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CS 753 BIOLOGY AND BIOCHEMISTRY J. O. Fening PhD.

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1 CS 753 BIOLOGY AND BIOCHEMISTRY J. O. Fening PhD

2 COURSE OBJECTIVES Expose students to theoretical and practical concepts of soil microbial flora and fauna The ecology and role of soil flora and fauna in soil health improvement; Build capacity to assess and monitor soil biological status in relation to soil quality and productivity.

3 LEARNING OUTCOMES Improved student capacity of understand the role of soil biota in soil-plant productivity Use of available strategies for optimum exploitation of soil biota in sustainable agricultural practices.

4 COURSE CONTENT An overview of Soil Microbiology; (overview of soil micro-, meso-and macro-fauna of agricultural significance, function and economic importance); The place of microorganisms in nature; Microbial community diversity and ecosystem functioning Humification (biodegradation effects of humification, humic and fulvic acid-formation and decomposition, Organic resource quality characteristic; Microbial decomposition of organic matter;

5 Pesticides and soil microorganism (effects on soil productivity, pesticide degradation; Mechanisms used by plant-growth promoting microbes Nitrogen fixing organisms, biological nitrogen fixation in tropical cropping systems) Mycorrhiza (overview of the role of mycorrhizas in tropical agriculture Taxonomy and classification of mycorrhiza; Management of VA in tropical crop production for sequestration of iron; Production of phytohormones or phytostimulators (stimulation of plant defense path, improved P uptake efficiency);

6 Introduction to molecular techniques (overview of molecular biology methods in microbial ecology, structure and function of nucleic acids, DNA replication and DNA extraction procedures, physical analysis of DNA, Genetic manipulation of DNA, development and use of nucleic acid probes and genetic makers); Biotransformation of nutrients and nutrient cycles.

7 T HE M ICROBIAL P OPULATION OF THE S OIL Most obvious property of soil – physical constituents. Biological element most considered – Macro fauna e.g. earthworm. Why overlooked the diversity, complexity and continual activity of the living organisms in the soil. Due to microscopic nature of the soil inhabitants. Microscopic nature compensated for by large numbers (10 7 – 10 9 ) per gram soil. An Overview

8 Without the vital process of the biological activity of soil microorganisms The soil would become a repository of dead plant remains. There would be no facility for the recycling of C, N, P for plant growth. A naturally fertile soil – A soil in which soil organisms are releasing inorganic nutrients from organic reserves at a rate sufficient to sustain rapid plant growth.

9 M ICROBIAL POPULATION OF THE SOIL Microflora ( bacteria, actinomycetes, fungi and algae) Microfauna (protozoa, nematode worms) Mesofauna (collembola, mites) Macrofauna (Earthworm, ants, termites, soil – inhabiting mammals)

10 S OIL F AUNA Compared to microbial studies. The soil Fauna until recently received little attention. Relative neglect has been due partly to: Difficulty of isolating the animals from the soil Problem of systematic classification Little was known about the actual food of many of the members

11 A DVANCES IN F AUNA S TUDIES Fixing and preparing casts and sections of soil without disturbing its structure. Identification of food sources by examination of gut contents, using microscopical and immunological methods.

12 E NUMERATION OF S OIL F AUNA Soil Monolith Procedure Tullsgren method Floating in salt solution Watering soil with emulsion of o – dichlorobenzene.

13 N EMATODES The soil nematodes – eelworms Small – segmented worms with their spindle – shaped bodies, between 1.5mm in length and µm in width. Most species are plant feeding. Live in association with roots or other underground structures as ecto-endo parasites. Characterised by the possession of mouth spears or styles use to penetrate the cells of host structures. Nematodes cause intractable problems as pest of economic crops.

14 L ARGER A RTHROPODS Soils normally support a diverse assemblage of larger arthropods. These include woodlice, centipedes and millipedes, beetles, ants, termites etc spiders and related forms. Some are partly considered as soil animals. These soil animals are important burrowers and channellers in the soil. Others such as beetles use the soil and particularly the litter layer, as a refuge. Most of these animals present in the soil attack the roots of agricultural crops so are considered as pest (beetles, wireworms, millipedes).

15 O THERS Slugs and snails - to some extent regarded as soil animals. Slugs can cause extensive damage to seedling crops in damp conditions. Populations can be as high as 1.4 x 10 9 ha -1.

16 S OIL I NHABITING M AMMALS Various small mammals – mice, voles, shrews rabbits, moles, gophers, are present in appreciable numbers in some undisturbed soils. Though their total weight per ha is usually small, they can cause loosening of the surface layers of the soil by their often extensive excavations.

17 F UNCTIONS OF S OIL F AUNA Decomposition of organic matter and cycling of nutrients – Numerous enzymes in gut including protease, lipase, amylase, cellulose and chitinase Provide well comminuted medium for microbial activity which greatly influence the total soil metabolism Burrowing activities have some importance in relation to soil drainage and aeration Direct drilling by slugs particularly in heavy clay soils provide positions for readily attack bon germination seeds

18 B ACTERIA Most numerous organisms in soil, with viable populations estimated at up to 200 millions individual cells per gram of soil. Most diverse in terms of physiology and nutrition. Identification and classification of a bacterial isolate requires information based on: Its morphology Nutrition Physiology In certain cases its immunological reactions Bases in its constituent nucleic acid. Number of bacteria in the soil is not evenly distributed. Usually correlated to the amount of organic matter. Numbers of bacteria immediately adjacent to roots are much higher than in the surrounding regions – Rhizosphere effect. T HE S OIL M ICROFLORA

19 F UNGI Form the second major group of soil microorganism. Depending on local conditions, particularly pH and ambient moisture content. Fungi or bacteria may predominate. Despite the fact that bacterial are more numerous many times fungi constitute the largest proportion of microbial protoplasm in cultivated soils. The numbers of fungi are greatest on the soil surface and decrease with increase in depth.

20 A CTINOMYCETES Are typically aerobic organisms and are uncommon in waterlogged soils. They are largely intolerant of acidity. Usually classed within the same taxonomic family as bacteria. But in many aspects of their morphology and growth patterns resemble those of fungi. The major importance of actinomycetes lies in their production of antibiotics e.g. streptomycetes.

21 A LGAE The algae are photosynthetic micro-organisms which contain chlorophyll and in some cases other pigments. In many soils the algae make no significant impact. Their importance is found in certain situations where they play unique roles. Where raw materials are exposed Where drastic erosion has occurred Where severe burning has taken place. In these situations, the photosynthetic ability of algae permits their growth as primary colonizers.

22 Retaining inorganic nutrients that might otherwise be leached away. Bringing them to the matrix the initial impact of organic material necessary for the transformation into soil to begin. In the most severe conditions where algae might not be able to survive. Association with fungi – lichen symbiosis allows initial colonization. The algae provides carbon substrates. All algae can contribute organic carbon and the cyanobacteria can acquire N 2 from the atmosphere.

23 I DENTIFICATION & E NUMERATION OF SOIL MICROFLORA Identification of soil microflora in situ is limited to the observation of certain algal forms such as diatoms and fungal sporing structures. For most purposes identification required removal from the soil – isolation. Isolation – Mechanical e.g. removal of fungal spores or physical followed by cultivation.

24 Cultivation involves sterilization, dilution and culturing If an organism has specific growth requirements. Selective culture methods are employed whereby a nutrient regime is designed to favour the desired organism and inhibit, or at least not encourage the growth of other microbes. Once isolation is achieved, identification is based on morphological characteristics for fungi, and algae and on physiological and biochemical characteristic for bacteria and actinomycetes.

25 P RESERVATION OF MICROBIAL CULTURES For further references Comparisons Re-examination Future usage Basic Principle: - Keep the morphological and physiological characteristic of organism intact Why culture.

26 P RESERVATION METHODS Culture tubes (agar slants) Preservation in soil (3 parts air dry soil + I part dried humus) Lyophilization

27 F UNCTIONS OF MICROFLORA Decompose plant and animal residues – liberate nutrients necessary for plant growth Oxidize and otherwise transform into forms readily available to plants various minerals such as ammonium salts Enter into various associations with plants that are highly important in the growth of plants Formation of various organic acids results in a greater solubility of soil nutrients, particularly the carbonates and phosphates They exert a highly favourable effect upon germination of seeds and subsequent

28 D ETECTION AND MEASUREMENT OF ACTIVITIES IN SOIL General activity measurements Respiration measurements (oxygen uptake or carbon dioxide evolution) Cell division rate Mycelial extension Enzyme activity or content Substrate utilization rate Product accumulation rate Radioisotope cycling studies

29 R ESPIRATION MEASUREMENTS Respiratory activity as a general activity measurement is well established. The Biometer flask is a common piece of apparatus used to measure the activity of soil samples. Soil is incubated at the required temperature, moisture content, etc. in the main flask and air can be added through the CO 2 absorber in the tube on top of the main flask. During incubation the CO 2 produced by respiration is absorbed into the alkali in the side tube.

30 This is then titrated to discover the amount of alkali neutralized by the evolved CO 2 More air can be added and the alkali replaced to continue the process of analysis. To obtain reproducible results from assays of respiration rate the soils are usually sieved to remove plant materials, insects, etc. This destroys aggregate structure and may stimulate activity due to release of nutrients previously protected inside the aggregates. Disturbance of any kind usually causes a temporary burst in respiratory activity.

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32 CO 2 evolved: Milligrams C or CO 2 = (B - V)NE Where V = volume (ml.) of acid to titrate the alkali in the CO 2 collectors from treatments to the end point, B = volume (ml.) of acid to titrate the alkali in the CO 2 collectors from controls to the end point, N = normality of acid, and E = equivalent weight. If the data are expressed in terms of carbon, E = 6; if expressed as CO 2, E = 22.

33 C ELL DIVISION RATE Cell division rates can be estimated by various counting methods (direct observation or plate counts) during a given time. If in situ rates are required, the very slow growth rates of soil microorganisms makes this a very difficult measurement to obtain. Autoradiographic tracer experiments can be used to trace cell division rates. Nalidixic acid inhibits cell separation after cell growth so that applying this antibiotic to samples leads to long filamentous cells of bacteria (cell growth but no division). These indicate cell division rates. Vital stains such as ANS or Calcofluor can be used to trace microbial cell division (or mycelial extension of fungi or actinomycetes).

34 M YCELIAL EXTENSION Fungi grow by extension of mycelium; if this can be observed, it can be used to estimate growth rates. Direct observation or extraction from soil (followed by observation and measurement) can be used.

35 E NZYME ACTIVITY OR CONTENT The dehydrogenase enzymes can be extracted from soil samples, reacted with tetrazolium dyes to form colouerd products and the colour measured by colorimertic methods. This gives an estimate of the dehydrogenase activity in the soil sample and thus an estimates of the organisms present. Various sieving techniques can be used to separate small soil animals, fungi and bacteria from soil samples, but they are all somewhat unsatisfactory - i.e. they do not completely separate the organisms even in a well-mixed soil suspension. Other enzymes that have been used to estimate activitiy include oxido-reductases, transferases, phosphaases, cellulases, and proteases.

36 S UBSTRATE UTILIZATION RATE Any compound that is a substrate for microorganisms can be used to estimate their activity if the decrease in substrate concentration can be measured. Examples are: carbohydrates - detected and measured by the anthrone reaction amino acids -detected and measured by the ninhydrin reaction proteins - detected and measured by the Biuret reaction (Folin) etc., etc. There are always problems of interference with assays, extraction problems, change in conditions as the substrates are used by the microorganisms, etc.

37 R ADIOISOTOPE CYCLING STUDIES Many elements can be obtained in the form of a radioactive isotope ( 14 C, 32 P, 3 H [tritium] etc.). They can be used to measure the activities of microorganisms growing and metabolizing compounds that include these isotopes. For example, glucose can be labelled with 14 C in specific positions in the glucose molecule and the fate of the radiolabelled atom can be traced through standard chemical analysis followed by detection of the radioactivity in specific fractions. If the radiolabelled atom has been evolved as CO 2 through respiratory of the cells, it will make CO 2 radioactive and this can be detected.

38 A UTORADIOGRAPHY If uniformly labelled 14 C-glucose (all C atoms are the radioactive isotope 14 C) is added to a soil and incubated, the rate of uptake of 14 C into cells can be detected using the technique of autoradiography. In this technique, the cells are added in suspension to the surface of a photographic emulsion on a plate. During incubation in the dark, the radioactivity in the cells causes the silver salts in the emulsion to become "exposed" (in a similar manner to exposure to light) and the silver grains produced show as dark spots on the emulsion. Presence of dark spots signals the presence of 14C. Any radioisotope can be used in this process to detect uptake. Different compounds can be purchased already labelled with specific radioisotopes of many different kinds.

39 If the cell suspension or soil sample with added glucose is incubated longer, the glucose will be metabolized and can be detected after absorption in apparatus similar to that described under Respiration Measurements above. An organic liquid is used to absorb the CO 2 so that an organic scintillation liquid can be added directly to the absorbing fluid. This can then be placed in a scintillation counter and the flashes of light emitted by the scintillation fluid molecules as they are impacted by emitted particles from the decay of the radioisotope can be measured.

40 In this manner, the amount of radioactivity can be measured. Stable isotopes (i.e. non-radioactive) can be used in a similar manner to trace microbial activity). For an example see the use of 15 N to trace nitrogen fixation later in the course. The incorporation of 3 H- labelled thymidine into DNA by cells is a popular measure of growth rates and activity.

41 M EASUREMENT OF B IOMASS A measure of the total microbial biomass in soils is often required when studying productivity or fertility of soils. Sometimes the biomass of specific parts of the microbiota is required - for example fungal biomass versus bacterial biomass. If possible, the metdod should yield results comparable to the results used for plant and animal determinations so that the overall cycling of material in the system can be compared. Early methods for biomass determination relied on counting microbial propagules and other cells directly by microscopy or by viable plate counting and converting these to biomass via some conversion factor.

42 More recently, newer methods have been developed that use measures of components of all cells (e.g. ATP) to estimate biomass. These methods include:

43 1. Soil fumigation method - A sample of soil is sieved and placed in a container and CO 2 output measured over 20 days. There is typically a period of very rapid respiration followed by a much lower but stable respiration rate. If the organisms are killed by fumigation with chloroform, this initial flush of activity does not occur and the cells are killed. If the chloroform is removed there will be bigger flush or rapid respiration because dead microbial cells will contribute to the total substrates available (they were killed by the chloroform treatment). The difference between the normal rapid respiration and the greater amount after chloroform treatment is due to the amount of microbial biomass originally in the system.

44 2. Determination of ATP content of soils - ATP is extracted from the cells in the soil and measured by its reaction with the enzyme system luciferin + luciferinase. The enzyme luciferinase is extracted from firefly tails and emits light when ATP reacts with its substrate luciferin. The emitted light is measured in a scintillation counter. Specific machinery is available for this ATP determination that integrates the reaction/enzyme/substrate system into one vial of dedicated small scintillation counter. The amount of ATP per gram a cell material varies, but averages 10.0 moles g -1 resting biomass. It has been proposed that the ratio of ATP to biomass C content is 1:120 in soil samples and this is very close to the ratio in exponentially growing microorganisms and eucaryotic cells.

45 3. Determination of cell wall components of bacteria - Bacteria contain specific cell wall components such as muramic acid that can be released by acid hydolysis and analyzed by High Pressure Liquid Chromatography (HPLC). The amounts of muramic acid in bacterial cell walls varies depending on whether the cells are gram negative or gram positive (12 g mg -1 average in Gram negative and 44 g mg -1 in Gram positive cells). Thus, unless the proportion of Gram positive to Gram negative cells is known, this technique has serious limitations. Bacterial spores also contain up to 4 times the normal levels of muramic acid.

46 4. Dilution plate counts and direct microscopical counting – Dilution plates usually only are able to culture between 1 and 10% of the viable organisms in soil samples. Direct microscopic observation methods (FITC, acridine orange staining, etc.) usually overestimate the number of cells because they include dead organisms or other particles in their count.

47 T HE PLACE OF MICROORGANISMS IN NATURE - THE NITROGEN CYCLE All life requires nitrogen-compounds, e.g., proteins and nucleic acids. Air, which is 79% nitrogen gas (N 2 ), is the major reservoir of nitrogen. But most organisms cannot use nitrogen in this form. Plants must secure their nitrogen in "fixed" form, i.e., incorporated in compounds such as: nitrate ions (NO 3 − ) ammonia (NH 3 ) urea (NH 2 ) 2 CO Animals secure their nitrogen (and all other) compounds from plants (or animals that have fed on plants).

48 Four processes participate in the cycling of nitrogen through the biosphere: nitrogen fixation decay nitrification denitrification Microorganisms play major roles in all four of these.

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51 N ITROGEN F IXATION The nitrogen molecule (N 2 ) is quite inert. To break it apart so that its atoms can combine with other atoms requires the input of substantial amounts of energy. Three processes are responsible for most of the nitrogen fixation in the biosphere: atmospheric fixation by lightning biological fixation by certain microbes — alone or in a symbiotic relationship with some plants and animalssymbiotic industrial fixation

52 A TMOSPHERIC F IXATION The enormous energy of lightning breaks nitrogen molecules and enables their atoms to combine with oxygen in the air forming nitrogen oxides. These dissolve in rain, forming nitrates, that are carried to the earth. Atmospheric nitrogen fixation probably contributes some 5– 8% of the total nitrogen fixed.

53 I NDUSTRIAL F IXATION Under great pressure, at a temperature of 600°C, and with the use of a catalyst, atmospheric nitrogen and hydrogen (usually derived from natural gas or petroleum) can be combined to form ammonia (NH 3 ). Ammonia can be used directly as fertilizer, but most of its is further processed to urea and ammonium nitrate (NH 4 NO 3 ).urea

54 B IOLOGICAL F IXATION The ability to fix nitrogen is found only in certain bacteria and archaea. Some live in a symbiotic relationship with plants of the legume family (e.g., soybeans, alfalfa). Some establish symbiotic relationships with plants other than legumes (e.g., alders). Some establish symbiotic relationships with animals, e.g., termites and "shipworms" (wood-eating bivalves). Some nitrogen-fixing bacteria live free in the soil. Nitrogen-fixing cyanobacteria are essential to maintaining the fertility of semi-aquatic environments like rice paddies. Biological nitrogen fixation requires a complex set of enzymes and a huge expenditure of ATP. Although the first stable product of the process is ammonia, this is quickly incorporated into protein and other organic nitrogen compounds.

55 D ECAY The proteins made by plants enter and pass through food webs just as carbohydrates do. At each trophic level, their metabolism produces organic nitrogen compounds that return to the environment, chiefly in excretions. The final beneficiaries of these materials are microorganisms of decay. They break down the molecules in excretions and dead organisms into ammonia.trophic level

56 N ITRIFICATION Ammonia can be taken up directly by plants — usually through their roots. However, most of the ammonia produced by decay is converted into nitrates. This is accomplished in two steps: Bacteria of the genus Nitrosomonas oxidize NH3 to nitrites (NO2−). Bacteria of the genus Nitrobacter oxidize the nitrites to nitrates (NO3−). These two groups of autotrophic bacteria are called nitrifying bacteria. Through their activities (which supply them with all their energy needs), nitrogen is made available to the roots of plants. Both soil and the ocean contain archaeal microbes, assigned to the Crenarchaeota, that convert ammonia to nitrites. They are more abundant than the nitrifying bacteria and may turn out to play an important role in the nitrogen cycle. Many legumes, in addition to fixing atmospheric nitrogen, also perform nitrification — converting some of their organic nitrogen to nitrites and nitrates. These reach the soil when they shed their leaves.

57 D ENITRIFICATION The three processes above remove nitrogen from the atmosphere and pass it through ecosystems. Denitrification reduces nitrates to nitrogen gas, thus replenishing the atmosphere. Once again, bacteria are the agents. They live deep in soil and in aquatic sediments where conditions are anaerobic. They use nitrates as an alternative to oxygen for the final electron acceptor in their respiration. Thus they close the nitrogen cycle.

58 T HE C ARBON C YCLE All living things are made of carbon. Carbon is also a part of the ocean, air, and even rocks. Because the Earth is a dynamic place, carbon does not stay still. It is on the move! In the atmosphere, carbon is attached to some oxygen in a gas called carbon dioxide. Plants use carbon dioxide and sunlight to make their own food and grow. The carbon becomes part of the plant. Plants that die and are buried may turn into fossil fuels made of carbon like coal and oil over millions of years. When humans burn fossil fuels, most of the carbon quickly enters the atmosphere as carbon dioxide. Carbon dioxide is a greenhouse gas and traps heat in the atmosphere. Without it and other greenhouse gases, Earth would be a frozen world. But humans have burned so much fuel that there is about 30% more carbon dioxide in the air today than there was about 150 years ago, and Earth is becoming a warmer place. In fact, ice cores show us that there is now more carbon dioxide in the atmosphere than there has been in the last 420,000 years.

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60 S ULFUR CYCLE Sulfur is one of the constituents of many proteins, vitamins and hormones. It recycles as in other biogeochemical cycles. The essential steps of the sulfur cycle are: Mineralization of organic sulfur to the inorganic form, hydrogen sulfide: (H 2 S). Oxidation of sulfide and elemental sulfur (S) and related compounds to sulfate (SO 4 2–). Reduction of sulfate to sulfide. Microbial immobilization of the sulfur compounds and subsequent incorporation into the organic form of sulfur.

61 These are often termed as follows: Assimilative sulfate reduction in which sulfate (SO 4 2–) is reduced to organic sulfhydryl (otherwise known as thiol) groups (R–SH) by plants, fungi and various prokaryotes. The oxidation states of sulfur are +6 in sulfate and –2 in R–SH. Desulfuration in which organic molecules containing sulfur can be desulfurated, producing hydrogen sulfide gas (H 2 S), oxidation state = –2. Oxidation of hydrogen sulfide produces elemental sulfur (So), oxidation state = 0. This reaction is done by the photosynthetic green and purple sulfur bacteria and some chemolithotrophs. Further oxidation of elemental sulfur by sulfur oxidizers produces sulfate.

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63 Potassium in nature occurs only as ionic salt. As such, it is found dissolved in seawater, and as part of many minerals. seawaterminerals Potassium ion is necessary for the function of all living cells, and is thus present in all plant and animal tissues. It is found in especially high concentrations in plant cells, and in a mixed diet, it is most highly concentrated in fruits. P OTASSIUM CYCLE

64 Potassium ions are an essential component of plant nutrition and are found in most soil types. Its primary use in agriculture, horticulture and hydroponic culture is as a fertilizer as the chloride (KCl), sulfate (K 2 SO 4 ) or nitrate (KNO 3 ). Potassium content of most plants typically ranges from 1/2 to 2 percent of the harvested weight of crops, expressed as (K 2 O), which is the conventional way fertilizer analysis is shown, in the order N, P, K. Modern high yield agriculture removes potassium from soils at a much faster rate than it can be replenished from weathering soil K containing minerals, which may not present in sufficient quantity. In animal cells, potassium ions are vital to cell function. They participate in the Na-K pump. In the form of potassium chloride, it is used to stop the heart, e.g. in cardiac surgery and execution by lethal injection.

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66 P HOSPHORUS CYCLE Unlike many other biogeochemical cycles, the atmosphere does not play a significant role in the movements of phosphorus, Because phosphorus and phosphorus-based compounds are usually solids at the typical ranges of temperature and pressure found on Earth. Soil microorganism acts as sink and source of available P in biogeochemical cycle. However, the major transfers in the global cycle of P are not driven by microbial reactions.

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68 M ICROBIAL D IVERSITY AND SOIL FUNCTIONS General concern to conserve biodiversity and its role in maintaining a functional biosphere. The tacit assumptions in many current studies are that (i)by characterizing diversity one will be able to understand and manipulate the working of ecosystems and (ii) the ability of an ecosystem to withstand serious disturbances may depend in part on the diversity of the system.

69 There is now experimental evidence that most organisms are functionally redundant The functional characteristics of component species are at least as important as the number of species per se for maintaining essential processes It is believed that at least some minimum number of species is essential for ecosystem functioning under steady conditions A large number of species is probably essential for maintaining stable processes in changing environments, the so-called ‘insurance hypothesis’

70 To provide a comprehensive view of the complex relations between microbial diversity and soil functionality we consider: The complexity of soil as a biological system; The problems in measuring microbial diversity and microbial functions in soil and the meaning of these measurements; Current ideas concerning the link between microbial diversity and soil functions; Instances when measurements of microbial diversity are unnecessary for a better understanding of soil functionality; and The research needed for a better evaluation and manipulation of microbial diversity and soil functionality.

71 S OIL AS A MICROHABITAT Soil is a complex microhabitat for the following distinctive properties. 1. The microbial population in soil is very diverse. Presence of about 6000 different bacterial genomes per gram of soil 2. Microbial biomass is large: in a temperate grassland soil the bacterial and fungal biomass amounted to 1-2 and 2-5t ha -1, respectively 3. Soil is a structured, heterogeneous and discontinuous system, generally poor in nutrients and energy sources (in comparison with the concentrations optimal for nutrient microbial growth in vitro ), with microorganisms living in discrete microhabitats

72 4. Another distinctive characteristic of soil as microhabitat is the property of the solid phase to adsorb important biological molecules such as proteins and nucleic acids. In this way some extracellular enzymes adsorbed by clay minerals or entrapped by humic molecules can maintain their activity, being protected against proteolysis, and thermal and pH denaturation. 5. The surfaces of soil mineral components can themselves catalyse many reactions. Clay minerals and Mn (III and IV) and Fe (III) oxides catalyse electron transfer reactions, such as the oxidation of phenols and polyphenols with formation of humic substances

73 D EFINITION OF MICROBIAL DIVERSITY Microbial diversity is a general term used to include genetic diversity, that is, the amount and distribution of genetic information, within microbial species; Diversity of bacterial and fungal species in microbial communities; Ecological diversity, that is, variation in community structure, complexity of interactions, number of trophic levels, and number of guilds. Here we consider microbial diversity simply to include the number of different fungal and bacterial species (richness) and their relative abundance (evenness) in soil microflora.

74 M AGNITUDE The number of formally named and accepted microorganisms is currently around 157,000 species Current conservative estimates of the probable world species totals are as high as 1.8 million The fungi and virus arguably being the least well known Group Known Species Total Species Percentage Known (%) Algae Bacteria Fungi Protozoa Viruses Total

75 M EASUREMENT OF MICROBIAL DIVERSITY Microbial diversity is measured by various techniques such as traditional plate counting and direct counts as well as the newer molecular- based procedures and fatty acid analysis. 1. Plate and direct count eg plate count technique or MPN technique 2. Molecular techniques The molecular techniques generally involve extraction of nucleic acid, directly or indirectly, from soil. They are independent of culture, and according to their sensitivity can detect species, genera, families or even higher taxonomic groups.

76 P HOSPHOLIPID FATTY ACID (PLFA) ANALYSIS This technique is based on the extraction, fractionation, methylation and chromatography of the phospholipid component of soil lipids.

77 M ICROBIAL AND BIOCHEMICAL FUNCTIONS IN SOIL Microbial and biochemical characteristics are used as potential indicators of soil quality, even if soil quality depends on a complex of physical, chemical and biological prosperities Microbial activity is a term used to indicate the vast range of activities carried out by microorganisms in soil Biological activity reflects not only microbial activities but also the activities of other organisms in the soil, including plant roots

78 Generally microbe-mediated processes are the most sensitive to perturbations in the soil For this reason the capacity of soil to recover from perturbations can be assessed by monitoring microbial activities The links between microbial diversity and soil functions have been studied by approaches based on the use of 1. soil with the same texture but different microbial composition 2. repeated CHC1 3 fumigations of soil to decrease microbial diversity 3. specific biocides for killing specific soil microorganism; and 4. sterile soils inoculated with soil microorganisms. The effect of microbial diversity on microbial functions in soil depends on the measured function.

79 S IGNIFICANCE OF MICROBIAL DIVERSITY AND ECOSYSTEM FUNCTION Individual level What may to a non-microbiologist appear to be a single individual of a plant or animal species Is in reality often an intimate functional biocosm involving a variety of microorganisms For instance herbivorous mammals such as cattle, could not function alone to digest the cellulosic and other materials on which they depend They rely on bacteria,fungi and protozoa resident in their guts About 85% of flowering plants and tress form intimate root associations with fungi, mycorrhizas which play a critical role in the absorption of nutrients most limiting their growth

80 Endophytic fungi ramify through healthy plant tissues at least in some cases producing chemicals which render the plants resistant to insect pest About fungi harness photosynthetic algae or cyanobacteria to form liches, swards of which dominate the ground in boreal and artic regions The of many wood boring beetles and termites always contain microorganisms especially anaerobic bacteria and protozoa essentially for their digestion of food stuffs

81 Ecosystem level Food pyramid of more complex organisms rest on wide microorganism bases eg numerous beetles feed on fungi A variety of microorganisms start to break down wood even before it is ingested and subjected to a termite’s gut biota without which the food web could not exit] Microorganisms are responsible for the break down of plant and animal remains and consequently for the cycling of nutrients in nature in their absence ecosystems would be swamped by their own debris The role of nitrogen fixation is especially important in ecosystem functioning and involves a considerable variety of microorganisms

82 Microorganisms that are pathogens are important in ecosystem maintenance by limiting the populations of a particular species that night otherwise dominate or disrupt the community The formation of soil it self through the geochemical and mechanical weathering of newly exposed rock involves microorganisms Perhaps the most important function of microorganisms in ecosystem is providing mechanisms for resilience to climate and other environmental changes.(microorganisms with similar functions renders them less vulnerable to change)

83 Global level Many of the green house gases (methane, hydrocarbons, ammonia, carbon monoxide) are all of microbial origin Microorganisms are also a carbon sink. Trapping substantial amounts of carbon within their tissues

84 PESTICIDE MICROBIOLOGY An important reserve for raising productivity and increasing the gross output of agricultural products is the elimination of losses of the harvest due to pest, plant diseases and weeds. This is achieved by integrated pest management including agrotechnical, quarantine, biological and chemical methods. Chemical Methods Based on the use of pesticides – herbicides, insecticides rodenticides, molluscicide, nematicides, fungicides etc

85 E NTRY OF P ESTICIDES INTO THE S OIL Introduced into the soil for destroying soil- dwelling pests, nematodes and pathogens of bacterial and fungal diseases. After treatment of green plants-pesticides are washed off by precipitation. Residues contained in leaves, roots etc. Disposal of expired products. Washing of pesticides equipment.

86 M OVEMENT IN THE S OIL Pesticides and their metabolites are found in the soil as a labile state with all three of its phases and can therefore migrate along the soil profile in a horizontal and vertical directions.

87 P ROCESSES OF M OVEMENT Molecular diffusion with capillary moisture Descending flow of gravitational water The root system of plants Displacement during tillage

88 B EHAVIOUR OF PESTICIDES IN THE SOIL Depending on the conditions, pesticides may remain in the soil unchanged and retain their toxicity for a more or less prolonged period. The property of pesticides to withstand the decomposing action of physical, chemical, and biological (biochemical and microbiological) processes characterizes their persistence. The persistence of pesticides in the soil depends on their chemical and physical properties, the dose, formulation (powder, liquid, etc.), the type of soil, its moisture content, temperature, and physical properties, the composition of the soil microflora, the specific composition of the growing plants, and the features of soil tilling.

89 With respect to the rate of their decomposition in the soil, pesticides can be divided into the following groups. Organochlorine insecticides – a decomposition period over 18 months. Derivatives of triazine, urea, and picloram-about 18 months. Derivatives of benzoic acid and amides of various acids-about 12 months. Phenoxyalkylcarboxylic acids, nitriles, derivatives of toluidine – 6 months. Derivatives of carbamic acid-up to 3 months. Organophosphorus substances – less than 3 months.

90 It must be noted that in many cases the type of soil and especially its microflora are the main factors determining the duration of decomposition of most pesticides. Even very persistent substances under the influence of certain microorganisms may rapidly decompose with complete destruction of the molecules. Pesticides incorporated into the soil in the form of granules persist in it for a longer time than powers or liquid substances. Pesticides, as a rule, are most persistent in soils with a high content of organic matter and a silt fraction.

91 D EGRADATION OF P ESTICIDES Physicochemical processes – Decomposition under solar radiation Hydrolytic and oxidation transformation adsorption by soil colloids owing to cation exchange Absorption by higher plants – translocation into stems, leaves & root vegetables. Microbiological decomposition

92 P HYSICOCHEMICAL PROCESSES Solar radiation. A marked role is played by the ultraviolet rays in the process of photooxidation. Many herbicides, especially dipyridyl preparations such as diquat, lose their toxicity under the action of solar radiation. The resulting metabolites have a low toxicity

93 H YDROLYTIC AND OXIDATION TRANSFORMATION Hydrolytic and oxidation transformations of many pesticides in the soil appreciably lower their toxic action. An important role is played by the chemical structure of the pesticide and its properties. The kind and number of halogen atoms and their arrangement in a molecule affect the rate of decomposition of pesticides that are derivatives of halogenated and aromatic carboxylic acids.

94 ADSORPTION BY SOIL COLLOIDS OWING TO CATION EXCHANGE Pesticides incorporated into the soil lose a part of their activity because of their being adsorbed by the soil colloids. The degree of adsorption of most pesticides largely depends on the humus content in the soil. The nature of adsorption of a pesticide will vary depending on whether an anion or a cation is the active part of a pesticide molecule, or whether its molecule is ampholytic or electrically neutral and does not dissociate. The degree of adsorption of pesticides by the soil depends greatly on its moisture content.

95 The larger the amount of water absorbed by the colloids, the smaller is the amount of free space remaining for the sorption of poisonous chemicals. The nature of adsorption depends on the chemical structure of the pesticide, its basicity, and on the properties of its functional groups to form hydrogen and dipole bonds The adsorption of pesticides in the soil also depends on its temperature. This is of a practical significance because triazine herbicides incorporated into the soil in cold and damp weather are adsorbed in the top layer of the soil, which prevents their being washed out or decomposed. Becoming desorbed when the weather gets warmer, they again exhibit their activity

96 M ICROBIAL DECOMPOSITION The mechanism of metabolism of pesticides in the soil under the influence of microorganisms consists in the following main reactions: dehalogenation, dealkylation, amide or ether hydrolysis, oxidation, reduction, breaking of an ether bond, hydroxylation of an aromatic ring, and breaking thereof. Catabolism – direct degradation in which the pesticide serve as energy source This involves: synthesis of inducible Presence of constitutive enzymes Conjugation Cometabolism

97 A BSORPTION AND D ETOXICATION BY P LANTS. The accumulation of persistent pesticides in the soil in a number of cases leads to their translocation into the stems, leaves, and root of vegetables. The level of the content of a pesticide in a plant is determined by absorption, the supply, and decomposition of the toxicant in the plant and the soil. As a whole, the intensity of migration of a pesticide from the soil into a plant and its accumulation in the productive organs Depend on its content in the soil although there is not always a direct relation between these parameters.

98 SOIL ORGANIC MATTER Soil Organic Matter Organic matter consists of: Undecayed plant and animal tissues. Fairly stable brown to black material bearing no trace of the anatomical structure from which it was derived. – Normally defined as humus. SOM also includes the organisms that live in soil and the soil biomass. SOM is measured by determining the organic carbon SOM = x OC

99 Elemental ratios in SOM SOM is composed of C 1 H 1 O 2 N 1 S 1 P The C: N ratio of SOM is relatively constant for different soils under a wide range of management conditions. Excluding strongly acid pH < 5 and poorly drained soils C:N ratios of top soils fall within surprisingly narrow limits, Most lying between 10 and 14. C:N ratio greater than 14 is a strong indication that A soil contains much partially decomposed plant material. Decomposition is often slowed under acid or anaerobic conditions

100 Hence acid soils and poorly drained soils have the widest C:N ratios Soils can develop under the most diverse conditions – temperate grassland, deciduous forest, tropical rainforest, and yet show similar C:N ratios. The C:N ratio often decrease down the soil profile. This is due to the presence of fixed NH 4 + The OC content of most soils decrease with depth but the content of fixed NH 4 + remains constant or increases so that the C:N ratio narrows sharply.

101 Extraction of organic matter from soil Extraction of O.M. from the soil with alkali Separate the major materials into: Humic acid Fulvic acid Humin These classes are different from one another in terms of their differential solubility in alkaline and acids. Their differential solubility relate to the molecular complexity of the substances. e.g. fulvic acids have lower molecular weight and higher oxidation states than humic acids and humin.

102 Humic substances are chemically reactive molecules because they have a number of functional groups such as: Carboxyl (CWH) Hydroxyl (OH) Ketone (C = O) Aldehyde (CHO) Because of the interactions of these radicals with other soil organic components, Decomposition may be slowed down due to the formation of large complex humic compound. The interaction of these radicals with the inorganic component of the soil may slow down the decomposition rate of O M. The living component of O. M. is responsible for catalyzing the O. M. transformation which is necessary for a stable productive ecosystem development.

103 O RGANIC MATERIAL RESOURCE QUALITY CHARACTERISTICS AND M ICROBIAL DECOMPOSITION The quantity of traditional organic inputs, such as crop residues and animal manures, has declined in many farming systems Due to reduced yields and other uses for animal feed, fuel and fiber Farmers are now faced with finding alternative or supplementary sources of nutrients. The variety of tropical agro-ecosystems and the diversity of organic inputs used in those systems, including trees, shrubs, cover crops, and composts Present a challenge for research and extension activities in soil fertility management.

104 The resource quality of plant materials varies with the plant species, plant parts and their maturity so it is essential that these are known for each plant material. Plant materials are classified by taxonomic family, genus, and species and whether they are able to nodulate and fix N or not. The material is further described according to plant part; leaf, stem, root, or stover and whether the material is fresh or litter.

105 To allow valid comparisons regarding the quality characteristic of organic materials They should be sampled, prepared, and analyzed by comparable methods. A minimum set of organic resource quality parameters that influence decomposition and nutrient release, and standard methodologies for measuring these parameters includes macronutrients, total C, lignin, soluble C, soluble polyphenolics, a-cellulose, and ash.

106 Organic resource quality of a given material may be influenced by the environmental conditions under which the plants grew therefore, information on the climate and soil from where the material was collected may assist with interpretation. In addition, site characteristics, such as, soil texture (% and, silt and clay) and climate variables (monthly maxima, minima and annual average temperature and rainfall) are needed for most decomposition and crop simulation models

107 Many of the organic resource entries include information on dry matter decomposition, and N and phosphorus release from the soil incubation or litter bag experiments. The methodologies used include field litterbag studies and laboratory incubations, including those with leaching or non-leaching conditions. Decomposition and nutrient release rates are reported by a variety of means and units such as decomposition constants on a per day, week, or yearly basis And N mineralization or immobilization based on the amount or percent of N released

108 To allow for comparisons a standard format and including cumulative mass or nutrient lost (or immobilized) at each sampling time as a percent of the initial amount is used. In addition to the time series data, a single exponential decay model is fitted to the data y = 100 exp(- kt), where y is the percent remaining, k the rate constant per week, and t the time in weeks.

109 Where data are available, k values are calculated for the different sampling times A decision tree has been developed for selecting organic materials as N source in biomass transfer systems. The decision tree results in four categories of materials that can be used 1. applying directly to the soil as an immediate source of N (category 1) 2. mixing with fertilizers (category 2), 3. compositing (category 3) 4. surface mulching for erosion control (category 4) The decision tree can be used to identify materials that fall into the various categories

110

111 Crop residues and organic wastes commonly are added to soils as sources of plant nutrients and to improve the physical properties of the soil. These materials do not contain the same quantity of nutrients. In fact, incorporating some organic materials into the soil can induce nitrogen deficiencies in plants. The composition of the added material determines whether nitrogen is released for plant growth or tied up in an unavailable form by the microorganisms that decompose the organic fertilizers.

112 R ATIO OF C ARBON TO N ITROGEN An important property of an organic residue that influences the immediate availability of nitrogen is the ratio of carbon to nitrogen (C:N). The addition of an organic fertilizer provides carbon that can serve as an energy source for most soil microorganisms. The residue not only will increase microbial activity but also nitrogen needs of the organisms. The microbes use the carbon to build cells and the nitrogen to synthesize proteins. If the organic residue has a C:N less than about 20:1 (high nitrogen content

113 then the microorganisms will obtain adequate nitrogen for their needs and will convert the excess organic nitrogen to ammonium (NH 4 + ). This conversion is called mineralization and is summarized in the following equation: organic N (e.g., protein) --> microbial activity --> NH 4 + (1) Ammonium is a form of nitrogen that plants can absorb organic nitrogen cannot be used by plants.

114 If the organic material has a C:N greater than approximately 20:1 (low nitrogen content) then the microorganisms whose activity increases because of the addition of the carbon will not obtain enough nitrogen from the residue. Consequently, the microbes absorb the plant- available sources of nitrogen in the soil. This process probably would cause a nitrogen deficiency in plants where a high C:N compound had been added to the soil.

115 The loss of plant-available nitrogen is called immobilization, which can be represented by the equation below: NO 3 - or NH > microbial activity --> organic N (unavailable nitrogen) (2) Immobilization could tie up the nitrate (NO 3 -) and ammonium (NH 4 +) for a number of months. After this time, the nitrogen will be released by mineralization of the organic nitrogen found in the residue and microbial tissue.

116 O THER C ONSIDERATIONS Other important considerations when adding organic compounds to soils are the rates of decomposition and the addition of toxic materials. Sawdust and wood chips decompose much more slowly than crop residues or animal manures. Some wood materials release toxic compounds upon decomposition. Biosolids such as sewage sludges could contain toxic metals and organic compounds; therefore, they must be managed carefully when applied to soils. Animal manures could increase soil salinity and could add large amounts of weed seeds to the soil.

117 B IOLOGICAL N ITROGEN F IXATION The 2 nd most important process apart from photosynthesis, in the manufacture of plant food. Nitrogen gas comprises about 80% of all gases in the atmosphere. This N 2 gas cannot be used directly by plants because of the N = N bond and has to be converted to a form that could be used through the process of BNF. All organisms that are able to fix N 2 share two properties in common. The ability to carry out this important and difficult reaction. And that all such organisms are prokaryotes.

118 N 2 - FIXING S PECIES IN T ROPICAL C ROPPING S YSTEMS The predominant group are the rhizobia – bacteria that form symbiotic associations with legume plants. These bacteria contribute the greatest amounts of biologically fixed N in agriculture The second most economically important group is the cyanobacteria (blue-green algae), which are found both as free-living species and in associations with a variety of plants, most notably the aquatic fern Azolla. This symbiotic association is deliberately introduced into rice paddy fields in China and Vietnam and contributes significant amounts of fixed N to rice crops

119 A third group of important N 2 -fixers are actinomycete Frankia species, which form symbiotic associations with flowering plants from a number of different families. Almost all of their host plants are woody perennials (trees and shrubs) And their importance in agroforestry is increasingly being realized. While most of known host plants for Frankia are temperate species, Frankia do form active N 2 -fixing symbioses with some tropical plants. The fast-growing tree genus Casuarina is the most prominent example, and is widely used in agroforestry in the tropics.

120 A fourth group of N 2 -fixers is more loosely associated with plants. This group includes Azospirillum species, which colonize the root epidermis of host species including wheat, maize and rice. Species of Herbaspirillum and Acetobacter, H. seropedicae and A diazotrophicus, have been found endophytically within the roots, shoots and stems of a variety of graminaceous plants, including sugarcane, wheat, maize and rice The last group of N 2 -fixing organisms that contribute to the N balance in tropical cropping systems is the free-living N 2 -fixers. Organisms such as Klebsiella and Azotobacter live in the soil and fix N 2 when other forms of N are unavailable.

121 E STIMATION OF RHIZOBIA POPULATION IN SOIL The plant infection count (also called the most probable number (MPN) count) is used to determine the number of viable r hizobia in the presence of other microorganisms. This indirect method is commonly applied in determining the quality of inoculants produced from nonsterile carrier materials. The MPN is calculated from the most likely number (m) found in the MPN tables. To find this number, use this procedure

122 Record nodulation as shown in Table Take note of the number of replications used (n=4) 1. Count the number of dilution steps used (s=10) 2. Add up the total number of + units (22) 3. Find this number (22) in Table A. 10 (calculated for 10 old dilutions) 4. Locate the most likely number (m) in column s=10, on the same line as 22, which is x 10 4

123 The MPN may now be calculated from “m” by using the formula below: The estimated number of rhizobia per gram or per ml is given by: x = m x d = (5.8 x 10 4 ) x 10 1 = 5.8 x 10 5 rhizobia /g inoculant v 1 m = likely number from the MPN table for the lowest dilution of the series d = lowest dilution (first unit) v = volume of aliquot applied to plant

124 T ABLE : E XAMPLE FOR RECORDING NODULATION FOR THE MPN COUNT. Nodulation (+) or ( - ) ReplicationsTotal Number of DilutionIIIIIIIVNodulated Units

125 NODULE DEVELOPMENT Root nodule formation can be divided into three stages 1. Pre-infection 2. Infection and nodule organogenesis and 3. Nodule function and maintenance The pre-infection starts when rhizobia are attracted by chemotaxis to the organic compounds excreted by root hairs, followed by the attachment of the bacteria to the root hairs leading to root hairs deformation and root hair curling. Attachments of rhizobia to the surface of root hairs produce many deformations including the characteristics shepherd crook.

126 At the center of the crook, disruption of the plant cell wall occurs which enables the rhizobia to enter the root hairs. As they do so, a new structure, the infection thread forms within the plant cell and encloses the rhizobia. The rhizobia themselves proliferate in their cells until they have almost filled them. These proliferating cells remain within the root endodermis and form the nodule.

127 T YPES OF NODULES The types of nodule that develops depends on the host plant, not on the rhizobial strain. There are two main nodule types; the determinate and the indeterminate. In general, temperate legumes such as Pisum, Vicia, Trifolium, and Medicago develop indeterminate nodules, while tropical legumes such as Vigna, Phaseolus and Glycine develop determinate nodules. Both types are composed of similar tissues, all formed from the nodule meristem. Indeterminate nodules are characterised by a persistent apical meristem whilst determinate nodules do not have a persistent meristem.

128 Determinate nodules grow for a fixed period, all parts of the nodule essentially differentiating at the same time and have a finite life span. In contrast, indeterminate nodules have an apical meristem that continues to be active throughout the lifetime of the nodule, Producing zones of new infection, and so giving rise to a gradient of differentiating progressing back towards the root. Nodules differ in shape and size, partly as a response to soil conditions and partly as a characteristic of the particular bacterial strain-plant variety interaction. They may be spherical, cylindrical, flattened and often bidentate or with coralloid branching, or may have an entirely irregular shape.

129 Their size may vary from that of a pinhead to over 1cm. The larger nodules are never spherical but have shapes giving a high ratio of surface area to volume, possibly to ensure and adequate supply of nitrogen gas to the active nodule cells and an adequate means of disposal of carbon dioxide produced in the nodule. In general, a nodule will only contain a single strain of Rhizobium, although dual occupancy of nodules is well documented

130 I NFECTIVENESS AND E FFECTIVENESS The ability of rhizobia to produce infections on the legume root and form nodules is called infectiveness. This property is restricted to specific groups of rhizobia and the hosts where the infections are induced. Infectiveness does not imply effectiveness in N 2 fixation. The term ‘symbiotic effectiveness’ provides an indication of a nodulated plant’s ability to fix nitrogen. To measure effectiveness in quantitative terms, growth and/or nitrogen fixation for the nodulated plants Is compared with the growth of plants receiving sufficient combined nitrogen or with the growth and nitrogen fixation of plants nodulated by a known superior inoculants strain.

131 As a general rule, nodules having white or green tissue pigmentation are considered inactive in nitrogen fixation. Ineffective nodulation caused by an incompatible or nonspecific rhizobial strain poses a problem to the plant as it demands photosynthate and renders little or no nitrogen fixation. Usually the symbiotic capacity of isolates is compared with that for plants inoculated with ‘standard’ highly effective strains and those treated with mineral nitrogen as controls.

132 The symbiotic effectiveness (S.E.) can be expressed as: S. E. = Dry mass or N content of plant inoculated with test strain x 100 Dry mass or N content of plant inoculated with standard strain Or, if a nitrogen-fed control plat is used: S. E. = Dry mass or total N of the plant inoculated with isolate x 100 Dry mass or total N of the N-supplied control plant

133 Occasionally the term ‘efficiency’ is used instead of ‘effectiveness’. Is more properly expressed as a measure, e.g., mg N 2 fixed per g nodule mass, or per mg carbon molecules utilized by the nodules fixing N 2. Although ‘highly effective’ associations are usually ‘efficient’, the two terms have a different basis of expression.

134 M ETHODS FOR ANALYZING DIVERSITY OF RHIZOBIA Cross inoculation The cross inoculation group concept is based on the ability of Rhizobium strains to specifically nodulate a group of legume host species. Based on this concept, rhizobial strains have long been described as specific for strains apparently restricted in their host range or promiscuous for strains with a very broad host range. The concept of cross-inoculation groups is of practical use in choosing which rhizobial strains to inoculate onto particular legume crops. It is also of some scientific value in raising the issue that there are particular groups of legume plants that tend to share common bacterial symbionts. However, the idea that all rhizobia-legume associations could be classified into neat, non-overlapping cross- inoculation groups has long been discredited

135 Host range may be heavily biased by the choice of host plants tested. For example, rhizobial strains have long been described as ‘specific’, for strains apparently restricted in their host range, Or ‘promiscuous’, for strains with a very broad host range. R. meliloti has always been considered specific, and yet a recent survey of field isolates of R. meliloti found 18 strains that could also induce ineffective nodules on Phaseolus vulgaris, Leucaena leucocephala and Macroptilium atropurpureum Many Rhizobium and Bradyrhizobium strains are so promiscuous that their host ranges do not even consist of closely- related legumes, But may include legume plants that are so distantly related as to be placed in different sub-families within the Leguminosae. For example, the fast-growing Rhizobium strain NGR234 has been shown to elicit nodules on over 37 genera of legumes, Including members of different sub-families such as Lablab purpureus (Papilionoideae,) and Leucaene leucocephala (Mimosoideae).

136 Another pitfall of the cross-inoculation concept is that it refers only to the capacity to nodulate a host plant, And pays no attention to fixation abilities. It is common to find strains of rhizobia which can elicit nodules on, say, ten different legume host species and yet in association with perhaps five of those host plants fix N 2 only weakly or not at all, A phenomenon that can be referred to as ‘host-specific fixation’. It indicates clearly that plant-bacterial recognition and specificity do not end with the initial induction of nodules, But continue right through nodule development All these stages of specificity, which are crucial from the practical viewpoint of aiming for enhanced symbiotic yields of fixed N Are overlooked by the cross-inoculation concept, at least in its simplest form.

137 The cross-inoculation group concept also fails to encompass the several known examples of specificity within a legume host species For example, R. fredii strains can effectively nodulate wild soyabean cultivars found in China, but are frequently either unable to induce nodules, or the nodules formed are ineffective, on the improved soyabean cultivars found in North America. Although the germplasm from which the North American cultivars have been bred was originally imported from the Far East

138 C ULTURAL AND METABOLIC CHARACTERISATION Cultural and metabolic characteristics have been described as useful guides for the recognition of rhizobial groups at the species level. Rhizobial strains may be recognised as such by a combination of a large number of traits such as growth characteristics, carbon source utilisation, stimulation by sugars or vitamins, limits of pH, temperature tolerance and production of hydrogen sulphide Cultural and metabolic parameters are used for a phenotypic characterisation that is frequently carried out in combination with an analysis of the genotype.

139 S EROLOGY Serological analysis characterises rhizobia according to their reactions with antisera produced against strains having some agronomic or particular interest. The most common serological methods currently used are agglutination, fluorescent antibody techniques And various forms of the enzyme-linked immunosorbent assay.

140 M OLECULAR ANALYSIS PCR is basically a biochemical amplification process where a single target DNA segment can be amplified a million-fold or more in several hours. The main feature of PCR is that, if the sequences of DNA flanking an unknown region of a DNA molecule are known, The unknown DNA can selectively be copied repeatedly to generate large quantities of DNA copies for further analysis. Analysis of the amplification product is usually done by standard agarose or polyacrylamide gel electrophoresis.

141 C OMPETITION FOR NODULE OCCUPANCY Competition in broad terms refers to interactions between two or more organisms struggling in order to gain advantage over limited resources such as nutrients, water, light and space, That are present in the environment in an amount insufficient to meet the biological demand. In the case of rhizobia, competition is most commonly used to refer to struggle for supremacy in nodule occupancy. Relative success in achieving module occupancy is affected by environmental factors, host plant species and cultivar, initial population size and distribution in the soil and by competition from other organisms.

142 M OLECULAR GENE MARKERS IN COMPETITION STUDIES Low symbiotic nitrogen fixation in plants is in many cases a result of competition between effective and ineffective rhizobia for nodule occupancy. In such cases, solving the rhizobial competition problem is essential in order to improve the symbiotic interaction between bacteria and plants. Apparently, the lack of suitable methodology to properly identify rhizobial strains has been the greatest barrier. Evaluation of the competitive ability of rhizobial strains has been done by employing intrinsic Or induced antibiotic resistances as identifying markers. Other markers used include the enzyme linked immunosorbent assay (ELISA). Analysis of plasmid profiles has also been used in rhizobial competition studies Techniques also have been developed by which a specific marker gene can be introduced into the genome of the organism to be studied.

143 The marker gene codes for an enzyme that gives rise to a coloured product following incubation with a histochemical substrate. The marker gene thus allows the visual detection of the marked organism. Such a marker gene in current use in ecological studies or rhizobia is the gus A gene encoding the enzyme B – glucuronidase (GUS) GUS is a hydrolase that cleaves a wide range of substrates – almost any aglycone conjugated to D – glucuronic acid in the configuration. Frequently used substrates are X-gluc and Magenta-gluc giving rise to a blue or red colour. Nodules occupied by gusA- marked rhizobia are detected by virtue of a simple colour change The greatest advantage of GUS is the nearly complete lack of endogenous activity in plants and most agriculturally important bacteria.

144 Another marker gene that has been used for the detection of bacterial strains and that can be used in combination with gusA, is the celB gene. It encodes the enzyme B-glucosidase with a high galactosidase activity that is thermostable and themoactive and has a half time of 85 hours at C. Assays for the detection of celB activity within a nodule or on plant sample are simple. The washed legume root is incubated in phosphate buffer at 70 0 C in order to destroy endogenous enzymes. The roots are then incubated in the presence of a chromogenic substrate for celB product such as X- gal (5-bromo-4-chloro-3-B-D-galactoside) giving rise to a blue colour.

145 I NTRO TO M OLECULAR BIOLOGY TECHNIQUES The realization that DNA lies behind all the cell’s activities led to the development of molecular biology Molecular biology aims to explain biological processes in terms of the structures and interactions between nucleic acids and proteins. Although it is a relatively young discipline, it has already transformed our understanding of the way in which cells store and express their genetic information, and has had an enormous impact on many fields of study, such as immunology, medicine, plant breeding, microbiology and forensic science. Molecular biology has also led directly to the immensely powerful, and potentially profitable, techniques of genetic engineering.

146 DNA STRUCTURE DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides: adenine, cytosine, guanine, and thymine. A nucleotide is a mono-, di- or triphosphate deoxyribonucleoside; that is, a deoxyribose sugar is attached to one, two or three phosphates. Chemical interaction of these nucleotides forms phosphodiester linkages, creating the phosphate- deoxyribose backbone of the DNA double helix with the bases pointing inward. Nucleotides (bases) are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine and cytosine pairs with guanine.

147 DNA strands have a directionality, and the different ends of a single strand are called the "3' (three-prime) end" and the "5' (five-prime) end." These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. In addition to being complementary the two strands of DNA are antiparallel: they are orientated in opposite directions. This directionality has consequences in DNA synthesis, because DNA polymerase can only synthesize DNA in one direction by adding nucleotides to the 3' end of a DNA strand. The pairing of bases in DNA through hydrogen bonding means that the information contained within each strand is redundant. The nucleotides on a single strand can be used to reconstruct nucleotides on a newly synthesized partner strand.

148 DNA replication

149 DNA POLYMERASE DNA polymerase adds nucleotides to the 3' end of a strand of DNA. If a mismatch is accidentally incorporated, the polymerase is inhibited from further extension. Proofreading removes the mismatched nucleotide and extension continues. DNA polymerases are a family of enzymes that carry out all forms of DNA replication. A DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. To begin synthesis of a new strand, a short fragment of DNA or RNA, called a primer, must be created and paired with the template strand before DNA polymerase can synthesize new DNA.

150 Once a primer pairs with DNA to be replicated, DNA polymerase synthesizes a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from two of the three total phosphates attached to each unincorporated base. When a nucleotide is being added to a growing DNA strand, two of the phosphates are removed and the energy produced creates a phosphodiester (chemical) bond that attaches the remaining phosphate to the growing chain. DNA polymerases are generally extremely accurate, making less than one error for every 107 nucleotides added.

151 DNA REPLICATION DNA replication, is the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. This process is "replication" in that each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules have been produced from a single double-stranded DNA molecule In a cell, DNA replication begins at specific locations in the genome, called "origins. Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork.

152 In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, A number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis. DNA replication can also be performed in vitro (outside a cell). DNA polymerases, isolated from cells, and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs such artificial synthesis in a cyclic manner to amplify a specific target DNA fragment from a pool of DNA.

153

154 POLYMERASE CHAIN REACTION ( PCR ) The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.

155 Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting.

156

157 Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size. A basic PCR set up requires several components and reagents. These components include: DNA template that contains the DNA region (target) to be amplified. Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C. Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis Monovalent cation potassium ions.

158 The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

159 Procedure Figure

160 Schematic drawing of the PCR cycle. ( 1) Denaturing at 94–96 °C. (2) Annealing at ~65 °C (3) Elongation at 72 °C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses. Typically, PCR consists of a series of repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three (Fig. ). The cycling is often preceded by a single temperature step (called hold ) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters.

161 These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers. Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. Annealing step: The reaction temperature is lowered to 50– 65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

162 Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'- phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment. Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction

163 P HYSICAL ANALYSIS OF DNA Agarose gel electrophoresis is a simple and highly effective method for separating, identifying, and purifying DNA fragments. The protocol can be divided into three stages: (1) a gel is prepared with an agarose concentration appropriate for the size of DNA fragments to be separated (2) the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation: and (3) the gel is stained or, if ethidium bromide has been incorporated into the gel and electrophoresis buffer, visualized directly upon illumination with UV light. Voltage applied at the ends of an agarose gel generates an electric field with a strength defined by the length of the get and the potential difference at the ends (V/cm). DNA molecules exposed to this electric field migrate toward the anode due to the negatively charged phosphates along the DNA backbone.

164 T RACKING DYES. The most common means of monitoring the progress of an electrophoretic separation is by following the migration of tracking dyes that are incorporated into the loading buffer. Two widely used dyes displaying different electrophoretic mobilities are Bromphenol Blue and Xyano Cyanol.

165 E THIDIUM BROMIDE. Ethidium bromide is commonly used for direct visualization of DNA in gels. The dye intercalates between the stacked bases of nucleic acids and fluoresces red-orange (560 nm). When illuminated with UV light (260 to 360nm). This allows very small quantities of DNA to be detected (< 5 ng) (Sharp et al., 1973).

166 M OLECULAR WEIGHT MARKERS. Among the samples loaded onto a gel, at least on lane should contain a series of DNA fragments of known sizes so that a standard curve can be constructed to allow the calculation of the sizes of unknown DNA fragments. The most commonly used molecular weight markers are restriction digests of phage λ DNA or, for smaller fragments, the plasmid and pBR322 that are frequently used as molecular weight markers.

167

168 Figure : Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.


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