Plant-Microbes Association

CHAPTER: 5

Plant-Microbes Association

Ø Symbiotic, Associative & Non-symbiotic Nitrogen Fixation.

Ø Azolla

Ø Blue Green Algae

Ø Mycorriza

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Symbiotic Nitrogen Fixation:

Nitrogen fixation:

•      It is a process by which nitrogen (N₂) in the atmosphere is converted into ammonia (NH₃).

•       Atmospheric nitrogen or molecular nitrogen (N₂) is relatively inert: it does not easily react with other chemicals to form new compounds.

•      Fixation processes free up the nitrogen atoms from their diatomic form (N₂) to be used in other ways.

•      Nitrogen fixation is essential for all forms of life because nitrogen is required to biosynthesize basic building blocks of plants, animals and other life forms, e.g., nucleotides for DNA and RNA and amino acids for proteins.

•      Therefore nitrogen fixation is essential for agriculture and the manufacture of fertilizer.

Rhizobia Bacteria:

•      The first Rhizobia were isolated from root nodules by M. Beijerinck, and shown to have the ability to re-infect their legume hosts, and to fix N₂ in symbiosis.

Rhizobium leguminosarum:

•      It is aspecies of gram-negative, aerobic, rod shaped bacteria that is found in soil and which causes formation of root nodules on some, but not all, types of field pea,  kidney bean, and clover.

Leguminous plants:

•      Leguminous plants are included under the family Fabaceae or Leguminosae, most of them have symbiotic nitrogen-fixing bacteria, Rhizobia in structures called root nodules.

Mechanisms of Symbiotic Nitrogen Fixation:

•      Symbiotic nitrogen fixation occurs in plants that harbor nitrogen-fixing bacteria within their tissues.

•      The best-studied example is the association between legumes and bacteria in the genus Rhizobium.

•      A symbiotic relationship in which both partners benefits is called mutualism. A mutualistic symbiosis is an association between two organisms from which each derives benefit.

•      It is usually a longer-term relationship, and with symbiotic nitrogen (N₂) fixation, often involves a special structure to house the microbial partner.

•      Each N₂-fixing symbiotic association involves an N₂-fixing prokaryotic organism, the microsymbiont (eg. Rhizobium, Klebsiella, Nostoc or Frankia) and a eukaryotic, usually photosynthetic, host (e.g., leguminous or nonleguminous plant, water fern or liverwort).

Basic steps in symbiotic nitrogen fixation:

1.      Root Nodule Formation

2.      Nitrogenase Activity

3.      Gene regulation in root nodules

1.      Root Nodule Formation:

•      Rhizobium bacteria stimulate leguminous plants to develop root nodules, which the bacteria infect and inhabit. The nodules develop in a complex series of steps:

Ø  Rhizobia can infect their hosts and induce root nodule formation using following mechanisms:

•      Root hair penetration and infection thread formation,

•      Entry via wounds or sites of lateral root emergence,

•      Penetration of root primordia

Steps:

i.            Rhizosphere organisms convert tryptophan in the root exudates to Indole Acetic Acid (IAA) which results in non-specific curling of the root-hairs

ii.            The capsular polysaccharides of rhizobia play a role in inducing “Shepherd’s Crook”, the site of infection.

iii.            Invagination of the root hair takes palce due to the production of polygalactouronase by rhizobia.

iv.            After this, infection threads are formed, through which rhizobia enter the host.

v.            When the infection thread reaches a cell deep in the cortex, it bursts out and the bacteria are liberated out in the cytoplasm.

vi.            Each bacterium so liberated gets surrounded by a membrane, pinched off from the infection thread.

vii.            Mitotic divisions of the host cell results in the development of a nodule.

viii.            Bacteria multiply rapidly and surrounded by membranous envelope.

ix.            Motile rhizobia, enlarges in size and lose their flagella and power of division. This state of bacteria is known as Bacteroids.

Ø  The space between the bacteroids and the membrane gets filled with leghaemoglobin. Leghaemoglobin helps bacteria in synthesis of ATP and Nitrogenase activity.

Ø  Nitrogen fixation takes place in these nodules and the effective nodules are pink in color.

Ø  Legume –Rhizobium  symbiosis is influenced by a variety of factors like host, bacterial strains, temperature, light, soil pH, phosphorous, etc.

2. Nitrogenase Activity:

•      Biological nitrogen fixation can be represented by the following equation, in which two moles of ammonia are produced from one mole of nitrogen gas, at the expense of 16 moles of ATP and a supply of electrons and protons (hydrogen ions):

                                         N₂ + 8H+ + 8e^- + 16 ATP = 2NH₃ + H₂ + 16ADP + 16 Pi

•      This reaction is performed exclusively by prokaryotes (the bacteria and related organisms), using an enzyme complex termed Nitrogenase. This enzyme consists of two proteins – an iron protein(nitrogenase reductase) and a molybdenum-iron protein (nitrogenase).

•      The reactions occur while N₂ is bound to the nitrogenase enzyme complex. The Fe protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe protein binds ATP and reduces the molybdenum-iron protein, which donates electrons to N₂, producing HN=NH. In two further cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to H₂N-NH₂, and this in turn is reduced to 2NH₃.

3. Gene regulation in root nodules:

                                         Various genes are involved in regulation process during entire nitrogen fixation mechanisms:

a.      nod-genes:

•      In the initial stage, host specificity becomes evident. This is controlled by nodulation (nod) gene.

•      Some of the nod genes induce the host plant to react by producing nodulins

•      Some node genes are required for root-hair curling and for cell divisions.

b.      nif-genes:

•      The genes that regulate the nitrogen fixation are called nif-genes.

•       These are found both in symbiotic and free living system

c.       fix-genes:

•      The symbiotic activation of nif-genes in the Rhizobium  is dependent on low oxygen concentration, which in turn is regulated by another set of genes called fix-genes.

Non- Symbiotic Nitrogen Fixation:

•      There are several free living microorganisms which fix nitrogen in soil.

•      Azotobacter spp. is the main organisms that fixes nitrogen freely.

•      other organisms includes: Clostridium, Klebsiella, Chloropseudomonas, Rhodospirrilum, Anabaena etc

•      Nitrogenase is the key enzyme involved in nitrogen fixation

•      The mode of action of nitrogenase is same as symbiotic nitrogen fixation.

•      The difference between symbiotic and non-symbiotic nitrogen fixation is that in this method there is no formation of root nodules.

•      The genetic determinants for nitrogen fixation is nif-genes.

•      The nif-genes which are present in the bacterium function as the controlling agents of nitrogen fixation.

•      There is another genes ntr-genes present in the free-living nitrogen-fixing bacterai which control the genes that allow growth on poor nitrogen sources.

Associative Nitrogen Fixation:

•      In this type of nitrogen fixation microbes are surface- associated on the roots  of plants.

•      Azospirrilum is associated with the roots of grasses and is capable of fixing atmospheric nitrogen.

•      Azospirrilum participates in all steps of the nitrogen cycle except nitrification .

•      It can fix atmospheric nitrogen in pure culture & under microaerophilic conditiion too.

•      A cluster of nif-genes has been identified in Azospirillum, which are considered to homologues to those of Klebsiella pneumoniae.

•      In some cases there has been root hair deformation due to the association with bacterium.

•      The bacterium invade the cortical and vascular tissues of the host, and lead to enhancement of the number of lateral roots and root hairs which help in increasing the mineral uptake by plant.

Blue-Green Algae:

•      The BGA abundantly distributed in the tropics play important roles in agriculture.

•      The first and foremost role is nitrogen fixation.

•      Most of the Nitrogen- fixing BGA are:

                                         Anabaena, Anabaenapsis, Aulosira, Cylindrospermum, Nostoc, Calothrix, Scytonema, etc.

•      BGA also add a bulk of organic matter to soil.

•      It also synthesize vitamins and growth substance ( Vit. B12, auxins and ascorbic acids.)

•      Cyanobacteria are able to survive in extreme environments because of unique adaptation in which the fix nitrogen.

•      These days mats of BGA are widely used as biofertilizer.

Mechanism of BGA in Nitrogen Fixation:

•      Cyanobacteria possess unique mechanisms for the protection of nitrogenase, the nitrogenfixing enzyme, against O2 .

•      Since diazotrophic cyanobacteria have the ability simultaneously to generate O2 and fix N2, the mechanisms that N2-fixers use in order to protect nitrogenase from O2 may be particularly well-developed in cyanobacteria.

1.      Heterocystous cyanobacteria :

•      Heterocystous cyanobacteria are those bacteria which produces heterocysts.

•   Heterocysts are specialized nitrogen fixing cells formed during nitrogen starvation by some filamentous cyanobacteria such as Nostoc, Cylindrospermum, & Anabaena.

•    Upon nitrogen starvation, some filamentous cyanobacteria start a programme of differentiation that leads to the formation of heterocysts.

•      These cells appear at semi-regular intervals along the filaments and are the sites of N2 fixation.

•      Heterocysts protect nitrogenase from inactivation by O2 by several mechanisms, including a high rate of respiration and decreased permeability to O2.

2.    Non-heterocystous cyanobacteria:

•      Typically, they fix N2 in the dark, and photosynthesise in the light. However, it is now emerging that different non-heterocystous cyanobacteria achieve this in different ways.

•      In some, Nitrogenase proteins are turned over only when bacteria are fixing nitrogen.

•      In other, the pattern of nitrogen fixation is endogenous.

Azolla-Anabaena Symbiosis:

•      The association between Azolla & Anabaena azollae is a symbiotic one, where in the eukaryotic partner Azolla houses the prokaryotic endosymbiont in its leaf cavities & provides carbon sources and in turn obtains its nitrogen requirements.

•      This mutual exchange of activities helps in quick growth and multiplication  of the fern under optimal environmental conditions.

•      This symbiosis helps to grow successfully in habitats lacking or having low levels of nitrogen under waterlogged conditions.

•      Mutually, they grow together at the surface of quiet streams and ponds throughout tropical and temperate region of the world.

•      Under microscopic examination, every Azolla sample will have filaments of Anabaena living within ovoid cavities inside the leaves.

Significances of Azolla- Anabaena Symbiosis:

•      Useful in rice paddies farming.

•      Capacities of fixing nitrogen become cheaper and faster

•      Help in enrichment and maintaining soil fertility

•      Offers sound ecological sustainability on a long term basis

•      Can be uses as green manure, water purifier, animal feed etc.

Mycorrhiza:

•      The symbiotic association of fungi with roots of higher plants is known as mycorrhiza,

•      In a mycorrhizal association, the fungus colonizes the host plant's roots, either intracellularly as in arbuscular mycorrhizal fungi (AMF or AM), or extracellularly as in ectomycorrhizal fungi.

•      The structure and development of mycorrhizal fungus hyphae is substantially altered in the presence of roots of host plants. These root-borne hyphae are distinct from hyphae which are specialised for growth in soil.

•      All mycorrhizas have intimate contact between hyphae and plant cells in an interface where nutrient exchange occurs.

•      The primary role of mycorrhizas is the transfer of mineral nutrients from fungus to plant. In most cases there also is substantial transfer of metabolites from the plant to fungus.

Types of Mycorrhiza:

                             There are four main types of mycorrhizae:

1.      Ectomycorrhiza:

•      In ectomycorrhiza , the fungus forms a compact mantle or sheath over the root surface and the hyphae grow out into the soil.

•      They are mostly found in the forest tress.

•      Basidiomycetes fungi producing mushroom (e.g. Amanita, Boletus) or puffball (Rhizopogon, Pisolithus) type fruiting bodies are the common fungal symbionts.

•      The mycorrhizal  hyphae assume at least partly the functions of root hairs.

•      Radioactive- labelling studies have shown that nitrogen, P, Ca, applied to soil can enter the plants through mycorrhiza.

•      The ectomycorrhizal fungi help in the phosphorous nutrition of plants through increased surface area of absorption, offer protection against some plant pathogens, and enhance rooting and survival of cutting through production of growth hormones.

2.      Vesicular arbuscular (VA) mycorrhiza:

•      The VA mycorriza have a loose network of hyphae in soil and a extensive growth within the cortex of the plant.

•      In the host cells they produce highly branched hyphal structures, called arbuscules and also vesicles. 

•      They are found in a wide variety of host plants, including most of the crop plants.

•      The fungi involved are Glomus, Acaulospora, Sclerocystis,Entrophospora etc.

•      These mycorriza improve plant growth through better uptake of P & Zn from soil.

•      It also penetrates the outermost cortex region, when the plant is well supplied with Phosphorous, but in phosphorous- deficient plants they penetrate deep into cortex and help plant to obtain nutrient from the soil.

•      It also stimulate beneficial organisms like Rhizobium, Azotobacter, and phosphate solubilizers in the rhizossphere and suppress the growth of root pathogenic fungi and nematodes.

3.      Ericoid Mycorriza:

•      Ericoid mycorrhizas are the third of the three more ecologically important types.

•      They have a simple intraradical (grow in cells) phase, consisting of dense coils of hyphae in the outermost layer of root cells.

•      It is seen mostly in the family like blueberry and Erica plants.

•      Pezizella ericae, an ascomycetes is most common fungal symbiont.

•      Ericoid mycorrhizas have also been shown to have considerable saprotrophic capabilities, which would enable plants to receive nutrients from not-yet-decomposed materials via the decomposing actions of their ericoid partners.

4.      Orchidaceous Mycorrhiza:

•      All orchids are infected at some stage in their life cycle by the orhidaceous mycorrhizal fungi.

•       After establishment of a mycorrhiza, organic carbon and other nutrients are passed from the fungus to the  orchid seed. 

 Biological interaction( Microbial Association in Soil)

Biological interactions are the effects of  organisms in a community on one another. In the natural world no organism exists in absolute isolation, and thus every organism must interact with the environment and other organisms. An organism's interactions with its environment are fundamental to the survival of that organism and the functioning of the ecosystem as a whole.

In ecology, biological interactions can involve individuals of the same species (intraspecific interactions) or individuals of different species (interspecific interactions). These can be further classified by either the mechanism of the interaction or the strength, duration and direction of their effects. Species may interact once in a generation (e.g.pollination) or live completely within another (e.g. endosymbiosis). Effects range from consumption of another individual (Predation, herbivory, or cannibalism), to mutual benefit (mutalism). Interactions need not be direct; individuals may affect each other indirectly through intermediaries such as shared resources or common enemies.

In soil, many microorganisms live in close proximity and interact among them-selves in a different ways. Some of the interactions or associations are mutually beneficial, or mutually detrimental or neutral.

The various types of possible interactions/associations occurring among the microorganisms in soil can be:

a) Beneficial:

i) mutualism ii) commensalisms and iii) proto-cooperation or

b) Detrimental / harmful –

i) amensalism, ii) antagonism,    iii) competition iv) Parasitism and v) predation

A.Beneficial Association:

1. Mutualism:

                        Mutualism is an interaction between two or more species, where species derive a mutual benefit, for example an increased carrying capacity. Similar interactions within a species are known as cooperation. Mutualism may be classified in terms of the closeness of association, the closest being symbiosis, which is often confused with mutualism. One or both species involved in the interaction may be obligate, meaning they cannot survive in the short or long term without the other species. Though mutualism has historically received less attention than other interactions such as predation, it is very important subject in ecology. Examples include, pollination an seed dispersal, gut flora, and nitrogen fixation by bacteria in the root nodules of legumes.

   2. Commensalism:

Commensalism benefits one organism and the other organism is neither benefited nor harmed. It occurs when one organism takes benefits by interacting with another organism by which the host organism is not affected. For example, many fungi can degrade cellulose to glucose, which is utilized by many bacteria. Lignin which is major constituent of woody plants and is usually resistant to degradation by most of the microorganisms but in forest soils, lignin is readily degraded by a group of Basidiomycetes fungi and the degraded products are used by several other fungi and bacteria which cannot utilize lignin directly. 

   3.  Proto-cooperation:

It is mutually beneficial association between two species / partners. Unlike symbiosis, proto-cooperation is not obligatory for their existence or performance of a particular activity. In this type of association one organism favor its associate by removing toxic substances from the habitat and simultaneously obtain carbon products made by the another associate/partner. Nutritional proto-cooperation between bacteria and fungi has been reported for various vitamins, amino and purines in terrestrial ecosystem and are very useful in agriculture.

Proto-cooperative associations found beneficial in agriculture are : i) synergism between VAM fungus-legume plants and Rhizobium in which nitrogen fixation and phosphorus availability / uptake is much higher resulting in higher crop yields and improved soil fertility, ii) synergism between PSM-legume plants and Rhizobium.

B.  Detrimental / harmful:

1. Ammensalism:

Amensalism is an interaction where an organism inflicts harm to another organism without any costs or benefits received by the actor. In this interaction /association one partner suppress the growth of other partner by producing toxins like antibiotics and harmful gases like ethylene, HCN, Nitrite etc. For e.g. many types of bacteria and fungi are perfectly capable of growing on bread under the right conditions. The bread mold Penicillium commonly grows on any bread that has passed its shelf life. This mold is capable of producing penicillin, which destroys many of the forms of bacteria that would also like to grow on this bread. It is this understanding of the bacteria-killing properties of penicillin that led to the use of it as an antibiotic medicine. ThePenicillium does not benefit from the death of the other bacteria, making this an example of antibiosis amensalism.

2. Antagonism:

In antagonistic interactions, one species benefits at the expense of another. Predation is an interaction between organisms in which one organism captures biomass from another. In such antagonism, one organism may directly or indirectly inhibit the activities of the other. Antagonistic relations are most common in nature and are also important for the production of antibiotics. The phenomenon of antagonism may be categorized into three i.e. antibiosis, competition and exploitation.

In the process of antibiosis, the antibiotics or metabolites produced by one organism inhibits another organism. An antibiotic is a microbial inhibitor of biological origin. Innumerable examples of antibiosis are found in soil. For example, Bacillus Species from soil produces an antifungal agent which inhibits growth of several soil fungi.

3. Competition:

Competition is a mutually detrimental interaction between individuals, populations or species. As soil, is inhabited by many different species of microorganisms, there exists an active competition among them for available nutrients and space. The limiting substrate may result in favoring one species over another. Thus, competition can be defined as “the injurious effect of one organism on another because of the removal of some resource of the environment”. This phenomenon can result in major fluctuations in the composition of the microbial population in the soil.

For example, chlamydospores of Fusarium, Oospores of Aphanomyces and conidia of Verticillium dahlae require exogenous nutrients to germinate in soil. But other fungi and soil bacteria deplete these critical nutrients required for spore germination and thereby hinder the spore germination resulting into the decrease in population.

4.Parasitism:

It is an association, in which one organism lives in or on the body of another. The parasite is dependent upon the host and lives in intimate physical contact and forms metabolic association with the host. So this is a host -parasite relationship in which one (parasite) is benefited while other (host) is adversely affected, although not necessarily killed. Parasitism is widely spread in soil communities, for example, bacteriophages (viruses which attack bacteria) are strict intracellular parasites Chytrid fungi, which parasitize algae, as well as other fungi and plants; there are many strains of fungi which are parasitic on algae, plants, animals parasitized by different organisms, earthworms are parasitized by fungi, bacteria, viruses etc.

5.Predation:

Predation is an association / exploitation in which predator organism directly feed on and kills the pray organism. It is one of the most dramatic inter relationship among microorganisms in nature, for example, the nematophagous fungi are the best examples of predatory soil fungi. Species of Arthrobotrytis and Dactylella are known as nematode trapping fungi. Other examples of microbial predators are the protozoa and slime mold fungi which feed on the bacteria and reduce their population. The bacteriophages may also be considered as predators of bacteria.