Stikstofbinding voor kleine boeren in Afrika
Giller, K.E. - \ 2015
Vork 2 (2015)3. - ISSN 2352-2925 - p. 16 - 21.
tuinbouw - kleine landbouwbedrijven - afrika - stikstofbindende bacteriën - rhizobium - bodemvruchtbaarheid - inkomen van landbouwers - agrarische bedrijfsvoering - peulgewassen - sojabonen - voedselproductie - projecten - teeltsystemen - horticulture - small farms - africa - nitrogen fixing bacteria - rhizobium - soil fertility - farmers' income - farm management - legumes - soyabeans - food production - projects - cropping systems
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.
Nationale invulling vergroening GLB vanuit het perspectief van biodiversiteit
Doorn, A.M. van; Vullings, L.A.E. ; Breman, B.C. ; Elbersen, B.S. ; Korevaar, H. ; Meijer, M. ; Naeff, H.S.D. ; Noij, I.G.A.M. ; Kuhlman, J.W. ; Polman, N.B.P. - \ 2013
Wageningen : Alterra, Wageningen-UR (Alterra-rapport 2478) - 71
gemeenschappelijk landbouwbeleid - agrarische bedrijfsvoering - landschapselementen - akkerranden - graslandbeheer - groenbemesters - stikstofbindende bacteriën - inventarisaties - cap - farm management - landscape elements - field margins - grassland management - green manures - nitrogen fixing bacteria - inventories
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.
De kracht van de stikstofbinders
Giller, K.E. ; Bisseling, T. - \ 2012
WageningenWorld 2012 (2012)1. - ISSN 2210-7908 - p. 30 - 37.
peulgewassen - stikstoffixatie - wortelknolletjes - stikstofbindende bacteriën - rhizobium - veldgewassen - afrika - legumes - nitrogen fixation - root nodules - nitrogen fixing bacteria - rhizobium - field crops - africa
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.
Receptor kinases regulating rhizobial infection
Limpens, E.H.M. - \ 2004
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - [S.l.] : S.n. - ISBN 9789085040590 - 125
wortelknolletjes - stikstofbindende bacteriën - genen - genetische regulatie - signaaltransductie - root nodules - nitrogen fixing bacteria - genes - genetic regulation - signal transduction
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 from
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.
|Nitrogen fixation in tropical cropping systems
Giller, K.E. - \ 2001
Wallingford : CAB International - ISBN 9780851994178 - 423
stikstoffixatie - stikstofbindende bacteriën - stikstofbindende bomen - bodembiologie - knobbelvorming - wortelknolletjes - rhizobium - teeltsystemen - peulgewassen - stikstof - nitrogen fixation - nitrogen fixing bacteria - nitrogen fixing trees - soil biology - nodulation - root nodules - cropping systems - legumes - nitrogen
Biological nitrogen fixation of soybean in acid soils of Sumatra, Indonesia
Waluyo, S.H. - \ 2000
Agricultural University. Promotor(en): W.M. de Vos; L. 't Mannetje; L.T. An. - S.l. : S.n. - ISBN 9789058082954 - 151
glycine max - sojabonen - bodembiologie - stikstoffixatie - stikstofbindende bacteriën - rhizobium - bradyrhizobium - inoculatie - entstof - biochemische technieken - dna-fingerprinting - stamverschillen - stammen (biologisch) - zaadbehandeling - omhullen - zure gronden - bodemaciditeit - bekalking - sumatra - indonesië - glycine max - soyabeans - soil biology - nitrogen fixation - nitrogen fixing bacteria - rhizobium - bradyrhizobium - inoculation - inoculum - biochemical techniques - dna fingerprinting - strain differences - strains - seed treatment - pelleting - acid soils - soil acidity - liming - sumatra - indonesia
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:
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.
Onderzoek naar vermindering van de stikstofbemesting door toepassing van Rhizobium phaseoli bij stamslaboon (Phaseolus vulgaris L.) = Investigations in order to reduce nitrogen fertilization of snap beans (Phaseolus vulgaris L.) by inoculation with Rhizobia bacteria
Neuvel, J.J. ; Floot, H.W.G. ; Postma, S. - \ 1994
Lelystad : PAGV (Verslag / Proefstation voor de Akkerbouw en de Groenteteelt in de Vollegrond nr. 168) - 114
stikstof - stikstofbindende bacteriën - phaseolus vulgaris - rhizobium - symbiose - nitrogen - nitrogen fixing bacteria - phaseolus vulgaris - rhizobium - symbiosis
Population dynamics of bacteria introduced into bentonite amended soil
Heijnen, C. - \ 1992
Agricultural University. Promotor(en): N. van Breemen; J.A. van Veen. - S.l. : Heijnen - 119
bodembacteriën - stikstofbindende bacteriën - symbiose - rhizobium - bodemstructuur - verbetering - zand - bodemverbeteraars - natuurlijke hulpbronnen - kleimineralen - bentoniet - pseudomonas - soil bacteria - nitrogen fixing bacteria - symbiosis - rhizobium - soil structure - improvement - sand - soil conditioners - natural resources - clay minerals - bentonite - pseudomonas
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 < 3 μm and between 3 and 6 μm 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 15 μm 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 < 6 μm 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 < 6 μm) 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 < 6 μm 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.
Coupling of MgATP hydrolysis and electron transfer in the pre-steady-state phase of the nitrogenase reaction
Mensink, R.E. - \ 1992
Agricultural University. Promotor(en): C. Veeger; H. Haaker. - S.l. : Mensink - 125
nitrogenase - stikstofbindende bacteriën - nitrogenase - nitrogen fixing bacteria
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.
|Nitrogen fixation in tropical cropping systems.
Giller, K.E. ; Wilson, K.J. - \ 1991
Wallingford, UK : CAB International - ISBN 9780851986715 - 313
assimilatie - stikstof - stikstofbindende bacteriën - tropen - assimilation - nitrogen - nitrogen fixing bacteria - tropics
Inhibition of nodulation of lucerne (Medicago sativa L.) by calcium depletion in an acid soil.
Pijnenborg, J.W.M. ; Lie, T.A. ; Zehnder, A.J.B. - \ 1990
Plant and Soil 127 (1990). - ISSN 0032-079X - p. 31 - 39.
medicago - bodem - calcium - zure gronden - kattekleigronden - stikstofbindende bacteriën - symbiose - rhizobium - medicago - soil - calcium - acid soils - acid sulfate soils - nitrogen fixing bacteria - symbiosis - rhizobium
16S rRNA as molecular marker in ecology of Frankia
Hahn, D. - \ 1990
Agricultural University. Promotor(en): A.J.B. Zehnder; A.D.L. Akkermans. - S.l. : Hahn - 126
frankia - stikstofbindende bacteriën - symbiose - rhizobium - betulaceae - microbiële ecologie - frankia - nitrogen fixing bacteria - symbiosis - rhizobium - betulaceae - microbial ecology
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.
Initially, 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 strains
Screening 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.
Because 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.
The 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.
Distribution and population dynamics of Rhizobium sp. introduced into soil
Postma, J. - \ 1989
Agricultural University. Promotor(en): A.J.B. Zehnder; J.A. van Veen. - Wageningen : Wageningen UR Library - 121
rhizobium - bodeminoculatie - stikstofbindende bacteriën - knobbeltjes - symbiose - microbiologie - landbouw - populatiedynamica - bodembacteriën - rhizobium - soil inoculation - nitrogen fixing bacteria - nodules - symbiosis - microbiology - agriculture - population dynamics - soil bacteria
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>50 μm 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>50 μm 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.
Characterization of the nifA regulatory gene of Rhizonium leguminosarum PRE
Roelvink, P. - \ 1989
Agricultural University. Promotor(en): A. van Kammen; R.C. van den Bos. - S.l. : Roelvink - 126
stikstofbindende bacteriën - symbiose - rhizobium - micro-organismen - genetica - heritability - rhizobium leguminosarum - nitrogen fixing bacteria - symbiosis - rhizobium - microorganisms - genetics - heritability - rhizobium leguminosarum
This thesis describes the characterization of the nif A regulatory gene of the pea endosymbiont Rhizobiumleguminosarum PRE.
Chapter I gives a general overview on the regulation of nitrogen fixation in diazotrophs, with special focus on the regulatory NifA protein. The regulation of genes involved in nitrogen fixation in two bacteria is discussed in detail: the free living Klebsiellapneumoniae and the endosymbiont of alfalfa R . meliloti . Major differences exist between these organisms where the onset of nitrogen fixation is concerned. K . pneumoniae has a general nitrogen regulatory circuitry which senses an internal biochemical signal i.e. the level of available ammonia as defined by the glutamine to 2-ketoglutarate ratio, a high ratio indicating a surplus, a low ratio a deficit. Sensing of a N-deficit results is translated, through a chain reaction of protein modifications, into activation of the regulatory NtrC product by phosphorylation. The resulting NtrC-P activates transcription of the regulatory nif LA operon, which encodes the inhibitor NifL and the activator NifA. The Klebsiella NifA thereupon activates transcription of the genes involved in nitrogen fixation. In a recently published paper David et al. (1988) suggest that the onset of nitrogen fixation in R . meliloti starts with the sensing of the external oxygen level. The FixL protein is hypothesized to sense a decrease in oxygen level. This protein is thus activated and in turn activates the FixJ protein, which directly or indirectly activates transcription of the nif A gene. The Rhizobium NifA protein activates transcription of the nitrogen fixation genes. In this overview we hypothesize that the oxygen sensing protein FNR instead of FixL senses the internal oxygen level. FNR then activates transcription of the fix LJ operon. The FixL protein may be a moderator of the activity of FixJ, comparable to the role of NtrB in activating NtrC. To date all rhizobial NifA proteins, in contrast to Klebsiella NifA, were shown to be oxygen sensitive. The structural analysis of the NifA protein is described and possible functions ascribed to domains identified in this protein are discussed. A model for NifA activity emerging from data presented for K . pneumoniae is
In Chapter 2 the DNA sequence and deduced amino acid sequence of R . leguminosarum PRE are presented. The amino acid sequence differs in 30 amino acids from that published for R . leguminosarum 3855 (Grönger et al., 1987). A possible explanation for this difference is discussed. The NifA Open Reading Frame (ORF) reveals two potential translation start sites, which in a heterologous E.coli background appear to be used both. The second translation start, which leads to a 488 amino acids, 53 kD protein, is preferred over the first, which leads to a 519 amino acids, 56.1 kD protein. The R . meliloti (Weber et al., 1985, Buikema et al., 1985) and B . japonicumnif A genes (Thöny et al., 1987) also have two translation start sites. It was shown for R . meliloti NifA (Beynon et al., 1988) the full length protein is the active form in an E . coli background. It is discussed that a translational preference for the second translational start site, leading to the inactive protein, as was found in pulse labeling experiments in E . coli may also exist in Rhizobium . We therefore suggest that the experiments presented by Beynon et al. (1988) are not conclusive as to the size of the functional protein in a Rhizobium background. Primer extension experiments and S 1 -nuclease protection were used to identify the putative nif A promoter. A transcription terminator was identified by S1-nuclease protection.
Chapter 3 deals with a phenomenon reported by Hawkins and Johnston (1988) and Roelvink et al. (1988). A nif A::Tn 5 mutant can not be complemented by a plasmid having only the nif A coding DNA fragment. A detailed analysis of the nif A- nif B intergenic region is presented. The nif A gene has a transcriptional terminator typical of bacterial genes (Brendel et al., 1986) consisting of a four GC basepairs stem and a nine base loop followed by a thymidine rich DNA stretch. This terminator was sapped by S1-nuclease protection. The nif B gene has a RpoN dependent promoter, having all nucleotides thought to be crucial to its activity. The nif A terminator was fused to the Tet - promoter and this fusion was cloned in a low copytranscriptional lac Z vector. The results show that the nif A terminator allows 85% readthrough. RNA::DNA hybridisation studies show that the nif A gene is transcribed at a level twice of that of nif B. By using a plasmid, which has a DNA region encompassing nif A, nif B and a ferredoxin like gene downstream of nifB (Grönger et al., 1988, Klipp et al., 1988) it was shown that nif A::Tn 5 mutants can be fully complemented. Taken together these findings suggest that the nif A and the nifB gene are in one operon. The failure of plasmids having the nifA encoding DNA fragment alone to complement a nif A::Tn 5 mutant results from a polar effect of the Tn 5 transposon on nif B transcription.
Chapter 4 deals with the nif H promoter region of R . leguminosarum PRE as one of the target sites of the NifA protein. We determined the nucleotide sequence of this region and identified a pseudo upstream activator sequence (UAS), a pseudo promoter, a consensus UAS and a consensus promoter. The promoter, mapped by primer extension experiments, differs from the consensus in one of the nucleotides thought to be invariant (see Gussin et al., 1986). The function of the nif H promoter elements was tested in a heterolo gous E . coli and a homologous Rhizobium background. Fusions of the nif H promoter region to lac Z, and fusions of deleted nif H promoter regions to lac Z, were used in activation studies byE .pneumoniae NifA in E . coli . Both high and low copy (deletion) nif H:: lac Z fusions were conjugated to Rhizobium . The activation study in an E . coli background showed that the pseudo UAS and the pseudo promoter are not involved in the function of the promoter. A different result was obtained with low copy nif H:: lac Z constructs in a Rhizobium background. The construct having both pseudo and consensus UAS, when compared with a construct having the consensus UAS only, seems to delay the onset of nitrogen fixation by three days. We suggest that this indicates that the presence of one or more UAS's modulates the expression of nif and fix genes, as was suggested for UAS's of B . Japonicumnif and fix genes (Gubler and Hennecke, 1988). A nif promoter region holding a UAS, when cloned in a multi copy vector, can inhibit nitrogen fixation by capturing the NifA activator needed for expression of nif and fix genes. A multicopy Inhibition study with (deleted) nif H:: lac Z
The role of MgATP in nitrogenase catalysis
Cordewener, J. - \ 1987
Agricultural University. Promotor(en): C. Veeger; H. Haaker. - S.l. : Cordewener - 97
stikstofbindende bacteriën - nitrogenase - nitrogen fixing bacteria - nitrogenase
Naast water bepaalt de hoeveelheid stikstof die in de bodem aanwezig is in de meeste gevallen de produktiviteit van de landbouwgebieden op aarde. Het is van belang dat stikstof in een voor de plant bruikbare vorm beschikbaar is. op het eerste gezicht lijkt het vreemd dat er een tekort aan stikstof kan optreden, omdat 80% van de lucht om ons heen uit stikstofgas bestaat. Maar voor nagenoeg alle vormen van leven is de stikstof zoals die in de lucht voorkomt, namelijk als het molekuul N 2 , niet bruikbaar. Planten en de meeste mikroorganismen verkrijgen hun stikstof in het algemeen uit verbindingen zoals ammonia (NH 3 ) en nitraat (NO 3 ). Dieren verkrijgen de stikstof die ze nodig hebben om te kunnen groeien en funktioneren door het eten van planten en andere dieren. De reden waarom elk levend wezen stikstof nodig heeft is gelegen in het feit dat twee van de meest essentiële bestanddelen van de cel stikstof bevatten, namelijk eiwitten en nucleïnezuren (DNA, RNA). Slechts een beperkt aantal mikroorganismen is in staat om stikstofgas uit de lucht (N 2 ) om te zetten in ammonia. Dit proces noemt men stikstofbinding. Ammonia wordt als N-bron gebruikt voor de synthese van stikstofhoudende verbindingen. De stikstofbindende bakteriën komen in allerlei vormen voor, maar de belangrijkste voor de landbouw zijn die bakteriën die in de wortelknolletjes van vlinderbloemige planten leven in symbiose met de plant. Zonder de wetenschappelijke achtergrond te kennen maakten de Romeinen reeds gebruik van zogenaamde wisselbouw, dit wil zeggen, door het afwisselend verbouwen van vlinderbloemige gewassen als klaver en erwteplanten en niet-vlinderbloemige gewassen zoals graan, werd de vruchtbaarheid van de bodem op peil gehouden. Het duurde echter nog tot in de 19e eeuw voordat ontdekt werd dat wortelknollen bakteriën bevatten die stikstof uit de lucht om kunnen zetten in een stikstofverbinding die door de plant gebruikt kan worden.
In de huidige, intensieve landbouwgebieden wordt voor een belangrijk deel gebruik gemaakt van (stikstof) kunstmest om aan de stikstofbehoefte van de plant te kunnen voldoen. De Industriële produktie van kunstmest gebeurt volgens het Haber-Bosch proces: uitgaande van de grondstoffen N 2 en H 2 wordt bij hoge druk (350-1000 atmosfeer) en een hoge temperatuur (±350°C) ammonia gevormd In aanwezigheid van een ijzer bevattende katalysator. Door de hoge energiekosten die gepaard gaan met dit proces, evenals de hoge investeringskosten die nodig zijn voor het opzetten van een kunstmestfabriek (het is alleen grootschalig ekonomisch uit te voeren) en de noodzaak van opslag en transport van het geproduceerde kunstmest, is de produktie van kunstmest en dus het gebruik ervan in de landbouw voor de meeste ontwikkelingslanden ekonomisch niet haalbaar. Om het toenemende aantal mensen op deze wereld van voedsel te kunnen voorzien is het echter nodig dat de landbouwproduktie toeneemt. Dit houdt in dat ook de stikstofgift aan de bodem drastisch moet stijgen. Aangezien de huidige methoden van kunstmestproduktie gebruik maken van niet onuitputtelijke bronnen van energie (fossiele brandstoffen), is het belangrijk dat onderzoek gedaan wordt met als doel de biologische stikstoffixatie te verbeteren, zodat het gebruik van kunstmest beperkt kan blijven. Biologische stikstofbinding is een proces dat gedreven wordt door een onuitputtelijke energiebron namelijk zonlicht. Bovendien wordt het biologisch gebonden stikstof efficiënt gebruikt en spoelt niet direkt uit.
Het enzym, dat in stikstofbindende bakteriën de omzetting van N 2 in NH 3 katalyseert noemt men nitrogenase. Het in dit proefschrift beschreven onderzoek heeft zich voornamelijk gericht op het werkingsmechanisme van dit enzym. Omdat de eigenschappen van nitrogenase uit de verschillende soorten stikstofbindende bakteriën nauwelijks verschillen, en omdat de bakterie Azotobactervinelandii gemakkelijk op grote schaal gekweekt kan worden, zijn de in dit proefschrift beschreven eksperimenten uitgevoerd met nitrogenase uit deze bakterie. Tijdens de zuivering van nitrogenase wordt het enzym in twee komponenten gescheiden, vaak aangeduid met komponent 1 en komponent 2. Beide komponenten zijn nodig voor de omzetting van N 2 in NH 3 . Verder zijn er voor de reduktie van N 2 tot NH 3 , ATP en een sterk reduktiemiddel nodig.
In het Haber-Bosch proces wordt de energie die nodig is om het inert N 2 molekuul te reduceren geleverd door een hoge temperatuur. Daarentegen vindt de omzetting van N 2 in NH 3 door het enzym nitrogenase plaats bij kamertemperatuur. ATP is hierbij de leverancier van de benodigde energie. ATP is een in de cel voorkomende energierijke verbinding. Deze vastgelegde energie kan gebruikt worden in energie vereisende reakties. Hierbij wordt een fosfaatgroep afgesplitst en ontstaat er ADP. Voor de omzetting van één molekuul N 2 in twee molekulen NH 3 is de hydrolyse van 16 molekulen ATP nodig. De omzetting van N 2 in NH 3 , en ook de hydrolyse van ATP, treedt alleen op wanneer beide komponenten van nitrogenase aanwezig zijn. De afzonderlijke komponenten kunnen geen ATP hydrolyseren.
In de hoofdstukken 2 en 3 staan de eigenschappen beschreven van de binding van ATP en ADP aan komponent 2. Komponent 1 bindt geen adenine nukleotiden. De elektronen die nodig zijn voor de omzetting van N 2 in NH 3 worden via komponent 2 naar komponent 1 getransporteerd. Men neemt aan dat de substraat reduktie plaatsvindt op komponent 1 van nitrogenase. De hydrolyse van ATP tijdens de nitrogenase reaktie vindt plaats wanneer een elektron wordt overgedragen van komponent 2 naar komponent 1. Omdat er geen elektronen-overdracht plaatsvindt in afwezigheid van ATP, wordt aangenomen dat de ATP hydrolyse nodig is voor deze elektronen-overdrachtsreaktie. In hoofdstuk 4 wordt aangetoond dat nitrogenase ook ATP hydrolyseert wanneer er geen elektronen-overdracht tussen beide komponenten plaatsvindt. Dit was een eerste aanwijzing dat de ATP hydrolyse mogelijk nog een andere funktie heeft behalve een direkte deelname in de elektronenoverdrachtsreaktie. Een tweede aanwijzing werd verkregen uit eksperimenten waarin aangetoond werd dat, alhoewel komponent 2 slechts twee bindingsplaatsen bezit voor ATP, er vier molekulen ATP binnen 60 millisekonden gehydrolyseerd worden door het nitrogenase kompleks. Dit suggereert dat er twee ekstra ATP bindingsplaatsen ontstaan, wanneer komponent 1 en komponent 2 een kompleks vormen. Deze ATP's zouden mogelijk rechtstreeks betrokken kunnen zijn bij de omzetting van N 2 in NH 3 op komponent 1.
In hoofdstuk 5 wordt verder Ingegaan op de betrokkenheid van ATP in de nitrogenase reaktie. In het huidige model wordt aangenomen dat de langzaamste stap van de nitrogenase katalyse de snelheid is waarmee het nitrogenase kompleks verplicht moet dissociëren na de overdracht van elektronen van komponent 2 naar komponent 1. De eksperimenten beschreven in hoofdstuk 5 suggereren dat de snelheidsbepalende reaktiestap onder bepaalde kondities anders is, namelijk de dissociatiesnelheid van ADP van Av 2 .
Omdat een aantal kenmerken van de ATP hydrolyse door nitrogenase terug te vinden zijn in die van een ander eiwit dat ATP hydrolyseert/synthetiseert, de zogenaamde proton ATPase, worden in hoofdstuk 6 de mogelijke overeenkomsten tussen beide eiwitten wat betreft het mechanisme van ATP hydrolyse bediskussieerd.
Non-ionic nitrogen nutrition of plants : nutrient uptake and assimilation and proton extrusion during utilization of urea or symbiotically fixed nitrogen
Beusichem, M.L. van - \ 1984
Landbouwhogeschool Wageningen. Promotor(en): G.R. Findenegg. - Wageningen : Beusichem - 141
assimilatie - stikstof - stikstofbindende bacteriën - rhizobium - symbiose - assimilation - nitrogen - nitrogen fixing bacteria - rhizobium - symbiosis
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 - was the form of N-nutrition.
In soils, mineralized N may confuse the comparison between NO - -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 - 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 - -supplied pea plants no phloem transport of cation-organate is necessary for. the regulation of Intracellular pH and electroneutrality.
|Isolation, cultivation and characterization of Frankia strains from actinorhizal root nodules
Burggraaf, A.J.P. - \ 1984
Leiden : Burggraaf - 179
frankia - planten - symbiose - stikstofbindende bacteriën - rhizobium - frankia - plants - symbiosis - nitrogen fixing bacteria - rhizobium
Carbon and nitrogen metabolism of free-living Frankia spp. and of Frankia-alnus symbioses
Blom, J. - \ 1982
Landbouwhogeschool Wageningen. Promotor(en): E.G. Mulder, co-promotor(en): A.D.L. Akkermans. - Wageningen : Blom - 91
frankia - stikstofbindende bacteriën - symbiose - rhizobium - betulaceae - assimilatie - stikstof - wortelknolletjes - knobbelvorming - biochemie - metabolisme - polymeren - frankia - nitrogen fixing bacteria - symbiosis - rhizobium - betulaceae - assimilation - nitrogen - root nodules - nodulation - biochemistry - metabolism - polymers
The research reported in this thesis deals with the symbiosis of Frankia spp. and Alnus glutinosa. Frankia spp. are actinomycetes giving rise to the formation of nitrogen-fixing nodules on the roots of a number of non-leguminous plants. In these nodules Frankia spp. live within the plant cells and obtain all sources of carbon and energy from the plant, giving fixed nitrogen in exchange.
To answer the question what compounds Frankia spp. obtain from the plant, insight into the metabolism of this microorganism is required. To obtain this insight, researches have been made with the free-living Frankia AvcI1, isolated from root nodules of Alnus viridis ssp crispa by Baker et al. (1979). This organism has been isolated and cultivated on complex media. In order to obtain insight into the C- and N-source requirements of Frankia AvcI1, a simple and well-defined growth medium is needed. The composition of this medium and the C- and N-metabolism of Frankia AvcI1 are the subjects of Chapter II of this thesis.
In Chapter II.1 it is shown that Frankia AvcI1 is able to grow on a medium containing Tween 80 (an oleate ester of polyethyleneglycol sorbitan) as sole C-source and either glutamic acid or NH 4 Cl as sole N-source. The growth yield of Frankia AvcI1 on various media is reported. It is shown that the doubling time of Frankia AvcI1 growing on QMOD/Tween medium is about 2 days, which is slow, even for an actinomycete. When growing with Casamino acids as nitrogen source, Frankia AvcI1 selectively takes up glutamic acid and aspartic acid, leaving the concentrations of the other detectable amino acids unchanged. The mechanism of this selection is still unclear.
Chapter II.2 contains further data on the C-sources utilized by Frankia AvcI1 It is shown that Frankia AvcI1 can utilize as C-source also other Tweens, viz. Tweens 20, 40, 60 and 85, and in addition several fatty acids, viz. acetic, propionic, butyric, valeric, caproic, caprylic, capric, palmitic and stearic acids. No growth of Frankia AvcI1 was observed on media containing triglycerides as C-sources.
The dependence of the growth yield on the nature of the carbon source and the concentration of NH 4+ in the media is shown. Utilization of NH 4+ as nitrogen source of Frankia AvcI1 growing in the Tween/NH 4+ medium is confirmed by incorporation experiments with 15 N-NH 4 Cl.
The results reported in Chapter II.3 show that Frankia AvcI1 does not take up glucose from a medium containing both glucose and Tween 80. In agreement with this observation, it is demonstrated that the activities of isocitrate lyase and malate synthase in cells grown on the QMOD/Tween medium (containing both glucose and Tween 80) are of the same order of
magnitude as in cells grown on the Tween/NH 4+ medium (containing Tween 80 as sole C-source).
Organisms growing on fatty acids, and degrading these compounds to acetyl-CoA, start gluconeogenesis with the action of the glyoxylate cycle (Kornberg and Krebs, 1957) leading to the conversion of 2 acetyl-CoA molecules to 1 molecule of succinate. The presence of the glyoxylate cycle enzymes isocitrate lyase and malate synthase in Frankia AvcI1 cells grown with Tween 80 as C-source is therefore not surprising. The succinate formed in the glyoxylate cycle can be converted to phosphoenolpyruvate by the subsequent action of the enzymes succinate dehydrogenase, fumarase, malic enzyme and pyruvate orthophosphate dikinase, which are found in cell-free extracts of Frankia AvcI1 From phosphoenolpyruvate, gluconeogenesis can continue with the action of phosphoglycerate kinase and 3-phosphoglyceraldehyde dehydrogenase.
For energy generation, Frankia AvcI1 can oxidize the acetyl-CoA derived from fatty acid breakdown in the citric acid cycle, although the low activity of the enzyme succinyl-CoA synthetase leaves room for the presumption that the citric acid cycle is not very operative in Frankia AvcI1 This is not impossible since the oxidation of fatty acids yields many reducing equivalents.
The data contained in Chapter II.4 show that Frankia AvcI1 does not take up succinate from a medium containing acetate plus succinate or succinate alone. In accordance, no repressing effect of succinate on the activities of the glyoxylate cycle enzymes was observed, whereas in cells grown on propionate these enzymes were not found, indicating that they are hot constitutive in Frankia AvcI1
Frankia AvcI1 is able to utilize several amino acids as sole nitrogen source, viz . alanine, γ-aminobutyric acid, aspartic acid, glutamic acid, glycine, leucine, phenylalanine, serine, threonine, tyrosine and valine.
No differences in C- and N-source requirements were observed for the three Frankia strains AvcI1, CPI1 (isolated by Callaham et al., 1978, from Comptonia peregrina root nodules) and AgSp+1 (isolated by Quispel and Burggraaf, 1981, from Alnus glutinosa spore-(+) type root nodules). These three strains were shown to utilize either Tween 80 or acetate, but no ethanol, lactate, glucose or succinate as sole C-source, and either NH 4 Cl, Casamino acids, aspartic acid or alanine as N-source.
From the data given in Chapter II it will be clear that Frankia AvcI1 is able to grow on a well-defined medium, which is a prerequisite for obtaining a clear insight into the physiology of the organism. The alcoholic root extract (Quispel and Tak, 1978) or the soybean lecithin (Lalonde and Calvert, 1979), added to the medium as growth factors, can be replaced by Tween 80 or fatty acids which are utilized as carbon source, while NH 4 Cl or amino acids can be utilized as nitrogen source.
It is unknown whether Frankia spp. grow with the same growth rate on media with different constituents. The growth yields presented in Chapter II are usually small as compared to the amount of carbon present in the media. This suggests that either an additional component of the medium is present in limiting concentrations or that Frankia spp. can only grow for a limited time after inoculation.
The only group of compounds known so far to be utilized as C-source by free-living Frankia AvcI1 are fatty acids, either free or esterified. The question what carbon source Frankia spp. living symbiotically in the nodule obtain from the plant, still remains to be answered. Based on the data obtained for free-living Frankia spp. it is not unlikely that some fatty acid functions as C-source under such conditions. Other possible candidates for this function are plant lipids like the alcoholic root extract (Quispel and Tak, 1978) or the soybean lecithin (Lalonde and Calvert, 1979). It is unlikely that sugars like glucose or dicarboxylic acids like succinate are playing this role, unless the ability of Frankia spp. to take up one or more of these compounds should alter during the transition from the free-living to the symbiotic stage.
The cultivation of free-living Frankia spp. is a powerful tool in discovering symbiotic interactions of the endophyte and the host. In Chapter II.4 of this thesis it is shown that by replacing acetate as C-source of the medium by propionate, the activities of the glyoxylate cycle enzymes are repressed. Other authors (Tjepkema, Ormerod and Torrey, 1980 and 1981, Gauthier, Diem and Dommergues, 1981) reported a medium in which free-living Frankia spp. show vesicle formation and N 2 -ase activity. The present knowledge thus enables one to influence regulation in freeliving Frankia spp., which is important in studying the symbiotic interactions mentioned above.
In Chapter III attention is paid to the assimilation of the ammonia produced by the endophyte living symbiotically in the root nodules of Alnus glutinosa grown in the greenhouse from seeds collected in Wageningen. In Chapter III.1 the composition of the pool of free amino acids in root nodules and the xylem tissue of stems is reported. It is shown that citrulline is the predominant free amino acid in nodules, while serine also occurs in relatively large amounts. In xylem tissue citrulline and glutamic acid are prominent.
The activities of N 2 -ase, GS, GDH and OCT in root nodule homogenates are reported. From the K m values of GDH for NH 4+ (16 mM) and glutamate (0.9 mM) and the concentrations in the nodule of NH 4+ (1.5 mM) and glutamate (0.5 mM) it is concluded that GDH in alder nodules probably is responsible for the deamination of glutamate and not for the synthesis of this key amino acid. The important function of GDH in the nitrogen metabolism of alder nodules is confirmed by the much higher activity of this enzyme in homogenates of nodules as compared to that in homogenates of root-tips and leaves.
The vesicle clusters, which contain the N 2 -ase activity, did not show activity of GS, GDH and OCT. The cytoplasm of the host cells was shown to possess the GS activity, while GDH and OCT are localized in the organelles of the host cells. No activity of NADH-dependent GOGAT was observed.
In Chapter 111.2 it is shown that the activity of NADH-dependent GOGAT from root nodules of lupins is inhibited by some compound in the homogenate of alder nodules.
Simultaneously with and independently of the present research, Schubert et al. (1981) analyzed the composition of the pool of free amino acids in nodules and the xylem tissue of Alnus glutinosa grown in the field in East Lansing, Michigan. The only difference with respect to these amino acid pools between the American alders and the European alders studied in the present research, is the relatively high amount of serine in nodules of the latter, whereas in nodules of the former this amino acid is not found. The results of both Schubert et al. (1981) and the present research confirm the hypothesis that in alder,citrulline is the main transport vehicle of fixed nitrogen (Miettienen and Virtanen, 1952; Leaf, Gardner and Bond, 1958; Wheeler and Bond, 1970).
From the results shown in Chapter 111.2 it can be concluded that our failure to find any GOGAT activity may be ascribed to the presence of an inhibiting compound in the homogenate of alder nodules, so that it is not excluded that GOGAT is active in alder nodules in vivo .
The data reported in Chapter III are in accordance with the following model: The nitrogen fixed as ammonia in the vesicle clusters of the endophyte, is assimilated in the cytoplasm of the host cell into glutamate by the action of GS and presumably GOGAT. Glutamate is in part deaminated in the plant organelles by the action of GDH to supply the NH 4+ required for the synthesis of carbamyl phosphate. Another part of the glutamate is converted to ornithine, which in the organelles of the host cell
reacts with carbamyl phosphate to form citrulline according to the OCT reaction. Citrulline is excreted from the nodule and serves as nitrogen source for the plant.
Actual and potential nitrogen fixation in pea and field bean as affected by combined nitrogen
Mil, M. van - \ 1981
Landbouwhogeschool Wageningen. Promotor(en): E.G. Mulder. - Wageningen : van Mill - 127
stikstofbindende bacteriën - symbiose - rhizobium - vicia faba - tuinbonen - pisum sativum - erwten - stikstofmeststoffen - assimilatie - stikstof - nitrogen fixing bacteria - symbiosis - rhizobium - vicia faba - faba beans - pisum sativum - peas - nitrogen fertilizers - assimilation - nitrogen
Actual nitrogen fixation of pea and field-bean plants, grown in soil in the open air, was determined as the acetylene reduction of nodulated roots. During the major part of the vegetative growth of these plants, actual nitrogen fixation was equal to the potential maximum nitrogenase activity of the bacteroids present in the nodules. This means that increase of the actual nitrogen fixation could be achieved only if the potential nitrogenase activity of the bacteroids would be enhanced or if more nodules would be present. During the generative growth phase, the potential nitrogenase activity of the bacteroids was not entirely utilized irrespective of the shoot mass of the host plant or the nitrogen-fixing capacity of the Rhizobium microsymbiont.
The addition of nitrate to nodulated plants sharply reduced actual nitrogen fixation but did not affect potential nitrogen fixation within 10 days. The nitrate effect was temporarily eliminated by treatment of the root nodules with benzyladenine, a synthetic plant-growth regulator with a photosynthate-attracting action. It is concluded that incomplete utilization of the nitrogenase present in the bacteroids is caused by an inadequate carbohydrate supply of the nodules upon supply of the leguminous plant with nitrate.
In root systems of pea plants growing in symbiosis with R. leguminosarum strains of poor nitrogen-fixing capacity, the N-limited growth led to an accumulation of carbohydrates. Upon the supply of such plants with nitrate, the carbohydrate level was decreased concomitantly to an increase in alternative (i.e. cyanide-resistant) respiration. Low rates of alternative respiration were found when nitrate was added to pea plants inoculated with highly effective strains, as the N-limitation of the plants was less severe. The carbohydrates respired in the nodules amounted to 6.3 mg C/mg N fixed with a moderately effective strain but only 3.1 mg C/mg N fixed with a highly effective strain.
Some rhizobial strains possess a hydrogenase that is capable of recirculating part of the hydrogen evolved in air by nitrogenase simultaneously with nitrogen fixation. However, the energy gain by hydrogen oxidation was very low, viz.
0.6-4.3% Of the costs of nitrogen fixation. As hydrogen oxidation did not delay nodule senescence or increase nitrogenase utilization, the presence of hydrogenase in rhizobial strains seems to be an unimportant factor in determining the nitrogen-fixing capacity of the symbiosis.
Strains of R. leguminosarum with a low nitrogen-fixing capacity showed a distinctly different nodule formation pattern on primary and lateral roots of pea plants as compared with highly effective strains. The estimates of the yield of fixed nitrogen, derived from the acetylene-reduction method, of plants inoculated with rhizobia] strains of different nitrogen-fixing capacity, equally deteriorated during the growth period. When rates of hydrogen production in air were subtracted from the acetylene reduction rates, the estimates of nitrogen fixation were severely biased in favour of a hydrogenase-containing strain.
Nitrogen fixation of pea plants infected with a highly effective strain of R. leguminosarum was decreased by the supply of combined nitrogen to a greater extent than that of plants with a moderately effective strain. In plants inoculated with a strain of poor nitrogen-fixing capacity, nitrogen fixation per plant was even stimulated by a low dose of combined nitrogen. This increase was probably due to an enhanced photosynthetic capacity which counteracted the adverse effect of combined nitrogen. Seed yields were increased by nitrate dressings, regardless of the rhizobia] strain. Seed yields of plants inoculated with moderately effective strains were slightly higher than those of highly effective strains at high levels of combined nitrogen, owing to higher nitrate uptake, higher nitrogen fixation and increased photosynthesis.
Effect of combined nitrogen on symbiotic nitrogen fixation in pea plants
Houwaard, F. - \ 1979
Landbouwhogeschool Wageningen. Promotor(en): E.G. Mulder. - Wageningen : Houwaard - 95
biochemie - kunstmeststoffen - mest - metabolisme - micro-organismen - stikstof - stikstofbindende bacteriën - knobbeltjes - erwten - pisum sativum - plantenvoeding - rhizobium - bodeminoculatie - symbiose - synthese - chemische analyse - blootstelling - milieuafbraak - kinetica - ecotoxicologie - biochemistry - fertilizers - manures - metabolism - microorganisms - nitrogen - nitrogen fixing bacteria - nodules - peas - pisum sativum - plant nutrition - rhizobium - soil inoculation - symbiosis - synthesis - chemical analysis - exposure - environmental degradation - kinetics - ecotoxicology
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.