approach in Medicago to identify genes involved in rhizobial infection. In Chapter 3 we report the effective use of RNA interference (RNAi) via Agrobacterium rhizogenes mediated root transformation as a reverse genetic tool to knock down gene expression in the roots of both Arabidopsis and Medicago. A. rhizogenes mediated root transformation has the advantage that it is a relatively fast method to generate genetically transformed roots. It is further shown that the silencing signal does not spread to non co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, RNAi appeared to be cell-autonomous in the epidermis of Medicago roots.In Chapter 4 we used A. rhizogenes mediated RNAi to knock down candidate genes from the Medicago SYM2 othologous region and examined their role in rhizobial infection. We survey-sequenced the SYM2 orthologous region and strikingly many genes encoding receptor-like proteins appeared to be present. Knock down by RNAi of two genes, LYK3 and LYK4 , affected rhizobial infection. By using Sinorhizobiummeliloti strains that are mutated in their nod genes, a block of infection thread formation due to knock down of LYK3/4 was shown to be correlated to the structure of the produced Nod factors. The two identified genes encode LysM domain containing receptor-like kinases. The nature of the LysM domains together with the Nod factor structure dependent block of rhizobial infection strongly suggests that these genes represent Nod factor receptors.The formation of a functional root nodule requires the tight coordination of the infection process by the bacteria and the formation of the nodule organ. In all mutants currently characterized the loss of (all) Nod factor responses in the epidermis is always correlated with the loss of the cortical cell division (nodule primordium) response. This led to the hypothesis that Nod factor signalling in the epidermis results in the generation of a secondary signal that subsequently triggers cortical cell division. In Chapter 5 we show that the presence of an essential component of the Nod factor perception and transduction machinery, NODULATION RECEPTOR KINASE ( NORK ), exclusively in the epidermis is sufficient to trigger cortical cell divisions by external application of Nod factors. This shows that mitotic activation of cortical cells is triggered by Nod factors in a non cell-autonomous manner and implies the involvement of intercellular communication in Nod factor signalling.Several lines of evidence indicate that the Nod factor perception and transduction machinery also plays a role in the nodule. In Chapter 6 we show that NORK expression is highly upregulated in the nodule and the region where it is expressed coincides with rhizobial nod gene activity in the infection zone of the nodule where bacteria are released into the plant host cells. By mimicking allelic series of NORK via RNA interference and by expression of 35S::NORK constructs in the mutant background we show that NORK controls the switch from infection thread growth to release of bacteria in the nodule. This suggests a role for the Nod factor perception and signaling machinery in this process.Finally Chapter 7 summarizes and discusses the results obtained by the research described in this thesis.

Leguminous plants are able to establish a symbiotic interaction with nitrogen-fixing soil bacteria, generally called rhizobia. This host-specific interaction results in the formation of a completely new organ, the root nodule, where the bacteria are hosted intracellularly and are able to fix atmospheric nitrogen. A successful interaction requires the strict coordination of two processes: the formation of the nodule organ from root cortical cells and infection of this new organ by the bacteria through tubular structures, called infection threads, whose formation is started in the root hairs. To initiate these processes a molecular dialogue is required between the two partners. Upon perception of flavonoids secreted by the plant the rhizobia produce lipo-chitooligosaccharidic signalling molecules, the Nod factors, which are essential and in most cases sufficient for the induction of symbiotic responses in the host plant. These Nod factors have a common basic structure consisting of three to fiveb-1,4-linked N -acetyl-D-glucosamine subunits with an acyl chain linked to the non-reducing terminal sugar residue. Depending on the rhizobial species, the structure of the acyl chain can vary and specific decorations at the reducing and non-reducing terminal glucosamine residues can be present, which function as major determinants of the host-specificity of the interaction. Nod factors are biologically active at nano- to picomolar concentrations and their activity depends on their structure, which implies that they are perceived by specific receptors. The major goal of the research described in this thesis was to unravel the molecular basis of Nod factor perception. Therefore we focussed on the pea SYM2 gene, which was proposed to be involved in Nod factor perception. In the pea accession Afghanistana SYM2 A allele was identified, which controls rhizobial infection in a Nod factor structure dependent manner. Only Rhizobium leguminosarumbvviciae strains that contain the bacterial nodulation gene, nodX , which specifically acetylates the reducing terminal sugar residue of pentameric Nod factors, are allowed to successfully infect pea plants containing the SYM2 A allele fromAfghanistan. This suggests that SYM2 A is involved in recognizing these nodX modified Nod factors and it would be attractive to clone this gene.However, positional cloning in pea is severely hampered because of its large genome size (~5000 Mb) and it is difficult to transform this legume. Therefore we decided to use a synteny based positional cloning approach in the phylogenetically closely related model legume Medicago truncatula (Medicago).In Chapter 1 an overview is given of the different steps of nodulation with a focus on the molecular basis of Nod factor perception and transduction as it was known at the start of the research.In Chapter 2 the level of microsynteny between pea and Medicago was studied in the SYM2 region. This resulted in the delineation of a ~350 kb physical BAC contig representing the SYM2 orthologous region in Medicago.A conservedgene content was observed in the SYM2 orthologous regions, which supports the idea that Medicago can be used as intergenomic vehicle to clone pea genes.Because SYM2 represent a natural occurring variation in pea, sequence analysis could not be used to identify the SYM2 gene. Therefore we developed a reverse genetics approach in Medicago to identify genes involved in rhizobial infection. In Chapter 3 we report the effective use of RNA interference (RNAi) via Agrobacterium rhizogenes mediated root transformation as a reverse genetic tool to knock down gene expression in the roots of both Arabidopsis and Medicago. A. rhizogenes mediated root transformation has the advantage that it is a relatively fast method to generate genetically transformed roots. It is further shown that the silencing signal does not spread to non co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, RNAi appeared to be cell-autonomous in the epidermis of Medicago roots.In Chapter 4 we used A. rhizogenes mediated RNAi to knock down candidate genes from the Medicago SYM2 othologous region and examined their role in rhizobial infection. We survey-sequenced the SYM2 orthologous region and strikingly many genes encoding receptor-like proteins appeared to be present. Knock down by RNAi of two genes, LYK3 and LYK4 , affected rhizobial infection. By using Sinorhizobiummeliloti strains that are mutated in their nod genes, a block of infection thread formation due to knock down of LYK3/4 was shown to be correlated to the structure of the produced Nod factors. The two identified genes encode LysM domain containing receptor-like kinases. The nature of the LysM domains together with the Nod factor structure dependent block of rhizobial infection strongly suggests that these genes represent Nod factor receptors.The formation of a functional root nodule requires the tight coordination of the infection process by the bacteria and the formation of the nodule organ. In all mutants currently characterized the loss of (all) Nod factor responses in the epidermis is always correlated with the loss of the cortical cell division (nodule primordium) response. This led to the hypothesis that Nod factor signalling in the epidermis results in the generation of a secondary signal that subsequently triggers cortical cell division. In Chapter 5 we show that the presence of an essential component of the Nod factor perception and transduction machinery, NODULATION RECEPTOR KINASE ( NORK ), exclusively in the epidermis is sufficient to trigger cortical cell divisions by external application of Nod factors. This shows that mitotic activation of cortical cells is triggered by Nod factors in a non cell-autonomous manner and implies the involvement of intercellular communication in Nod factor signalling.Several lines of evidence indicate that the Nod factor perception and transduction machinery also plays a role in the nodule. In Chapter 6 we show that NORK expression is highly upregulated in the nodule and the region where it is expressed coincides with rhizobial nod gene activity in the infection zone of the nodule where bacteria are released into the plant host cells. By mimicking allelic series of NORK via RNA interference and by expression of 35S::NORK constructs in the mutant background we show that NORK controls the switch from infection thread growth to release of bacteria in the nodule. This suggests a role for the Nod factor perception and signaling machinery in this process.Finally Chapter 7 summarizes and discusses the results obtained by the research described in this thesis.

The aim of this study is to improve soybean cultivation in transmigration areas, especially in Sitiung, West Sumatra. However, these soils are very acid, and have a high P-fixing capacity. To reduce the amounts of fertilisers, normally 5 - 7 ton lime ha -1 and 100 kg P as TSP, seed, pelleted with lime (60 kg ha -1 ) and TSP (10 kg ha -1 ), was introduced. In this way only 2 ton lime ha -1 are required.Soybean can fix nitrogen (BNF) in symbiosis with ( Brady ) Rhizobium bacteria. However, these acid soils in general, have low numbers of ( Brady ) Rhizobium . By inoculating the soils with ( Brady ) Rhizobium , BNF of soybean, and yield, were considerably improved.A study was made of the indigenous ( Brady ) Rhizobium population in view of the following:Although at the beginning the numbers may be low, by repeated soybean cultivation, the numbers will increase, and they may interfere with inoculation of effective ( Brady ) Rhizobium strains.These indigenous ( Brady ) Rhizobium are adapted to local stress conditions, and they may be useful for the improvement of strains, to be used as inoculants.Using molecular techniques, indigenous strains derived from soil samples from old soybean areas (Java) and from new soybean areas (Sumatra) were classified in more detail. Most likely B. japonicum is the dominant strain in Java while in Sumatra B. elkanii is more present. A Sinorhizobium fredii -like strain was isolated from one soil sample from Java.

The aim of this study is to improve soybean cultivation in transmigration areas, especially in Sitiung, West Sumatra. However, these soils are very acid, and have a high P-fixing capacity. To reduce the amounts of fertilisers, normally 5 - 7 ton lime ha -1 and 100 kg P as TSP, seed, pelleted with lime (60 kg ha -1 ) and TSP (10 kg ha -1 ), was introduced. In this way only 2 ton lime ha -1 are required.Soybean can fix nitrogen (BNF) in symbiosis with ( Brady ) Rhizobium bacteria. However, these acid soils in general, have low numbers of ( Brady ) Rhizobium . By inoculating the soils with ( Brady ) Rhizobium , BNF of soybean, and yield, were considerably improved.A study was made of the indigenous ( Brady ) Rhizobium population in view of the following:Although at the beginning the numbers may be low, by repeated soybean cultivation, the numbers will increase, and they may interfere with inoculation of effective ( Brady ) Rhizobium strains.These indigenous ( Brady ) Rhizobium are adapted to local stress conditions, and they may be useful for the improvement of strains, to be used as inoculants.Using molecular techniques, indigenous strains derived from soil samples from old soybean areas (Java) and from new soybean areas (Sumatra) were classified in more detail. Most likely B. japonicum is the dominant strain in Java while in Sumatra B. elkanii is more present. A Sinorhizobium fredii -like strain was isolated from one soil sample from Java.

Eén van de belangrijkste moleculen in de plantenwereld is ammoniak (NH 3 ). Uitgaande van ammoniak synthetiseert de plant biomoleculen zoals eiwitten en nucleïnezuren. Planten zelf zijn niet in staat moleculair stikstof (N 2 ) te reduceren tot ammoniak. De biologische reductie van N 2 tot NH 3 wordt bewerkstelligd door diazotrope bacteriën. Deze bacteriën bevatten het enzymcomplex nitrogenase. Nitrogenase bestaat uit twee zuurstofgevoelige metalloproteïnen die afzonderlijk geïsoleerd kunnen worden.De grootste van deze metalloproteïnen is een α 2β2 -tetrameer. Elke αβ-unit functioneert als een onafhankelijke katalytische eenheid. De feitelijke reductie van N 2 vindt plaats op een ijzer-molybdeen cofactor (FeMoco). De voor de reductie benodigde elektronen worden in vivo geleverd door een ferredoxine of flavodoxine. Het MoFe-eiwit is niet in staat deze elektronen rechtstreeks op te nemen. In de katalytische cyclus van nitrogenase staat het ferredoxine of flavodoxine een elektron af aan de tweede component van het nitrogenase complex. Deze component is een Fe-eiwit, welk het elektron vervolgens aan het MoFe-eiwit doorgeeft.Reductie van een metalloprotein door een ander metalloprotein is een veel voorkomende reactie in de biochemie. Het bijzondere van nitrogenase is dat elektronoverdracht slechts in aanwezigheid van magnesium-adenosinetrifosfaat (MgATP) optreedt. Experimenten verricht onder steady-state condities hebben laten zien dat per elektron dat naar een substraat wordt getransporteerd, er twee MgATP moleculen worden gehydrolyseerd. Geruime tijd bestond er onzekerheid over de vraag waar in de katalytische cyclus MgATP wordt gehydrolyseerd. In 1978 publiceerde de Brighton groep een experiment waaruit werd geconcludeerd dat de hydrolyse van MgATP en elektrontransport van het Fe-eiwit naar het MoFe-eiwit direct gekoppeld zijn. De gevonden ratio mol MgATP gehydrolyseerd/mol elektron overgedragen was gelijk aan 1. De auteurs interpreteerden dit resultaat als een aanwijzing dat elders in de katalytische cyclus ook MgATP wordt gehydrolyseerd.De conclusie van de Brighton groep dat elektrontransport van het Fe-eiwit naar het MoFe-eiwit en de hydrolyse van MgATP gekoppeld zijn, werd algemeen aanvaard. Echter, een punt van discussie bleef de ratio mol MgATP gehydrolyseerd/mol elektron overgedragen. In 1980 verscheen een publikatie uit de groep van Burris waarin werd geconcludeerd dat deze ratio gelijk is aan 2, hetgeen impliceert dat de hydrolyse van MgATP alleen plaats vindt bij elektronoverdracht van het Fe-eiwit naar het MoFe-eiwit. Echter, in 1987 verscheen een publikatie van de vakgroep Biochemie uit Wageningen waarin werd gerapporteerd dat er 4 moleculen MgATP gehydrolyseerd kunnen worden per mol Fe- eiwit. Het Fe-eiwit heeft twee bindingsplaatsen voor MgADP of MgATP. Deze stoichiometrie impliceert dat er niet meer dan twee MgATP moleculen gehydrolyseerd kunnen worden per turnover van het Fe~ eiwit. Teneinde een ratio van 4 te kunnen verklaren, moest worden aangenomen dat twee extra bindingsplaatsen ontstaan wanneer het Fe-eiwit en MoFe-eiwit associëren.Het doel van dit proefschrift was duidelijkheid te krijgen in de rol die MgATP vervult in de pre-steady-state ATPase activiteit van nitrogenase. In hoofdstuk 2 wordt een onderzoek beschreven waarin 2-azido-analogen van MgAMP, MgADP, en MgATP worden gebruikt om de nucleotide bindingsplaatsen binnen het nitrogenase complex te karakteriseren. Met deze experimenten zijn geen aanwijzingen verkregen dat in het nitrogenase complex nucleotide bindingsplaatsen bestaan naast die op het Fe-eiwit. Wel is duidelijk geworden dat Av2(ox) twee verschillende soorten bindingsplaatsen heeft die van belang zijn voor labellings experimenten met azido-nucleotiden. Een van de bindingsplaatsen heeft een affiniteit voor de azidogroep en wordt geïdentificeerd als het [4Fe-4S] cluster. De tweede bindingsplaats is specifiek voor MgADP en MgATP. Een deel van de gevormde bindingen wordt gereduceerd door 2-mercaptoethanol, hetgeen resulteert in verlies van label door het gemodificeerde eiwit.In hoofdstuk 3 wordt aangetoond dat de door de Wageningse onderzoeksgroep gerapporteerde burst reactie van 4 moleculen MgATP gehydrolyseerd/mol Av2, veroorzaakt is door een apparaat artefact. Een herhaling van de experimenten met verbeterde rapid quench apparatuur gaf geen bevestiging van het algemeen aanvaard mechanisme waarin elektronoverdracht vooraf wordt gegaan door een volledige hydrolyse van MgATP. Er wordt ook op gewezen dat de gepubliceerde rapid-quench experimenten tegenstrijdige antwoorden geven op de vraag of elektronoverdracht voor dan wel na hydrolyse van MgATP plaats vindt. Tevens wordt beschreven hoe protonproduktie in de pre-steady-state fase van de nitrogenase reactie gevolgd kan worden met een stopped-flow spectrofotometrie techniek. Bij deze techniek wordt de kleursverandering van de pH- indicator cresol rood gebruikt om protonproduktie waar te nemen. De waargenomen snelheid van protonproduktie ligt lager dan die van elektronoverdracht.In hoofdstuk 4 wordt de temperatuursafhankelijkheid van de MgATP-afhankelijke elektronoverdracht van het Fe-eiwit naar het MoFe-eiwit bekeken. De amplitude van het stopped-flow signaal is afhankelijk van de reactietemperatuur. Deze waarneming wordt verklaard door aan te nemen dat elektronoverdracht van het Feeiwit naar het MoFe-eiwit reversibel is. De snelheid van elektronoverdracht vertoont een interessante temperatuursafhankelijkheid. De resultaten zijn geanalyseerd in het kader van de transition- state theorie. Dit laat zien dat het geactiveerde complex gekarakteriseerd wordt door een uitzonderlijk hoge activeringsentropie. Deze waarneming is een aanwijzing dat elektronoverdracht gepaard gaat met een grote conformatieverandering binnen het nitrogenase complex.In hoofdstuk 5 wordt bij 23 °C het effect bestudeerd van een hoge NaCl-concentratie op de extinctieverandering bij 430 nm, die gepaard gaat met elektrontransport van het Fe-eiwit naar het MoFe-eiwit. Net zoals een lage reactietemperatuur verlaagt NaCl deze extinctieverandering. Rapid-freeze EPR experimenten geven niet aan dat deze waarnemingen zonder meer verklaard mogen worden door een reversibele elektronoverdracht tussen beide nitrogenase eiwitten aan te nemen.In hoofdstuk 6 van dit proefschrift is een algemene discussie van de resultaten gegeven. Er wordt geconcludeerd dat er in dit proefschrift geen eenduidige aanwijzingen zijn verkregen die aantonen dat volledige hydrolyse van MgATP noodzakelijk is voor elektronoverdracht tussen de nitrogenase eiwitten. Daarom wordt een alternatief gegeven voor modellen die in de literatuur zijn verschenen.

Bacteria have frequently been introduced into the soil environment, e.g. for increasing crop production or for biological control purposes. Many applications require high numbers of surviving organisms in order to be effective. However, survival of bacteria after introduction into soil is generally poor, and numbers of introduced bacteria have been known to decrease from 10 9to approximately 10 3cells/g soil in 25 days. Thus, if bacteria are to be used as effective microbial inoculants to, a means to increase survival levels in soil needs to be found.Survival of Rhizobium leguminosarum biovar trifolii introduced into loamy sand was found to be greatly enhanced by amendments of bentonite, in amounts of 5 or 10%, to the soil. Bentonite appeared to offer introduced bacteria protection against protozoan predation, resulting in increased bacterial survival levels in bentonite amended soil as compared to unamended soil.Also in liquid cultures, protozoan activity was strongly hampered by the presence of bentonite, thereby also improving the survival of Rhizobium. Bentonite did not release any substances toxic to protozoa in liquid cultures, and presumably this would not occur in the soil environment either. Bentonite toxicity could therefore not explain the increased survival levels of bacteria introduced into bentonite amended soil. It was suggested that, in liquid cultures, bentonite clay increased the minimum level of bacteria for effective predation by protozoa.Changes in soil structure as a result of bentonite additions could explain the observed increases in bacterial survival levels. A mathematical relationship was found describing the log numbers of introduced rhizobia surviving in soil samples after an incubation period of 57 days using 3 pore size classes. Pores with necks < 3μm and between 3 and 6μm positively affected survival levels. These pores apparently were large enough to allow bacteria to enter, but were too small to be accessible to predating protozoa. Pores with necks between 6 and 15μm had a negative influence on rhizobial survival levels, because bacteria situated inside these relatively large pores could be reached and predated upon by protozoa. Therefore, pores < 6μm were found so serve as protective microhabitats for bacteria introduced into soil. A larger number of such protective microhabitats in bentonite amended loamy sand than in unamended loamy sand could explain the observed increase in survival levels of bacteria introduced into bentonite amended soil. The colonization potential of protective microhabitats was suggested to be determined largely by pore shape and the continuity of the water-filled pore system. Increased numbers of protective microhabitats (pores < 6μm) in bentonite-amended soil as compared to unamended soil were demonstrated visually by micro-morphoiogical studies using Cryo Scanning Electron Microscopy.The effectiveness of bentonite was strongly determined by the way in which the clay and the inoculum were added to the soil. When a bentonite suspension and bacteria were mixed together prior to inoculation, the clay offered more protection against predation than when bentonite powder and bacteria were added separately. This suggested that when the protective agent was present at the site of introduction, a more efficient use of the clay could be made, resulting in enhanced survival levels.Apart from survival, bentonite additions to soil also influenced bacterial respiration. The cumulative amount of CO 2 respired by rhizobia introduced into sterile bentonite- amended loamy sand was significantly higher than in unamended loamy sand. Carbon was used more efficiently during growth in bentonite-amended than in unamended loamy sand. The maintenance respiration of rhizobial cells was not influenced by the presence of bentonite clay. The growth rate of rhizobia introduced into sterile soil was increased by the presence of bentonite.Pseudomonas fluorescens was also found to survive at higher levels in bentoniteamended than in unamended soil, suggesting the bentonite effects were not limited to Rhizobium only. Pseudomonas fluorescens was used to study root colonization by bacteria introduced into bentonite amended soil. A rhizosphere effect (i.e. the occurrence of higher cell concentrations in rhizosphere soil than in bulk soil) was observed both in the absence and in the presence of bentonite clay, but it was less pronounced in the latter case. This finding suggested that bacteria were physically hindered by bentonite, making it more difficult to invade the immediate root environment. However, protection against predation by bentonite enhanced survival to such an extent, that overall survival in the rhizosphere was still higher in bentonite amended loamy sand than in the unamended soil.It can be concluded from this thesis that soil structure, and especially the pore size distribution of the soil is a key factor determining the survival, chances of bacteria introduced into soil. Application of introduced bacteria for e.g. biological control will probably stand a larger chance of being successful in soils with relatively high numbers of pores < 6μm However, it is unlikely that bentonite will ever be applied to soil in amounts of e.g. 5%, because of the large impact of bentonite additions on, for example, the moisture characteristics of the soil. However, the knowledge obtained on the importance of the pore size distribution of a soil, and the fact that the she of introduction of the bacteria will largely determine survival chances, will be of great importance for the future development of successful carrier materials for introducing bacteria into soil.

Bacteria have frequently been introduced into the soil environment, e.g. for increasing crop production or for biological control purposes. Many applications require high numbers of surviving organisms in order to be effective. However, survival of bacteria after introduction into soil is generally poor, and numbers of introduced bacteria have been known to decrease from 10 9to approximately 10 3cells/g soil in 25 days. Thus, if bacteria are to be used as effective microbial inoculants to, a means to increase survival levels in soil needs to be found.Survival of Rhizobium leguminosarum biovar trifolii introduced into loamy sand was found to be greatly enhanced by amendments of bentonite, in amounts of 5 or 10%, to the soil. Bentonite appeared to offer introduced bacteria protection against protozoan predation, resulting in increased bacterial survival levels in bentonite amended soil as compared to unamended soil.Also in liquid cultures, protozoan activity was strongly hampered by the presence of bentonite, thereby also improving the survival of Rhizobium. Bentonite did not release any substances toxic to protozoa in liquid cultures, and presumably this would not occur in the soil environment either. Bentonite toxicity could therefore not explain the increased survival levels of bacteria introduced into bentonite amended soil. It was suggested that, in liquid cultures, bentonite clay increased the minimum level of bacteria for effective predation by protozoa.Changes in soil structure as a result of bentonite additions could explain the observed increases in bacterial survival levels. A mathematical relationship was found describing the log numbers of introduced rhizobia surviving in soil samples after an incubation period of 57 days using 3 pore size classes. Pores with necks < 3μm and between 3 and 6μm positively affected survival levels. These pores apparently were large enough to allow bacteria to enter, but were too small to be accessible to predating protozoa. Pores with necks between 6 and 15μm had a negative influence on rhizobial survival levels, because bacteria situated inside these relatively large pores could be reached and predated upon by protozoa. Therefore, pores < 6μm were found so serve as protective microhabitats for bacteria introduced into soil. A larger number of such protective microhabitats in bentonite amended loamy sand than in unamended loamy sand could explain the observed increase in survival levels of bacteria introduced into bentonite amended soil. The colonization potential of protective microhabitats was suggested to be determined largely by pore shape and the continuity of the water-filled pore system. Increased numbers of protective microhabitats (pores < 6μm) in bentonite-amended soil as compared to unamended soil were demonstrated visually by micro-morphoiogical studies using Cryo Scanning Electron Microscopy.The effectiveness of bentonite was strongly determined by the way in which the clay and the inoculum were added to the soil. When a bentonite suspension and bacteria were mixed together prior to inoculation, the clay offered more protection against predation than when bentonite powder and bacteria were added separately. This suggested that when the protective agent was present at the site of introduction, a more efficient use of the clay could be made, resulting in enhanced survival levels.Apart from survival, bentonite additions to soil also influenced bacterial respiration. The cumulative amount of CO 2 respired by rhizobia introduced into sterile bentonite- amended loamy sand was significantly higher than in unamended loamy sand. Carbon was used more efficiently during growth in bentonite-amended than in unamended loamy sand. The maintenance respiration of rhizobial cells was not influenced by the presence of bentonite clay. The growth rate of rhizobia introduced into sterile soil was increased by the presence of bentonite.Pseudomonas fluorescens was also found to survive at higher levels in bentoniteamended than in unamended soil, suggesting the bentonite effects were not limited to Rhizobium only. Pseudomonas fluorescens was used to study root colonization by bacteria introduced into bentonite amended soil. A rhizosphere effect (i.e. the occurrence of higher cell concentrations in rhizosphere soil than in bulk soil) was observed both in the absence and in the presence of bentonite clay, but it was less pronounced in the latter case. This finding suggested that bacteria were physically hindered by bentonite, making it more difficult to invade the immediate root environment. However, protection against predation by bentonite enhanced survival to such an extent, that overall survival in the rhizosphere was still higher in bentonite amended loamy sand than in the unamended soil.It can be concluded from this thesis that soil structure, and especially the pore size distribution of the soil is a key factor determining the survival, chances of bacteria introduced into soil. Application of introduced bacteria for e.g. biological control will probably stand a larger chance of being successful in soils with relatively high numbers of pores < 6μm However, it is unlikely that bentonite will ever be applied to soil in amounts of e.g. 5%, because of the large impact of bentonite additions on, for example, the moisture characteristics of the soil. However, the knowledge obtained on the importance of the pore size distribution of a soil, and the fact that the she of introduction of the bacteria will largely determine survival chances, will be of great importance for the future development of successful carrier materials for introducing bacteria into soil.

The research described in this thesis focusses on the role of biotic factors encountered with the establishment of the symbiosis between black alder plants ( Alnus glutinosa ) and introduced Frankia strains. A selection of plant clones and Frankia strains that gave optimal nodulation and nitrogen fixation in forestry was made. For this reason nodulation tests with increasing complexity were set up. An attempt was made to investigate whether introduced strains behaved differently on plants grown under axenic and non-axenic conditions. Since Frankia strains were difficult to identify by conventional techniques, special attention was given to the development of new molecular techniques for identification of the strains at the nucleic acid level.Inoculation testsInitially, plant material of two physiologically different ecotypes of Alnus glutinosa, the forest ecotype "Bentheim" and the pioneer ecotype "Weerribben", respectively, was selected. Using tissue culture techniques plant material of both ecotypes was cloned in order to obtain genetically identical plants (Chapter 2). These micropropagated plants were used to set up a standardized inoculation system under axenic conditions in order to study the genetically determined nodulation ability and nitrogen-fixing capacity of Frankia strains and to select superior Frankia strains as source of inoculum (Chapter 3). The usefulness of selected Frankia strains as inoculum was further tested under more practical conditions, in perlite as model environment for nitrogen-limited conditions and in two soils, representing natural environments with different nutritional factors and different microbial populations. The results of the inoculation tests under axenic conditions were confirmed by studies under greenhouse conditions.The performance of the symbiosis was effected by many variables, e.g. the plant genotype, the Frankia strain and environmental conditions. The influence of the environmental conditions became more pronounced when plants were grown on either a sandy loam ("Bentheim") or a peat ("Weerribben") soil and inoculated with Frankia strains. Plant growth was positively influenced, e.g. by mycorrhizal fungi in "Bentheim" soil, or negatively influenced, e.g. by oomycetes in "Weerribben" soil. The effects of inoculation with Frankia on plant growth remained minimal. The establishment of the introduced Frankia strain was also dependent on the soil conditions. The introduced spore(-) Frankia strain was only able to compete with the natural spore(+) population of the "Weerribben" soil. Introduction of this strain to "Bentheim" soil did not show any establishment of the introduced strain. In contrast to the sandy loam of "Bentheim" which was rich in nutrients, the peat of "Weerribben" was a representative of poor soils. It could therefore be used for feasability studies in inoculation programmes. The use of pure cultures of Frankia as inoculum instead of soil or crushed nodules, has the advantage to prevent the contamination of the plant with root pathogens. Pure cultures did not result in a better symbiosis.Atypical Frankia strainsScreening of several isolates obtained from nodules of both alder ecotypes indicated the existence of atypical, ineffective Frankia strains. The alder clones used showed variable resistance against infection of the ineffective strains (Chapter 4). When compared with growth after the addition of a single strain dual inoculation of typical, effective Frankia strains and an ineffective Frankia strain to both alder clones showed growth increment of the plants (Chapter 5). The growth enhancing effect of the ineffective Frankia strain was not paralleled by increased number of nodules. Nothing is known yet about the growth stimulation by atypical Frankia strains. The results indicate that simultaneous inoculation of different Frankia strains to Alnus plants can be profitable for the host plant.Ribosomal RNABecause the ineffective Frankia strains lacked morphological and physiological characteristics of typical Frankia strains and because nodule formation on actinorhizal plants might be reduced or even absent, detection of the ineffective strains and studies on their competitive abilities were quite difficult. Reliable markers which could be used to detect both types of Frankia in nodules and in soil without reisolation had not been available at that moment. An attempt was made to find specific markers in a molecule which was commonly used to unravel evolutionary relationships: the 16S ribosomal RNA. New sequencing techniques allowed the rapid determination of total or almost total 16S rRNA sequences. Total 16S rRNA sequences indicated the presence of conserved and variable regions. Conserved regions had been used to investigate quantitative evolutionary relationships amoung bacteria. The conserved regions of the total 16S rRNA sequence of the effective Frankia strain Ag45/Mut15 were compared with aligned sequences of other actinomycetes and used to determine the position of the family Frankiaceae in the phylogenetic tree of the actinomycetes (Chapter 6).Analyses of variable regions of 16S rRNA of closely related organisms indicated sufficient variation, despite the fact that DNA/DNA homology studies suggested these two species might actually be one and the same. Large differences in DNA/DNA homology studies of Frankia which were also obtained between strains of one compatibility group suggested chances on large variation within the variable regions of different strains. Sequence analyses of variable regions of 16S rRNA of two ineffective Frankia strains (i.e. AgB1.9 and AgW1.1) and the effective strain Ag45/Mut1 5, all belonging to the Alnus -compatibility group, showed large differences in base composition. These sequences were used to design complementary synthetic oligonucleotides that could act as specific probes in hybridization experiments. The specificity of these probes was shown in hybridization experiments against immobilized rRNA from 23 Frankia strains belonging to different compatibiliy groups and of several related soil actinomycetes. The probes were able to distinguish between Nif +and Nif -strains, between several Nif -strains and between several Alnus compatible Nif +strains and strain AgKG'84/4 also belonging to the Alnus -compatibility group (Chapter 7). Strong strain specific sequences, however, were not obtained. The design of oligonucleotide probes opens up the possibility to investigate competitive abilities of selected strains under defined conditions, e.g. in model systems with perlite and defined Frankia strains. The question whether competition studies under these controlled conditions are ecologically relevant needs further investigations because little basic knowledge on Frankia population dynamics is yet available. The application of probes to identify introduced strains in soil remains restricted, due to the low specificity for strains. Up to now we are not able to design reliable strain specific probes that can be used to follow the establishment of introduced Frankia strains in natural environments. A much more . promising application of probes towards rRNA is concerned with the development of a genus-specific oligonucleoticle probe against Frankia (Chapter 9) that theoretically enables quantitative detection of total Frankia populations.RNA extractionThe application of oligonucleoticle probes in the detection of specific Frankia strains does not only depend on specificity of the probes but also on the development of a reliable isolation method for target sequences. Ribosomal RNA is preferable to DNA as target because of its relative abundance in large amounts in metabolically active cells. Actinorhizal nodules represent enrichments of Frankia, which are metabolically highly active and consequently contain large amounts of Frankia RNA. Our investigations resulted in the development of a rapid RNA extraction method that was sensitive enough to investigate strain composition also from very small nodules or lobes (Chapter 8). The detection of target sequences, however, remained limited by the design of specific probes and the ratios of different target sequences in one sample. For reliable signal expression in hybridization experiments quite similar amounts of target sequences per sample were needed.So far, the usefulness of rRNA sequences as targets for oligonucleoticle probes was only shown in combination with pure cultures of Frankia (Chapter 7) or in metabolically highly active enrichments, e.g. nodules (Chapter 8). Terrestrial environments like soil contain populations of many different microorganisms. These populations normally grow under suboptimal nutrition conditions. Bacteria adapt to these conditions by forming special starvation cells, which are metabolically inactive and contain only low amounts of rRNA. The starvation respons often results in viable, but non-culturable populations. The recalcitrant character of Frankia, which are difficult to isolate, makes it a useful model microorganism of soil bacteria. The application of oligonucleoticle probes for detection of Frankia in soil depends on the development of an extraction method for RNA. RNA directly isolated from soil as target for Frankia specific oligonucleoticle probes was useful in detection of Frankia (Chapter 9). Quantification of the obtained signals, however, is still unreliable because Frankia is a hyphae forming organism. It is also quite difficult to correlate cell numbers (theoretical estimation) to the amount of RNA. The concentration of these molecules in an organism is a function of the activity of the individual cell. Quantification of hybridization signals therefore depends on the availability of basic information of the metabolic activity of Frankia cells in soil. This information, however, is very difficult to obtain for recalcitrant microorganisms like Frankia. It is much easier for other microorganisms, e.g. for Streptomyces . Streptomyces spores are quite easy to isolate from soil and the establishment of Streptomyces cells, i.e. as spores or as mycelium in soil, is well studied. Quantification based on hybridization signals must be possible when this basic knowledge is available. In case of Frankia methods that enable quantification must still be developed. Similar to Streptomyces these quantification methods for Frankia can be of direct character, e.g. quantitative extraction of spores, or of indirect character, e.g. determination of mycelium by phage counts.The development of rapid and sensitive methods to detect Frankia on the basis of rRNA sequences opens up new ways to study other recalcitrant microorganisms in the environment. This molecular approach in microbial ecology can definitely further be explored when the advantages of rRNA as stable target and the rapid extraction of RNA from soil can be combined with in vitro amplification methods commonly used with DNA or mRNA. Promising approaches can also be expected in in situ studies using hybridization signal intensity of fluorescent dye labelled oligonucleotides and the amount of rRNA as criterium for bacterial activity.

The research described in this thesis focusses on the role of biotic factors encountered with the establishment of the symbiosis between black alder plants ( Alnus glutinosa ) and introduced Frankia strains. A selection of plant clones and Frankia strains that gave optimal nodulation and nitrogen fixation in forestry was made. For this reason nodulation tests with increasing complexity were set up. An attempt was made to investigate whether introduced strains behaved differently on plants grown under axenic and non-axenic conditions. Since Frankia strains were difficult to identify by conventional techniques, special attention was given to the development of new molecular techniques for identification of the strains at the nucleic acid level.Inoculation testsInitially, plant material of two physiologically different ecotypes of Alnus glutinosa, the forest ecotype "Bentheim" and the pioneer ecotype "Weerribben", respectively, was selected. Using tissue culture techniques plant material of both ecotypes was cloned in order to obtain genetically identical plants (Chapter 2). These micropropagated plants were used to set up a standardized inoculation system under axenic conditions in order to study the genetically determined nodulation ability and nitrogen-fixing capacity of Frankia strains and to select superior Frankia strains as source of inoculum (Chapter 3). The usefulness of selected Frankia strains as inoculum was further tested under more practical conditions, in perlite as model environment for nitrogen-limited conditions and in two soils, representing natural environments with different nutritional factors and different microbial populations. The results of the inoculation tests under axenic conditions were confirmed by studies under greenhouse conditions.The performance of the symbiosis was effected by many variables, e.g. the plant genotype, the Frankia strain and environmental conditions. The influence of the environmental conditions became more pronounced when plants were grown on either a sandy loam ("Bentheim") or a peat ("Weerribben") soil and inoculated with Frankia strains. Plant growth was positively influenced, e.g. by mycorrhizal fungi in "Bentheim" soil, or negatively influenced, e.g. by oomycetes in "Weerribben" soil. The effects of inoculation with Frankia on plant growth remained minimal. The establishment of the introduced Frankia strain was also dependent on the soil conditions. The introduced spore(-) Frankia strain was only able to compete with the natural spore(+) population of the "Weerribben" soil. Introduction of this strain to "Bentheim" soil did not show any establishment of the introduced strain. In contrast to the sandy loam of "Bentheim" which was rich in nutrients, the peat of "Weerribben" was a representative of poor soils. It could therefore be used for feasability studies in inoculation programmes. The use of pure cultures of Frankia as inoculum instead of soil or crushed nodules, has the advantage to prevent the contamination of the plant with root pathogens. Pure cultures did not result in a better symbiosis.Atypical Frankia strainsScreening of several isolates obtained from nodules of both alder ecotypes indicated the existence of atypical, ineffective Frankia strains. The alder clones used showed variable resistance against infection of the ineffective strains (Chapter 4). When compared with growth after the addition of a single strain dual inoculation of typical, effective Frankia strains and an ineffective Frankia strain to both alder clones showed growth increment of the plants (Chapter 5). The growth enhancing effect of the ineffective Frankia strain was not paralleled by increased number of nodules. Nothing is known yet about the growth stimulation by atypical Frankia strains. The results indicate that simultaneous inoculation of different Frankia strains to Alnus plants can be profitable for the host plant.Ribosomal RNABecause the ineffective Frankia strains lacked morphological and physiological characteristics of typical Frankia strains and because nodule formation on actinorhizal plants might be reduced or even absent, detection of the ineffective strains and studies on their competitive abilities were quite difficult. Reliable markers which could be used to detect both types of Frankia in nodules and in soil without reisolation had not been available at that moment. An attempt was made to find specific markers in a molecule which was commonly used to unravel evolutionary relationships: the 16S ribosomal RNA. New sequencing techniques allowed the rapid determination of total or almost total 16S rRNA sequences. Total 16S rRNA sequences indicated the presence of conserved and variable regions. Conserved regions had been used to investigate quantitative evolutionary relationships amoung bacteria. The conserved regions of the total 16S rRNA sequence of the effective Frankia strain Ag45/Mut15 were compared with aligned sequences of other actinomycetes and used to determine the position of the family Frankiaceae in the phylogenetic tree of the actinomycetes (Chapter 6).Analyses of variable regions of 16S rRNA of closely related organisms indicated sufficient variation, despite the fact that DNA/DNA homology studies suggested these two species might actually be one and the same. Large differences in DNA/DNA homology studies of Frankia which were also obtained between strains of one compatibility group suggested chances on large variation within the variable regions of different strains. Sequence analyses of variable regions of 16S rRNA of two ineffective Frankia strains (i.e. AgB1.9 and AgW1.1) and the effective strain Ag45/Mut1 5, all belonging to the Alnus -compatibility group, showed large differences in base composition. These sequences were used to design complementary synthetic oligonucleotides that could act as specific probes in hybridization experiments. The specificity of these probes was shown in hybridization experiments against immobilized rRNA from 23 Frankia strains belonging to different compatibiliy groups and of several related soil actinomycetes. The probes were able to distinguish between Nif +and Nif -strains, between several Nif -strains and between several Alnus compatible Nif +strains and strain AgKG'84/4 also belonging to the Alnus -compatibility group (Chapter 7). Strong strain specific sequences, however, were not obtained. The design of oligonucleotide probes opens up the possibility to investigate competitive abilities of selected strains under defined conditions, e.g. in model systems with perlite and defined Frankia strains. The question whether competition studies under these controlled conditions are ecologically relevant needs further investigations because little basic knowledge on Frankia population dynamics is yet available. The application of probes to identify introduced strains in soil remains restricted, due to the low specificity for strains. Up to now we are not able to design reliable strain specific probes that can be used to follow the establishment of introduced Frankia strains in natural environments. A much more . promising application of probes towards rRNA is concerned with the development of a genus-specific oligonucleoticle probe against Frankia (Chapter 9) that theoretically enables quantitative detection of total Frankia populations.RNA extractionThe application of oligonucleoticle probes in the detection of specific Frankia strains does not only depend on specificity of the probes but also on the development of a reliable isolation method for target sequences. Ribosomal RNA is preferable to DNA as target because of its relative abundance in large amounts in metabolically active cells. Actinorhizal nodules represent enrichments of Frankia, which are metabolically highly active and consequently contain large amounts of Frankia RNA. Our investigations resulted in the development of a rapid RNA extraction method that was sensitive enough to investigate strain composition also from very small nodules or lobes (Chapter 8). The detection of target sequences, however, remained limited by the design of specific probes and the ratios of different target sequences in one sample. For reliable signal expression in hybridization experiments quite similar amounts of target sequences per sample were needed.So far, the usefulness of rRNA sequences as targets for oligonucleoticle probes was only shown in combination with pure cultures of Frankia (Chapter 7) or in metabolically highly active enrichments, e.g. nodules (Chapter 8). Terrestrial environments like soil contain populations of many different microorganisms. These populations normally grow under suboptimal nutrition conditions. Bacteria adapt to these conditions by forming special starvation cells, which are metabolically inactive and contain only low amounts of rRNA. The starvation respons often results in viable, but non-culturable populations. The recalcitrant character of Frankia, which are difficult to isolate, makes it a useful model microorganism of soil bacteria. The application of oligonucleoticle probes for detection of Frankia in soil depends on the development of an extraction method for RNA. RNA directly isolated from soil as target for Frankia specific oligonucleoticle probes was useful in detection of Frankia (Chapter 9). Quantification of the obtained signals, however, is still unreliable because Frankia is a hyphae forming organism. It is also quite difficult to correlate cell numbers (theoretical estimation) to the amount of RNA. The concentration of these molecules in an organism is a function of the activity of the individual cell. Quantification of hybridization signals therefore depends on the availability of basic information of the metabolic activity of Frankia cells in soil. This information, however, is very difficult to obtain for recalcitrant microorganisms like Frankia. It is much easier for other microorganisms, e.g. for Streptomyces . Streptomyces spores are quite easy to isolate from soil and the establishment of Streptomyces cells, i.e. as spores or as mycelium in soil, is well studied. Quantification based on hybridization signals must be possible when this basic knowledge is available. In case of Frankia methods that enable quantification must still be developed. Similar to Streptomyces these quantification methods for Frankia can be of direct character, e.g. quantitative extraction of spores, or of indirect character, e.g. determination of mycelium by phage counts.The development of rapid and sensitive methods to detect Frankia on the basis of rRNA sequences opens up new ways to study other recalcitrant microorganisms in the environment. This molecular approach in microbial ecology can definitely further be explored when the advantages of rRNA as stable target and the rapid extraction of RNA from soil can be combined with in vitro amplification methods commonly used with DNA or mRNA. Promising approaches can also be expected in in situ studies using hybridization signal intensity of fluorescent dye labelled oligonucleotides and the amount of rRNA as criterium for bacterial activity.

The nitrogen-fixing activity of the symbiotic system of Pisum sativum with Rhizobium leguminosarum is adversely affected by combined nitrogen. Both ammonium chloride and potassium nitrate, when added to the roots, lower the nitrogenase activity (acetylene-reduction) of intact pea plants. During a 3-day treatment of the plants with combined nitrogen, when the in vivo nitrogenase activity falls to less than 50%, the nitrogenase activity of isolated bacteroids, treated with toluene and supplied with ATP and reductants, does not decrease. Thus, the potential nitrogenase activity of the root nodules is unaffected by short-term combined-nitrogen treatment of the plants. The adverse effect of ammonium chloride on the nitrogenase activity of pea plants is counteracted by the addition of sucrose or of methionine sulfoximine (an inhibitor of ammonia assimilation) to the rooting medium. A higher light intensity also diminishes the effect of ammonium chloride on nitrogenase activity.Ammonium chloride has no specific inhibitory effect on the nitrogenase activity of isolated pea bacteroids, neither in the anaerobic, nor in the aerobic assay. On the other hand, ammonium chloride does inhibit the nitrogenase activity of detached root nodules within a few hours. At a lower oxygen concentration in the assay this inhibition is more pronounced. The effect of ammonium chloride on detached nodules is relieved by simultaneous addition of methionine sulfoximine.Various carbon compounds (glucose and tricarboxylic acid cycle intermediates) stimulate the nitrogenase activity of detached nodules; only dicarboxylic acids of the tricarboxylic acid cycle support the nitrogenase activity of isolated bacteroids. Efficiencies of nitrogen fixation as to consumption of carbon compound are similar in both system , although lower than in the intact system. Ammonium chloride does not affect respiratory activities of detached nodules or of isolated bacteroids.It is concluded from the above-mentioned results that combined nitrogen, added to intact plants or detached nodules, does not affect nitrogenase activity directly. The results support the photosynthate theory, i.e. the photosynthate supply of the nodules is reduced and consequently the nitrogenase activity decreased, owing to the consumption of carbon compounds for the assimilation of the added combined nitrogen.

This thesis encompasses six papers, dealing with mainly ionic balance aspects of non-ionic nitrogen nutrition of plants. In most cases urea nutrition or symbiotic N 2 -fixation were compared with NH 4+ - or NO 3- -supply with respect to nutrient uptake and assimilation.From ionic balance and proton release data it was established that maize and sugar-beet plants are able to absorb urea as an undestructed molecule. Results of xylem sap analyses learned that urea, like NH 4+ , is almost quantitatively metabolized in the roots.Complete ionic uptake balances, including direct measurements of respective H + - and OH - /HCO 3- -release from the roots of N 2 -fixing and NO 3- -supplied pea plants are presented. Excess nutrient cation over anion uptake and hence H + -release by N 2 -fixing plants increased at higher pH of the nutrient solution. When such plants were grown in soil, cation uptake also exceeded anion uptake, but root growth was severely reduced at low soil pH. This effect could be eliminated completely by liming. Root growth was not inhibited when NO 3- was the form of N-nutrition.In soils, mineralized N may confuse the comparison between NO 3- -nutrition and N 2 -fixation. It is suggested that the relative contribution of N 2 -fixation to the total N- accumulation in plants reflects the point of time at which ( 15 N-)NO 3- in the soil was depleted and the N 2 -fixing process started.Different ionic uptake patterns of plants in relation to the form of nitrogen nutrition necessarily invoke essential differences in both inorganic and organic chemical composition of the xylem sap of these plants. Complete xylary ionic balances and data about partitioning of the nitrogenous compounds In xylem saps allowed the conclusion that N 2 -fixing pea plants belong to the group of amidetransporting legumes and that in NO 3- -supplied pea plants no phloem transport of cation-organate is necessary for. the regulation of Intracellular pH and electroneutrality.

In this thesis the population dynamics of bacteria introduced into soil was studied. In the introduction, the existence of microhabitats favourable for the survival of indigenous bacteria is discussed. Knowledge about the distribution of introduced bacteria over such microhabitats, however, is scarse. Nevertheless, it was hypothesized that upon introduction, bacteria reach other microsites in soil than bacteria which are already present for some time, thereby influencing the survival of introduced organisms. Methods to study the distribution of introduced bacteria in soil, as well as the effect of their distribution on the population dynamics, were assessed. A model organism, Rhizobium leguminosarum biovar trifolii and two different soils, a loamy sand and a silt loam, were used for this purpose.Two methods for the enumeration of bacteria introduced into soil were compared (Chapter 2). Although immunofluorescence was a very promissing method at the moment we started our work, selective plating proved to be more suitable for the enumeration of low numbers of introduced bacteria, since it had a lower detection limit than immunufluorescence. In addition, selective plating did not depend on flocculation processes which were shown to influence significantly the results obtained with the immunofluorescence technique. Moreover, only cells able to divide were counted. However, with the immunofluorescence technique we were able to determine cell lengths and we detected that the length of cells which were grown in a rich medium decreased after their introduction into soil.To study the micro-distribution of bacteria in soil, different fluorochromes were tested on their ability to stain bacteria in thin sections of undisturbed soil samples. Calcofluor white M2R in combination with acridine orange was successfully applied for the detection of bacteria in thin soil sections (Chapter 3). However, specific staining of the introduced rhizobia with conjugated antiserum was not successful. Therefore, an alternative method for the assessment of the distribution of introduced bacteria, a soil washing procedure, was used in Chapters 4, 5 and 6. With this method, free occuring bacteria and bacteria associated with soil particles or aggregates>50μm were distinguished.The bacterial distribution through soil could be manipulated by inoculating soils at different initial moisture contents. At a lower initial moisture content, only the narrowest pores are filled with water, so that inoculated rhizobial cells will reach narrower pores when they are transported passively by the waterflow. At a higher initial moisture content, water already present in the narrower pores prevent the introduced cells from entering these pores. With such an inoculation procedure, rhizobial cells were found to be associated to a larger extent with soil particles when soils were inoculated at lower initial moisture contents. In natural soils, this resulted in an improved survival of rhizobia during at least 100 days after inoculation (Chapter 4). Moreover, the number of particleassociated cells decreased less than the number of free occuring cells in natural soil. It was concluded that rhizobial cells associated with soil particles or aggregates>50μm occupied a more favourable microhabitat than free occuring cells. In sterilized soil, numbers of both particleassociated and free occuring cells increased and the initial differences in distribution did not result in different final population levels (Chapter 5). Therefore, it was concluded that the microhabitats in natural soil rendered protection to biotic rather than to abiotic factors.The influence of competitors and predators on the distribution and population dynamics of rhizobium was studied by the addition of specific groups of organisms to sterilized soils (Chapter 5). Previous to inoculation with rhizobia, sterilized soils were recolonized with several bacterial isolates which were obtained from these soils and part of the soil portions were inoculated with a flagellate precultered on rhizobial cells. In the presence of flagellates, which predate on bacteria, a higher percentage of particle-associated rhizobial cells was present than in the absence of flagellates. In recolonized soils, i.e. in the presence of competitors, the percentages of particle -associated rhizobial cells were lower than in soils that were not recolonized previous to inoculation. Thus, the presence of competitors made it more difficult for rhizobial cells to colonize the microsites where they can be associated with soil particles or aggregates. The total number of rhizobial cells was influenced only little (silt loam) or not at all (loamy sand) by the competitors or by the addition of flagellates alone. However, when both competitors and predators were present, numbers of rhizobial cells decreased drastically. This synergetic effect was explained by hypothesizing that after the predation of accessible bacterial cells by the flagellates, the regrowth of rhizobial cells will be limited by the presence of competitive microorganisms in many of the favourable microhabitats.The association of rhizobial cells with soil particles may be the result of enclosure in pores or attachment to soil surfaces of rhizobia. The role of attachment was studied with a R. leguminosarum strain and three Tn5 mutants which were altered in their cell surface properties (Chapter 6). Although the importance of association with soil particles or aggregates was affirmed, the results gave no evidence that attachment to soil particle surfaces was an important factor for the survival of introduced cells.The final population level of introduced rhizobia was studied in more detail by inoculating sterilized and natural soils with different inoculum levels (Chapter 7). In sterilized soils, populations reached, independent of the inoculum density, a final level which was suggested to represent the carrying capacity of the soils in terms of available habitable pore space, moisture and substrate for survival of the bacteria. In natural soil, however, the survival levels were dependent on the inoculum density. In this case, the chances of introduced cells to reach favourable microhabitats, determined the survival level of the entire population.In all experiments final population levels in natural and in sterilized soils were higher in the silt loam than in the loamy sand (Chapter 2-8). Pore space which is suitable for bacteria to survive (=habitable pore space) or which protects bacteria from predation (=protective pore space) was estimated for both soils. The occupancy by bacteria was in all cases lower than 0.5%, so that no serious space limitation could be expected. Therefore, the larger water-filled pore volume at the water potential used (pF 2) in the silt loam as compared to the loamy sand, could not explain the differences in population sizes. In sterilized soil substrate availability was suggested to determine the final population level. In natural soil, however, the survival of rhizobial cells was suggested to be dependent on the number of introduced bacteria that reached the protective pore space (Chapter 4 and 7).In this thesis it is shown that the soil washing procedure is useful for the study of the distribution of introduced bacteria. Immediately after introduction, only few bacteria were associated with soil particles. The number of particle-associated bacteria decreased less pronounced than the number of free occuring bacteria, giving evidence that the distribution of introduced bacteria in soil is indeed an important factor influencing its survival. Moreover, the distribution could be manipulated by inoculating soil at different moisture contents. Inoculation of dryer soils, as well as the use of higher inoculum densities, resulted in higher survival levels, which could be well explained with the concept of distribution of cells over protective and non-protective pore space. The occurance of different population levels under apparently the same environmental conditions during incubation, suggests that extensive translocation in natural soil is absent.The ability to manipulate the distribution and thereby to influence the survival of introduced bacteria, is important for the application of bacteria in soil. The availability of methods for biological control of soil-borne pathogens, nitrogen fixation and degradation of xenobiotics in soil, is depending on the possibility of introduced bacteria to establish in soil. The knowledge obtained in this research project can be used to improve the survival of bacteria introduced into soil.The spatial distribution of bacteria through the soil matrix might also be a useful concept for other areas in soil(micro)biology. The occurance, for example, of genetransfer in different soil systems might be better understood when more details about bacterial distribution are available. Also the preservation of organic matter and the activity of predators will depend on the distribution of bacteria in the soil matrix. In eerste instantie is in deze studie gezocht naar een geschikte methode om de ruimtelijke verdeling van in de grond geintroduceerde bacterien te onderzoeken i.v.m. hun overlevingskansen. En vervolgens is de invloed van de ruimtelijke verdeling van deze bacterien op hun overleving bestudeerd aan de hand van een model-organisme, Rhizobium leguminosarum biovar trifolii, uitgevoerd in de beide grondsoorten zwak lemig zand en kleigrond.

In this thesis the population dynamics of bacteria introduced into soil was studied. In the introduction, the existence of microhabitats favourable for the survival of indigenous bacteria is discussed. Knowledge about the distribution of introduced bacteria over such microhabitats, however, is scarse. Nevertheless, it was hypothesized that upon introduction, bacteria reach other microsites in soil than bacteria which are already present for some time, thereby influencing the survival of introduced organisms. Methods to study the distribution of introduced bacteria in soil, as well as the effect of their distribution on the population dynamics, were assessed. A model organism, Rhizobium leguminosarum biovar trifolii and two different soils, a loamy sand and a silt loam, were used for this purpose.Two methods for the enumeration of bacteria introduced into soil were compared (Chapter 2). Although immunofluorescence was a very promissing method at the moment we started our work, selective plating proved to be more suitable for the enumeration of low numbers of introduced bacteria, since it had a lower detection limit than immunufluorescence. In addition, selective plating did not depend on flocculation processes which were shown to influence significantly the results obtained with the immunofluorescence technique. Moreover, only cells able to divide were counted. However, with the immunofluorescence technique we were able to determine cell lengths and we detected that the length of cells which were grown in a rich medium decreased after their introduction into soil.To study the micro-distribution of bacteria in soil, different fluorochromes were tested on their ability to stain bacteria in thin sections of undisturbed soil samples. Calcofluor white M2R in combination with acridine orange was successfully applied for the detection of bacteria in thin soil sections (Chapter 3). However, specific staining of the introduced rhizobia with conjugated antiserum was not successful. Therefore, an alternative method for the assessment of the distribution of introduced bacteria, a soil washing procedure, was used in Chapters 4, 5 and 6. With this method, free occuring bacteria and bacteria associated with soil particles or aggregates>50μm were distinguished.The bacterial distribution through soil could be manipulated by inoculating soils at different initial moisture contents. At a lower initial moisture content, only the narrowest pores are filled with water, so that inoculated rhizobial cells will reach narrower pores when they are transported passively by the waterflow. At a higher initial moisture content, water already present in the narrower pores prevent the introduced cells from entering these pores. With such an inoculation procedure, rhizobial cells were found to be associated to a larger extent with soil particles when soils were inoculated at lower initial moisture contents. In natural soils, this resulted in an improved survival of rhizobia during at least 100 days after inoculation (Chapter 4). Moreover, the number of particleassociated cells decreased less than the number of free occuring cells in natural soil. It was concluded that rhizobial cells associated with soil particles or aggregates>50μm occupied a more favourable microhabitat than free occuring cells. In sterilized soil, numbers of both particleassociated and free occuring cells increased and the initial differences in distribution did not result in different final population levels (Chapter 5). Therefore, it was concluded that the microhabitats in natural soil rendered protection to biotic rather than to abiotic factors.The influence of competitors and predators on the distribution and population dynamics of rhizobium was studied by the addition of specific groups of organisms to sterilized soils (Chapter 5). Previous to inoculation with rhizobia, sterilized soils were recolonized with several bacterial isolates which were obtained from these soils and part of the soil portions were inoculated with a flagellate precultered on rhizobial cells. In the presence of flagellates, which predate on bacteria, a higher percentage of particle-associated rhizobial cells was present than in the absence of flagellates. In recolonized soils, i.e. in the presence of competitors, the percentages of particle -associated rhizobial cells were lower than in soils that were not recolonized previous to inoculation. Thus, the presence of competitors made it more difficult for rhizobial cells to colonize the microsites where they can be associated with soil particles or aggregates. The total number of rhizobial cells was influenced only little (silt loam) or not at all (loamy sand) by the competitors or by the addition of flagellates alone. However, when both competitors and predators were present, numbers of rhizobial cells decreased drastically. This synergetic effect was explained by hypothesizing that after the predation of accessible bacterial cells by the flagellates, the regrowth of rhizobial cells will be limited by the presence of competitive microorganisms in many of the favourable microhabitats.The association of rhizobial cells with soil particles may be the result of enclosure in pores or attachment to soil surfaces of rhizobia. The role of attachment was studied with a R. leguminosarum strain and three Tn5 mutants which were altered in their cell surface properties (Chapter 6). Although the importance of association with soil particles or aggregates was affirmed, the results gave no evidence that attachment to soil particle surfaces was an important factor for the survival of introduced cells.The final population level of introduced rhizobia was studied in more detail by inoculating sterilized and natural soils with different inoculum levels (Chapter 7). In sterilized soils, populations reached, independent of the inoculum density, a final level which was suggested to represent the carrying capacity of the soils in terms of available habitable pore space, moisture and substrate for survival of the bacteria. In natural soil, however, the survival levels were dependent on the inoculum density. In this case, the chances of introduced cells to reach favourable microhabitats, determined the survival level of the entire population.In all experiments final population levels in natural and in sterilized soils were higher in the silt loam than in the loamy sand (Chapter 2-8). Pore space which is suitable for bacteria to survive (=habitable pore space) or which protects bacteria from predation (=protective pore space) was estimated for both soils. The occupancy by bacteria was in all cases lower than 0.5%, so that no serious space limitation could be expected. Therefore, the larger water-filled pore volume at the water potential used (pF 2) in the silt loam as compared to the loamy sand, could not explain the differences in population sizes. In sterilized soil substrate availability was suggested to determine the final population level. In natural soil, however, the survival of rhizobial cells was suggested to be dependent on the number of introduced bacteria that reached the protective pore space (Chapter 4 and 7).In this thesis it is shown that the soil washing procedure is useful for the study of the distribution of introduced bacteria. Immediately after introduction, only few bacteria were associated with soil particles. The number of particle-associated bacteria decreased less pronounced than the number of free occuring bacteria, giving evidence that the distribution of introduced bacteria in soil is indeed an important factor influencing its survival. Moreover, the distribution could be manipulated by inoculating soil at different moisture contents. Inoculation of dryer soils, as well as the use of higher inoculum densities, resulted in higher survival levels, which could be well explained with the concept of distribution of cells over protective and non-protective pore space. The occurance of different population levels under apparently the same environmental conditions during incubation, suggests that extensive translocation in natural soil is absent.The ability to manipulate the distribution and thereby to influence the survival of introduced bacteria, is important for the application of bacteria in soil. The availability of methods for biological control of soil-borne pathogens, nitrogen fixation and degradation of xenobiotics in soil, is depending on the possibility of introduced bacteria to establish in soil. The knowledge obtained in this research project can be used to improve the survival of bacteria introduced into soil.The spatial distribution of bacteria through the soil matrix might also be a useful concept for other areas in soil(micro)biology. The occurance, for example, of genetransfer in different soil systems might be better understood when more details about bacterial distribution are available. Also the preservation of organic matter and the activity of predators will depend on the distribution of bacteria in the soil matrix.

The nitrogen-fixing activity of the symbiotic system of Pisum sativum with Rhizobium leguminosarum is adversely affected by combined nitrogen. Both ammonium chloride and potassium nitrate, when added to the roots, lower the nitrogenase activity (acetylene-reduction) of intact pea plants. During a 3-day treatment of the plants with combined nitrogen, when the in vivo nitrogenase activity falls to less than 50%, the nitrogenase activity of isolated bacteroids, treated with toluene and supplied with ATP and reductants, does not decrease. Thus, the potential nitrogenase activity of the root nodules is unaffected by short-term combined-nitrogen treatment of the plants. The adverse effect of ammonium chloride on the nitrogenase activity of pea plants is counteracted by the addition of sucrose or of methionine sulfoximine (an inhibitor of ammonia assimilation) to the rooting medium. A higher light intensity also diminishes the effect of ammonium chloride on nitrogenase activity.Ammonium chloride has no specific inhibitory effect on the nitrogenase activity of isolated pea bacteroids, neither in the anaerobic, nor in the aerobic assay. On the other hand, ammonium chloride does inhibit the nitrogenase activity of detached root nodules within a few hours. At a lower oxygen concentration in the assay this inhibition is more pronounced. The effect of ammonium chloride on detached nodules is relieved by simultaneous addition of methionine sulfoximine.Various carbon compounds (glucose and tricarboxylic acid cycle intermediates) stimulate the nitrogenase activity of detached nodules; only dicarboxylic acids of the tricarboxylic acid cycle support the nitrogenase activity of isolated bacteroids. Efficiencies of nitrogen fixation as to consumption of carbon compound are similar in both system , although lower than in the intact system. Ammonium chloride does not affect respiratory activities of detached nodules or of isolated bacteroids.It is concluded from the above-mentioned results that combined nitrogen, added to intact plants or detached nodules, does not affect nitrogenase activity directly. The results support the photosynthate theory, i.e. the photosynthate supply of the nodules is reduced and consequently the nitrogenase activity decreased, owing to the consumption of carbon compounds for the assimilation of the added combined nitrogen.