Nitrogen Fixation - University Of MarylandNitrogen Fixation 101 Jude Maul, USDA-ARS, Sustainable Agricultural Systems Lab Beltsville, MD, 20770 [email protected] And Julie Grossman,
35
Nitrogen Fixation 101 Jude Maul, USDA-ARS, Sustainable Agricultural Systems Lab Beltsville, MD, 20770 [email protected]And Julie Grossman, North Carolina State University Department of Soil Science [email protected]
Focus on legumes – nodulation and species specific relationships
How do we measure N-fixation?
Ways we are working on improving N-fixation in cropping systems. Quick example of our work with hairy vetch
Nitrogen Fixation
More than 99% of N on Earth is unavailable!
N2 must be “fixed” by prokaryotes into ammonia to be used for metabolic processes.
N is a primary constituent of proteins and nucleic acids, therefore essential for life.
Presenter
Presentation Notes
Although a huge amount of N is available in the atmosphere, soils, and water of Earth, more than 99% of this is unavailable! Typically reactive N has not accumulated globally. Why? Denitrificatoin happened at approximately the same rate. Now this is changing due to our human induced changes in N-cycle
Crop residue
Mineral Nitrogen
Decomposition
Legume based cropping system
N2
Biological Nitrogen Fixation
Presenter
Presentation Notes
Maize picture from free clip art: http://plants.phillipmartin.info/plants_corn_plant.htm
Who can fix nitrogen?
Associative
• Use C from rhizodeposits or decaying wood
• Genera represented: Azospirillum, Herbaspirillum, Burkholderia
• Many tropical grasses have them
Phototrophic
Symbiotic
N2
Presenter
Presentation Notes
BNF is a process exclusively restricted to the prokaryotes of the domains Bacteria and Archaea. Free-living; organisms that either photosynthetically fix their own C (photosynthetic diazatrophs, or phototrophs) or those that associate with other eukaryotes and use already fixed C (heterotrophic diazatrophs) released from roots as soluble C rhizodeposits, or where an actively decomposing high C:N ratio material is found (i.e. wood). Those that can fix their own C have an advantage as C is usually most limiting to microbial growth. Atmospheric – lightning + N2 + O2 = N2O Industrial – Haber-Bausch process Biological – Free-living – Clostridium Associative – Azotobacter, Azospirillum Symbiotic Rhizobium, Bradyrhizobium with legumes Frankia with Casuarina Nostoc and Anabaena with Azolla lichens
Who can fix nitrogen?
Associative
• Use C from rhizodeposits or decaying wood
• Genera represented: Azospirillum, Herbaspirillum, Burkholderia
• Many tropical grasses have them
Phototrophic
• Cyanobacteria in rice paddies
• i.e. Azolla water fern and Anabaena azollae symbiont
• High N-fixation ability - > 100 kg N ha-1 yr
• Biological soil crusts have phototrophic N-fixers too
Symbiotic
N2
Presenter
Presentation Notes
BNF is a process exclusively restricted to the prokaryotes of the domains Bacteria and Archaea. Free-living; organisms that either photosynthetically fix their own C (photosynthetic diazatrophs, or phototrophs) or those that associate with other eukaryotes and use already fixed C (heterotrophic diazatrophs) released from roots as soluble C rhizodeposits, or where an actively decomposing high C:N ratio material is found (i.e. wood). Those that can fix their own C have an advantage as C is usually most limiting to microbial growth. Atmospheric – lightning + N2 + O2 = N2O Industrial – Haber-Bausch process Biological – Free-living – Clostridium Associative – Azotobacter, Azospirillum Symbiotic Rhizobium, Bradyrhizobium with legumes Frankia with Casuarina Nostoc and Anabaena with Azolla lichens
Who can fix nitrogen?
Associative
• Use C from rhizodeposits or decaying wood
• Genera represented: Azospirillum, Herbaspirillum, Burkholderia
• Many tropical grasses have them
Phototrophic
• Cyanobacteria in rice paddies
• i.e. Azolla water fern and Anabaena azollae symbiont
• High N-fixation ability - > 100 kg N ha-1 yr
• Biological soil crusts have phototrophic N-fixers too
Symbiotic • Legumes and rhizobia • Actinorhizal plants and
Frankia
N2
Presenter
Presentation Notes
BNF is a process exclusively restricted to the prokaryotes of the domains Bacteria and Archaea. Free-living; organisms that either photosynthetically fix their own C (photosynthetic diazatrophs, or phototrophs) or those that associate with other eukaryotes and use already fixed C (heterotrophic diazatrophs) released from roots as soluble C rhizodeposits, or where an actively decomposing high C:N ratio material is found (i.e. wood). Those that can fix their own C have an advantage as C is usually most limiting to microbial growth. Atmospheric – lightning + N2 + O2 = N2O Industrial – Haber-Bausch process Biological – Free-living – Clostridium Associative – Azotobacter, Azospirillum Symbiotic Rhizobium, Bradyrhizobium with legumes Frankia with Casuarina Nostoc and Anabaena with Azolla lichens
Who can fix nitrogen?
Associative
• Use C from rhizodeposits or decaying wood
• Genera represented: Azospirillum, Herbaspirillum, Burkholderia
• Many tropical grasses have them
Phototrophic
• Cyanobacteria in rice paddies
• i.e. Azolla water fern and Anabaena azollae symbiont
• High N-fixation ability - > 100 kg N ha-1 yr
• Biological soil crusts have phototrophic N-fixers too
Symbiotic • Legumes and rhizobia • Actinorhizal plants and
Frankia
N2
Presenter
Presentation Notes
BNF is a process exclusively restricted to the prokaryotes of the domains Bacteria and Archaea. Free-living; organisms that either photosynthetically fix their own C (photosynthetic diazatrophs, or phototrophs) or those that associate with other eukaryotes and use already fixed C (heterotrophic diazatrophs) released from roots as soluble C rhizodeposits, or where an actively decomposing high C:N ratio material is found (i.e. wood). Those that can fix their own C have an advantage as C is usually most limiting to microbial growth. Atmospheric – lightning + N2 + O2 = N2O Industrial – Haber-Bausch process Biological – Free-living – Clostridium Associative – Azotobacter, Azospirillum Symbiotic Rhizobium, Bradyrhizobium with legumes Frankia with Casuarina Nostoc and Anabaena with Azolla lichens
Associative Fixers with tropical grasses: Colonization of the corn root surface by A. brasilense at the root elongation zone
Someday add slide about Cuba and their use of Azospirillum on sugar cane
Who can fix nitrogen?
Associative
• Use C from rhizodeposits or decaying wood
• Genera represented: Azospirillum, Herbaspirillum, Burkholderia
• Many tropical grasses have them
Phototrophic
• Cyanobacteria in rice paddies
• i.e. Azolla water fern and Anabaena azollae symbiont
• High N-fixation ability - > 100 kg N ha-1 yr
• Biological soil crusts have phototrophic N-fixers too
Symbiotic • Legumes and rhizobia • Actinorhizal plants and
Frankia Diazotroph (def): Bacteria that use N2 as their sole source of N.
N2
Presenter
Presentation Notes
BNF is a process exclusively restricted to the prokaryotes of the domains Bacteria and Archaea. Free-living; organisms that either photosynthetically fix their own C (photosynthetic diazatrophs, or phototrophs) or those that associate with other eukaryotes and use already fixed C (heterotrophic diazatrophs) released from roots as soluble C rhizodeposits, or where an actively decomposing high C:N ratio material is found (i.e. wood). Those that can fix their own C have an advantage as C is usually most limiting to microbial growth. Atmospheric – lightning + N2 + O2 = N2O Industrial – Haber-Bausch process Biological – Free-living – Clostridium Associative – Azotobacter, Azospirillum Symbiotic Rhizobium, Bradyrhizobium with legumes Frankia with Casuarina Nostoc and Anabaena with Azolla lichens
Photosynthetic diazatrophs: Cyanobacteria of different forms
•Common in aquatic areas
•Photosynthesize
•Protect O2 through membranes or heterocysts
filamentous
Presenter
Presentation Notes
(Blue green algae) / cyanobactera fix N. Common in aquatic areas
Cyanobacteria in association with plants
Azolla used in Asia for centuries in association with rice
Presenter
Presentation Notes
Färgfabriken, a center for contemporary art and architecture in Stockholm, has invited Erik Sjödin to work with his ongoing project Super Meal this summer. Super Meal is a project that revolves around growing, cooking and eating the water plant Azolla. Azolla is a fern that lives in a unique symbiosis with a cyanobacteria that enables it to fix atmospheric nitrogen. Under ideal conditions it can double its biomass in two days, which makes it one of the world’s fastest growing plants. For centuries Azolla has been used as organic fertilizer in rice paddies in China. Lately it has also been introduced as fertilizer and animal fodder in other parts of the world. However, despite being a rich source of nutrients, it is still more or less unexplored as an ingredient in food for humans. Recent research on Azolla as a component of a space diet for habitation on Mars is the inspiration for Super Meal. Among other things, the research shows that it is possible to grow all the food a human needs in an area of about two hundred square meters – less than one hundredth of the area that the average American’s food production occupies today. (http://www.cityfarmer.info/2010/07/15/growing-cooking-and-eating-azolla-a-water-fern/)
Who can fix nitrogen?
Associative
• Use C from rhizodeposits or decaying wood
• Genera represented: Azospirillum, Herbaspirillum, Burkholderia
• Many tropical grasses have them
Phototrophic
• Cyanobacteria in rice paddies
• i.e. Azolla water fern and Anabaena azollae symbiont
• High N-fixation ability - > 100 kg N ha-1 yr
• Biological soil crusts have phototrophic N-fixers too
Symbiotic • Legumes and rhizobia • Actinorhizal plants and
Frankia Diazotroph (def): Bacteria that use N2 as their sole source of N.
N2
Presenter
Presentation Notes
BNF is a process exclusively restricted to the prokaryotes of the domains Bacteria and Archaea. Free-living; organisms that either photosynthetically fix their own C (photosynthetic diazatrophs, or phototrophs) or those that associate with other eukaryotes and use already fixed C (heterotrophic diazatrophs) released from roots as soluble C rhizodeposits, or where an actively decomposing high C:N ratio material is found (i.e. wood). Those that can fix their own C have an advantage as C is usually most limiting to microbial growth. Atmospheric – lightning + N2 + O2 = N2O Industrial – Haber-Bausch process Biological – Free-living – Clostridium Associative – Azotobacter, Azospirillum Symbiotic Rhizobium, Bradyrhizobium with legumes Frankia with Casuarina Nostoc and Anabaena with Azolla lichens
Legume/rhizobia symbiosis
Most terrestrial N2-fixing symbioses involve a N2-fixing prokaryote and a
photosynthetic host.
Presenter
Presentation Notes
Nitrogen fixation in legumes attributed to a group of bacteria consisting of a number of genera collectively known as rhizobia.
How the nodules form
Chronological sequence between plant and bacteria
Highly specific! A specific bacterial species with one, or a limited number of, plant species
Early visible steps in infection and nodule formation by Rhizobium
Steps in root hair infection in clover: *Rhizobium attachment to root hairs, *Localized enzyme production causing wall softening and root-hair curling *penetration by ~20 rhizobia, with fresh cell wall material deposited around them *Infection thread formation and movement of the rhizobia down the root hair
Photos courtesy of J.Gen. Microbiol.
Soybean root nodules
Nodule morphology depends on the plant, not the bacteria
Clover root nodules are tiny!
Pea nodules large and round
Presenter
Presentation Notes
Nodule development and N fixation are complex processes involving a large number of genes. Often found on plasmids, most expressed only when come into contact with flavonoid extracts of plants. nodD gene products recognize flavonoids and bacteria secrete protein that initiates attachment and root hair curling. Once bacteria released into plant cell cytoplasm are enclosed by plant derived peribacteroid membrane and differentiate into what are called bacteroids. N-fixation takes place here and membrane controls flow of N nutrients (malate and succinate) as well as iron, molybdenum and sulfur required.
Infection thread
Presenter
Presentation Notes
Biochemistry of N-fixation. Nitrogenase enzyme complex is vital: made up of Fe-S proteins Protein one: Fe-S protein Protein two: Fe-S-Mo protein Electrons flow to ferrodoxin and then reduces the MO-Fe protein, which then reduces the N2 gas to NH3. Hydrogen evolution is a constant feature of nitrogenase activity due to coreduction of H+. High levels of nitrogen in soil environment will result in. lack of nitrogen fixation and nodulation.
C N
O2
Presenter
Presentation Notes
Biochemistry of N-fixation. Nitrogenase enzyme complex is vital: made up of Fe-S proteins Protein one: Fe-S protein Protein two: Fe-S-Mo protein Electrons flow to ferrodoxin and then reduces the MO-Fe protein, which then reduces the N2 gas to NH3. Hydrogen evolution is a constant feature of nitrogenase activity due to coreduction of H+. High levels of nitrogen in soil environment will result in. lack of nitrogen fixation and nodulation.
Mechanisms to avoid oxygen
High respiratory rate – Azotobacter
Specialized cells – heterocysts
Avoidance – anaerobic metabolism
O2 regulating proteins - leghemoglobin
Effective nodules contain leghemoglobin
Nodules vary in their morphology
Presenter
Presentation Notes
Used to be called leg-hemoglobin, but since occurs in actinorhizozal symbioses as well, is perhaps better called hemoglobin.
Marschner, Mineral Nutrition of Higher Plants
Energetics of N2 Fixation
Soil N pool
Fixed N
N fixation decreases as soil N fertility increases
N additions
Drinkwater, 2004
Presenter
Presentation Notes
Mention ‘look at the size of the arrows’. Change color of the arrows and the pool
Factors affecting BNF in the field
Acid soil factors
Temperature
Water availability
Soil nutrients (P, Mo, Fe, S, Ca, Co, B, Ni)
Competition and persistence
Contact of rhizobia with fertilizer or fungicide
Presence of suitable rhizobia (see Inoculation)
Photo by P. Graham
When is it desirable to inoculate?
When the legume hasn’t been previously cultivated in the region, or hasn’t been cultivated for quite some time
When environmental conditions are unfavorable and could limit rhizobial survival in soil
When soil analysis shows N deficiency
When previous sowings have shown good nodulation but little evidence of nitrogen fixation
When a species is new to a region, yield increases following inoculation can reach 30-40% .
Commercial inoculant often uses peat as a carrier
Qualities of a good inoculant strain
Nodulates and fixes N with all the varieties of legume for which it is recommended.
Competes well with native rhizobia in the soil
Persistent over time in the soil
Tolerant of environmental conditions in the soil
Genetically stable
Grows well in simple and economic culture media
Resident rhizobia can represent a barrier to inoculant nodule formation
Rhizobia in soil are from either Inoculant, or indigenous (native) soil populations
Native rhizobia can be beneficial, or problematic if they compete with inoculant strain and are less efficient in fixing N (Obaton et al., 2002; Botha et. al, 2004)
Competitiveness of a rhizobia strain is its capacity to form nodules on a legume in the presence of other rhizobia of the same specificity.
Presenter
Presentation Notes
Optimal nitrogen fixation efficiency requires that adequate numbers of compatible and effective rhizobia microsymbionts are present in the rhizosphere. Rhizobia occupying legume root nodules come from either commercial inoculant applied to legume seed at planting, or from resident indigenous populations of rhizobia in the field soil. In many places where legumes are cultivated, populations of rhizobia are already plentiful in the soil, arising either from past use of commercial inoculants, or from indigenous rhizobia that nodulate wild legumes. In some regions these indigenous rhizobia can be beneficial as they may allow for improved legume production without labor and time spent on inoculation. In contrast, soils containing an abundance of indigenous rhizobia can be problematic if they are competitive with the strain introduced through inoculation and prove to be less efficient in fixing nitrogen than the inoculant strain (Obaton et al., 2002; Maier and Triplett, 1996; Van Rensburg and Strijdom, 1985; (McDermott and Graham, 1990). The competitiveness of a strain is its capacity to form many nodules on a legume in the presence of other rhizobia of the same specificity. This capacity is linked to complex mechanisms involving genes of the bacteria and of the legume as well as a series of en- vironmental factors (reviewed by Stretter, 1994)ine if strains of this bacteria retained their genetic characteristics many years after being introduced. The present work began in 1973 and was intented to study the evolution of distinguishable strains of B. japonicum. After 16 or 20 years in soils, the strains (Obaton et al, 2002)
