Laboratory Exercise 3 - Rhizobium-Arbuscular Mycorrhizal-Legume Symbiosis PDF

Laboratory Exercise No. 3 Rhizobium-Arbuscular Mycorrhizal Fungi-Legume Symbiosis  Learning Outcomes To understand legume nodulation and study their nitrogen-fixing abilities Discuss significance of Tripartite Symbiosis Nitrogen in the Air Required by all living organisms...

Laboratory Exercise No. 3 Rhizobium-Arbuscular Mycorrhizal Fungi-Legume Symbiosis  Learning Outcomes To understand legume nodulation and study their nitrogen-fixing abilities Discuss significance of Tripartite Symbiosis Nitrogen in the Air Required by all living organisms for the synthesis of proteins, nucleic acids and other nitrogen-containing compounds Earth’s atmosphere contains almost 80% N2 Cannot be utilized in this form (N2) until it has been fixed Green plants, the main producers of organic matter, use this supply of fixed nitrogen to make proteins that enter and pass through the food chain. Microorganisms (the decomposers) break down the proteins in excretions and dead organisms, releasing ammonium ions. Nitrogen in the Air Nutrient element most frequently found limiting to the growth of green plants Results from the continual loss of nitrogen from the reserve of combined or fixed nitrogen Continually depleted by processes: Microbial denitrification Soil erosion Leaching Chemical volatilization Removal of nitrogen-containing crop residues  This replacement of soil nitrogen is generally accomplished by the addition of chemically fixed nitrogen in the form: commercial inorganic fertilizers activity of biological nitrogen fixation (BNF) systems The Nitrogen Cycle Series of processes that converts nitrogen gas to organic substances and back to nitrogen in nature. A continuous cycle that is maintained by the decomposers and nitrogen bacteria. Five Steps of Nitrogen Cycle 1. Nitrogen Fixation (N2 to NH3/NH4+or NO3-) 2. Nitrification (NH3 to NO3-) 3. Nitrogen Assimilation (Incorporation of NH3 and NO3- into biological tissues) 4. Ammonification (organic N compounds to NH3) 5. Denitrification (NO3- to N2) Nitrogen Fixation Nitrogen can be fixed in three ways: 1. Atmospheric fixation Occurs spontaneously due to lightning; a small amount only is fixed this way. 2. Industrial Fixation The Haber process, which is very energy inefficient, used to make N fertilizers. 3. Biological fixation Nitrogen-fixing bacteria fix 60% nitrogen gas. The Mechanism of Nitrogen Fixation Atmospheric nitrogen (N2) is a molecule composed of two atoms of nitrogen linked by a very strong triple bond. Large amounts of energy are required to break this bond and the molecule is therefore quite chemically unreactive. General chemical reaction for the fixation of nitrogen: N2 + 3H2 + Energy -> 2NH3 The Mechanism of Nitrogen Fixation Living organisms use energy derived from the oxidation ("burning") of carbohydrates to reduce molecular nitrogen (N2) to ammonia (NH3). The chemical process of nitrogen fixation involves "burning" of fossil fuels to obtain: Electrons Hydrogen atoms Energy Biological Nitrogen Fixation (BNF) The reduction of nitrogen gas to ammonia is energy intensive. It requires 16 molecules of ATP and a complex set of enzymes to break nitrogen bonds so that it can combine with hydrogen. N2 + 3H2 + Energy -> 2NH3  Fixed nitrogen is made available to plants by the death and lysis of free living nitrogen-fixing bacteria Another is symbiotic association of some nitrogen-fixing bacteria with plants. Magnitude of BNF It is estimated that BNF on a global scale may reach a value of 175 million metric tons of nitrogen fixed per year. Amount of nitrogen fixed in any given situation depend on the environmental conditions and the nature of biological system(s) present which are capable of nitrogen fixation. Diversity of BNF Systems BNF is known to occur to a varying degree in many different environments including: Soils Fresh water Salt water and sediments Within the plant roots, stems and leaves of higher plants Digestive tracts of some animals The potential for nitrogen fixation exists for any environment capable of supporting growth of microorganisms. Symbiotic Nitrogen Fixation The most important contribution to BNF comes from the symbiotic association of certain microorganisms with the roots of higher plants. (e.g., Rhizobium) Small nodules are formed on the roots and these become filled with an altered form of the bacteria (bacteroids) which fix appreciable amounts of nitrogen. This symbiosis alone accounts for 20% of global biological nitrogen fixed annually. Rhizobium sp. Gram negative, free living bacteria Most well known species of a group of bacteria that acts as the primary symbiotic fixer of nitrogen. Can infect the roots of leguminous plants, leading to the formation of lumps or nodules where N fixation takes place.  Rhizobium sp. Establish highly specific symbiotic associations with legumes Form root nodules Fix nitrogen within root nodules Nodulation genes are present on large plasmid  Rhizobium sp. The bacterium’s enzyme system supplies a constant source of reduced nitrogen to the host plant. The plant furnishes nutrients and energy for the activities of the bacterium About 90% of legumes can become nodulated. Rhizobium sp. Free living rhizobia cannot fix nitrogen and they have a different shape from the bacteria found in root nodules. Regular in structure – straight rods In root nodules the nitrogen-fixing form exists as irregular cells called bacteroids which are often club and Y-shaped. Root Nodule Formation Set of genes in the bacteria control different aspects of the nodulation process. One Rhizobium strain can infect certain species of legumes but not others Host plant Bacterial symbiont Alfalfa Rhizobium meliloti Clover Rhizobium trifolii Soybean Bradyrhizobium japonicum Beans Rhizobium phaseoli Pea Rhizobium leguminosarum Sesbania Azorhizobium caulinodans Root Nodule Formation Specificity genes - determine which Rhizobium strain infects which legume. Even if a strain is able to infect a legume, the nodules formed may not be able to fix nitrogen. Such rhizobia are termed ineffective Effective strains - induce nitrogen-fixing nodules Effectiveness is governed by a different set of genes in the bacteria from the specificity genes. Nod genes - direct the various stages of nodulation. Root Nodule Formation 1. Bacteria encounter root; they are chemotactically attracted toward specific plant chemicals (flavonoids) exuding from root tissue, especially in response to nitrogen limitation naringenin daidzein (a flavanone) (an isoflavone) Root Nodule Formation 2. Bacteria attracted to the root attach themselves to the root hair surface and secrete specific oligosaccharide signal molecules (nod factors). 3. In response to oligosaccharide signals, the root hair becomes deformed and curls at the tip; bacteria become enclosed in small pocket. Cortical cell division is induced within the root. Root Nodule Formation Nodule development Enlargement of the nodule, nitrogen fixation and exchange of nutrients Root Nodule Formation 4. Infection thread penetrates through several layers of cortical cells and then ramifies within the cortex. Cells in advance of the thread divide and organize themselves into a nodule primordium. 5. The branched infection thread enters the nodule primordium zone and penetrates individual primordium cells. 6. Bacteria are released from the infection thread into the cytoplasm of the host cells, but remain surrounded by the peribacteroid membrane. Failure to form the PBM results in the activation of host defenses and/or the formation of ineffective nodules. Root Nodule Formation 7. Infected root cells swell and cease dividing. Bacteria within the swollen cells change form to become endosymbiotic bacteroids, which begin to fix nitrogen. The nodule provides an oxygen-controlled environment (leghaemoglobin = pink nodule interior) structured to facilitate transport of reduced nitrogen metabolites from the bacteroids to the plant vascular system, and of photosynthate from the host plant to the bacteroids. Leghaemoglobin An oxygen carrier and hemoprotein found in the nitrogen-fixing root nodules of leguminous plants. The pink color of nodules indicates the activeness of nodules in nitrogen fixation due to the presence of leghaemoglobin, a protein capable of binding oxygen. Large nodules and dark pink centers indicate effective nitrogen fixation and small nodules indicate low or no nitrogen fixation. N2 Fixing Enzyme Dinitrogen is reduced to ammonia by an enzyme complex known as nitrogenase (occurs with the bacteroids) The enzyme, nitrogenase consists of two components which is highly conserved in sequence and structure are: Nitrogenase (large, containing molybdenum, iron and inorganic sulphur) Nitrogenase reductase (small, containing iron and inorganic sulphur) Nitrogenase reduces one molecule of N2 to two molecule of NH3 by the utilization of 16 molecules of ATP and release one molecule of H2 as a by-product. N2 Fixing Enzyme Availability of molybdenum (Mo) in the soil is essential because the enzyme, nitrogenase, present in root nodules contains it. Mycorrhizae (Root-Fungus Association) mykēs (fungus) and ῥíζα, rhiza (roots) Relationships between specific fungi and the roots of numerous plant genera Essential for ecosystem functioning and the survival of plants estimates of 80–90% of all plant life believed to engage in at least one of the seven types of mycorrhizae. Most important forms of mycorrhizae: 1. Arbuscular mycorrhizae 2. Ectomycorrhizae Basics of the Mycorrhizal Relationship In AM and EM associations: 1. The fungus colonizes the roots of an appropriate plant host. 2. It develops threads of fungal material (emanating, or extrametrical hyphae) into the surrounding soil. 3. Some of the water and nutrients are transported through the hyphae into the roots, where they are exchanged with the plant for sugars derived from photosynthesis. Arbuscular Mycorrhizae (AM) Occur in the roots of herbaceous plants and trees such as sweetgum and maple The fungi forming AM typically produce large resting spores, which can be used to identify AM fungal species AM produce organs of nutrient transfer (generally known as haustoria) within root cells. Technically known as Arbuscules (from Latin word for “tiny tree”) Arbuscular Mycorrhizae (AM) Sometimes AM fungi also produce storage organs (vesicles) between root cells, a feature that led them to be called vesicular-arbuscular mycorrhizal (VAM) fungi in the past. Arbuscular Mycorrhizae (AM) Ectomycorrhizae Occur on numerous tree genera, including those commonly found in the nursery and landscape Unlike the large resting spores formed by AM fungi, EM fungal spores are often small and wind dispersed from fleshy fruiting bodies we call mushrooms. EM fungi produce a sheath of fungal material on the root known as the mantle, and an intercellular organ of nutrient transfer called the “Hartig net.” Ectomycorrhizae In contrast to AM arbuscules, the Hartig net is found between, not within root cells Colonized root tips may be brightly colored, and are often swollen and highly branched Host Range of Mycorrhizae AM and EM fungi vary in their levels of host species specificity. EM fungal species tend to form associations with specific host plant species AM fungal species are “generalists” and can associate with hundreds of different host plant species. This characteristic is reflected in the number of recognized mycorrhizal fungal species: thousands of EM vs. about 200 AM fungal species. Peanut (Arachis hypogea L.) widely used for food, fodder, fuel and soil improvement ability to form symbioses with extremely broad range of beneficial soil microorganisms (Black et al., 2012) most important beneficial legume symbioses are with rhizobia and AMF Rhizobium sp. established symbiotic relationship with many leguminous plants and form nodules capable of fixing/converting atmospheric N2 into ammonia and export the fixed N to the host plant (Roy et al., 2006) To maximize the benefits of BNF, ample supply of P is necessary. AM Fungi legumes establish symbiosis not only with rhizobia but also with AMF (Wang et al., 2011; Tajini et al., 2012) studies have reported the positive benefits of AMF on plant P uptake and N2 fixation (Hamel, 2004; Jia et al., 2009) Dual inoculation provided synergistic effects on nodulation of peanut and N2 fixation in low P soils (Devi and Reddy, 2001) Tripartite Symbiosis Mycorrhiza benefits the legume host through improved P nutrition (Cardoso and Kuyper, 2006), whereas Rhizobia fixes N2. Sugars and flavonoids are being released by the roots (legume) for the growth, reproduction and survival of both AMF and Rhizobia