what is the meaning of Agriculture Microbiology 1.pptx

microbiology

1. Agricultural Microbiology Complied By Dr. Dawit getahun ( phd ) Assistant professor in Plant Pathology ODA BULTUM UNVERSITY

2. Course Description Definition and historical development of Microbiology Types and structure of microscopes Culture techniques; Classification of microorganisms into different groups; Characteristics of bacteria; Microbes in rhizosphere and phyllosphere Morphology , biology, nutrition, reproduction and classification of microorganism and other microorganisms of agricultural importance;

3. Microbiology of plant pathogenic microbes; Role of different microbes in nutrient transformations and nitrogen fixation Microbial interaction in the soil system; Like composting and decomposition of organic matter through microbes; biodegradation of pesticides; mycorrhizae and Their role in agriculture and commercial use of bio fertilizers in agriculture. Course Description

4. Objective of agricultural microbiology course By the end of the course, students will be able: To know microorganisms of agricultural importance and techniques of their handling; To describe the importance of agricultural microbiology; To recognize microorganisms of agricultural importance: bacteria, fungi and viruses; to understand laboratory techniques for culturing and identification of microorganisms; and to describe the roles of microorganisms in agriculture.

5. Chapter 1. Introduction of Microbiology what is Microbiology ? Microbiology is the science that deals with the study of microorganisms. The term microbiology derived from three Greek words mikros [small] bios [life] and logos [study]. . Its concerned with their form, structure, reproduction, physiology, metabolism and classification. It includes the study of their distribution in nature, their relationship to each other and other living organisms, their effects on human beings and on other animals and plants, their abilities to make physical and chemical changes in our environment and their reactions to physical and chemical agents.

6. what is Microorganisms? : Microorganisms are tiny and invisible to naked eye. So, they can be looked into and studied only with the help of microscope are an organism too small ( less than 0.1 mm in size) that cannot be clearly perceived (seen) by the human eye. E.g. Bacteria, fungi (yeast, mold), protozoa, algae, virus, phytoplasma and rickettsia. The human eye is not able to perceive (see) objects with a diameter of less than 0.1 mm size, therefore an object must be required to magnify at least 0.1mm to preferably 0.2mm for clear vision. Being too small to be seen with unaided eyes, these organisms are termed as microorganisms.

7. Among microorganisms , viruses are the smallest while algae are considered as largest microorganisms. Despite categorized under microorganisms, there are few microbes such as some fungi and algae that have visible sizes. Viruses are not considered as independent living cell because of their incapability of existence outside the living bodies. They are just genetic material surrounded by protein coat.

8. Bacteria, fungi, protozoa and algae are fairly simple organisms as most of them are single celled with no complex cellular organization even multicellular microorganisms do not have diverse range of cell types. Most microorganisms are unicellular in which all the life processes are performed by a single cell. All living cells contain protoplasm which is a colloidal organic complex consisting of largely proteins, lipids and nucleic acids.

9. Why study of Microorganisms is so important in Microbiology? Because some of them are useful and some are harmful to animals and us. Microorganisms has significant role in natural processes such as decomposition of organic matter, fixation of atmospheric nitrogen in soil, release of nutrients from soil and water to plants and animals. Some microbes help us in preparing our food; some help us in maintaining ecosystem and few can be eaten by us.

10. A number of industries use microbes for different purposes such as; in making of cheese and wine, in the production of penicillin, interferon and alcohol, in the processing of domestic and industrial wastes. C ause disease, spoil food;  Deteriorate materials like iron pipes, glass lenses and wood pilings. A number of enzymes, amino acids, hormones, and vitamins are prepared by using microorganisms.  Techniques of diagnosis and epidemiology of specific Microbes can be studied.

11. Scope of Microbiology Microbiology has an impact on medicine, agriculture, food science, ecology, genetics, biochemistry, immunology, and many other fields. Many microbiologists are primarily focus on gruoping of specific Microorganism ; Virologists - viruses Bacteriologists - bacteria  Phycologists – algae Mycologist -fungi  Protozoologists – protozoa

12. Microbiology– in context to agriculture Microbiology in general, has very diverse utility in agriculture, horticulture, animal sciences, fisheries and forestry and hence studied as a agriculture microbiology. A big number of harmful microorganisms called pathogens are responsible of plant diseases. these microbial pathogens are found in the soil, air and water and can infect the plant through the roots and leaves.

13. Therefore, getting inside into the causes, mode of dissemination, prevalence and control of those diseases requires basic understanding of microbiology under sub-discipline called plant pathology or phytopathology. A. Plant Pathology is basically the study of microorganisms that cause disease in plants . It also involves the understanding of interaction of environmental factors and host with infecting microorganisms. The microorganisms which cause diseases in the plants are called as plant pathogens .

14. B.Antagonism : In contrasting to plant pathogens, certain native microorganisms present in the soil feed upon (or antagonistic to) these pathogens and can prevent the infection of crop plants. This particular behaviour of microorganisms is called antagonism . Literally, antagonism is an interaction between two organisms where one organism benefits at the cost of harm to another organism. Example includes predation or a predator eating prey or parasitism. In case of plant pathogens , antagonism usually involves competition between two microorganisms for food, nutrients and production of inhibitory compounds such as antimicrobial metabolites, secondary metabolites, antibiotics and extracellular enzymes.

15. Terminology Secondary metabolites (SMs) are small organic molecules produced by an organism that are not essential for their growth, development and reproduction. extracellular enzymes :- Enzymes that are secreted to the outside the cell for the external chemical reactions are known as extracellular enzymes. Metabolites are chemical compounds of a low molecular weight that play a critical role in microbial metabolism. Intracellular metabolites are enclosed by cell structures such as the cell membrane or envelope, acting as a mechanical barrier. Antibiotics , also known as antibacterials, are medications that destroy or slow down the growth of bacteria. An antimicrobial agent is defined as a natural or synthetic substance that kills or inhibits the growth of microorganisms such as bacteria, fungi and algae.

16. C.Bio-pesticides: Some soil microorganisms produce compounds that stimulate the natural defense mechanisms of the plant and improve its resistance to pathogens. Biopesticides is deffined as the compounds derived from some living organisms and used to manage insect-pests by means of specific biological lethal effects. is contractions of ' biological pesticides ', include several types of pest management intervention: through predatory, parasitic, or chemical relationships. E.g. Bacillus thuringiensis ( Bt) , a bacterium capable of causing disease of Lepidoptera, Coleoptera and Diptera, is a well-known insecticide. The (Bt toxin) has been incorporated directly into plants through the use of genetic engineering.

17. D. Organic farming: Modern agriculture seeks to introduce agricultural practices that are health and maintain the long-term ecological balance of the soil ecosystem . In this context, use of biofertilizers, phytostimulators and biopesticides are examples. an environment friendly alternative of mineral fertilizers and chemical pesticides . E.Genetic engineering: is the way of transferring bacterial genes from one organism to another. In this direction, the specific genes from particular bacteria that can kill certain insects but do not cause harm to humans have been successfully transferred to plants as protein through Genetic engineering. This protein is toxic to the insects, so that when the insect feeds on the plant, the insect dies. E.g. Bt

18. F.Food and Fermentation technology Some of the promising examples where the microorganisms have been extensively used to aid up food industries are- Beer and wine production by yeast, bread making, processing of milk to dairy products by lactic acid bacteria and the production of vinegar by acetic acid bacteria. G.Soil Microbiology The soil is a favourable environment for a diverse range of microorganisms (Bacteria Fungi, algea and protozea. The activity of all these microorganisms is vital for the soil and is accountable for soil quality, texture, structure and other properties as well. H.Dairy Microbiology deals with microorganisms associated with milk and milk products. The science comprises of the study of the control and destruction of undesirable microorganisms leading to spoilage of the milk and milk products on one hand while on the other, also deals with intentional and directional introduction of beneficial microorganisms.

19. Historical development of Microbiology With the advent of advanced tools and microscopic techniques, the field of microbiology is developing day-by-day with the inclusion of practical application and aspects of human welfare. It is also interesting to mention here that relatively recent advances of microscopic tools have made certain old concepts and claims somewhat inaccurate. Many individuals have made significant contributions to the development of microbiology. Some benchmark points of microbiology are furnished as follow-

20. The ancient Babylonians: - are the first record of human using microbes. Over 8000 years ago, they were using yeast to make beer and Over 6000 years ago acetic acid bacteria to make vinegar . About 5000 years ago, Persia (Iran) recorded the wine making . The roman God of mold and mildew was Robigus and Robigo which means crop rust . (Rust is one of the plant diseases caused by fungus). God Robigus was very much feared because of crop lost. About 2000 years ago , Romans proposed that diseases were caused by tiny animals . The real microbiology history starts from 1600 s, when people began to make crude lenses and microscopes .

21. Robert Hooke was the first person to report seeing microorganism under a microscope. He saw cells in a piece of cork in 1665 , but his lenses were apparently too poor to “ see” bacteria . contributions of Antony von Leeuwenhoek :- he is dutch merchats He is t he discoverer of the microbial world (1632-1723) with his microscope . His microscopes were able to give clear images at magnifications from about 50 to 300 diameters. He’s place in the scientific history depends on the range and skill of his microscopic observations . He studied almost every conceivable object that could be looked through a microscope. He described the microbial world he observed as ‘animal cules’ or ‘little animals’.

22. All the main kinds of unicellular organisms that we know today – protozoa, fungi, algae, & bacteria were first described by Leeuwenhoek. He is regarded as Father of „ Bacteriology‟ and „Protozoology‟ , because of his contribution to the field of bacteria and protozoa. Th e origin of microbes He believes that microbes were cames from seeds or germs of these animalcules, which were always present in the air .

23. Important discoveries of Agricultural Microbiology S. A. Waksman and hes contributions published the book “ Principles of soil Microbiology " and He discovered the antibiotic "Streptomycin" produced by soil actinomycets (1944). Rossi (1929) and Cholondy (1930) they developed " Contact Slide / Buried slide" technique for studying soil micro flora. Van Niel - studied chemoautotrophic bacteria and bacterial photosynthesis. Kubo proved-the role and importance of “leghaemoglobin” (Red pigment) present in root nodules of legumes in nitrogen fixation.

24. Ruinen (1956) coined the term " Phyllosphere " to denote the region of leaf influenced by microorganisms . Jensen (1942) developed the method of studying nodulation on agar media in test tubes . Barbara Mosse and J. W. Gerdemann (1944) reported occurrence of VAM (vesicular-arbuscular Mycorrhiza) fungi (Glomus, Aculopora genera) in the roots of agricultural crop plants which helps in the mobilization of phosphate . Alexander Fleming developed the antibiotic "Penicillin " from the fungus Penicillium notatum (1929).

25. Hardy & Associates developed the technique of estimation of biological nitrogen fixation . Dobereiner and associates (1975) coined the term “ Associative Symbiosis ” to denote the association between nitrogen fixing Azospirillum and cereal roots. Dommergues & associates had discovered nodules on stem of Sesbania rostrata which could fix nitrogen. discovered N2 fixing stem nodules on Casuarina sp. caused by Frankia, an actinomycete. Brefeld Introduced the practice of isolating soil fungi by " Single Cell " technique and cultivating / growing them on solid media. He used gelatin (first solidifying agent) in culture media as solidifying agent.

26. The Doctrine of spontaneous generation (SG). is the belief of Spontaneous formations of life being from non-living matters. Spontaneous Generation also called abiogenesis , the theory of abiogenesis is defined the belief that all living things originated spontaneously from inanimate/non-living matter, without the need for a living progenitor to give them life . It became difficult to disprove this doctrine, because of lack of experimental proof. Later, Francesco Redi in 1665 performed experiments and showed that maggots that develop in putrefying meat are the larval stages of flies and will never develop in putrefying meat if it is protected from flies laying eggs. H e was the first to disprove SG of animals. Theories on the origin of life (Theory of Spontaneous Generation)

27. Red’s experiment Observation : flies land on meat that uncovered Flask and latter maggots appeared. Hypothesis: if flies land on the meat, then flies laid or produce the maggots. In his experment he need to disprove the theory of spontenous generation which belief (maggot cames from meat which is (dead), not from the life flies.) Then he prepared three flask having meat (Flask sealed, unsealed flask as control and flask sealed with gauze) as expermental unit as shown figure below. Conclusion: Maggots can only appears when flies come in contact with meat and he concluded spontenoues generation does not occur.

28. Lazzaro Spallanzani (1729-99) was the first to provide evidence that microorganisms do not develop spontaneously. He boiled beef broth for an hour and then sealed the flasks. No microbes appeared following incubation. John Needham (1713-81) insisted (take a firm stand) that air was essential for SG of microbes. By sealing the flasks, the air had been excluded. Louis Pasteur (1822-1895) the immortal French scientist, performed various experiments to exposed to air. Pasteur and Tyndall’s experiments finally disproved the Doctrine of Spontaneous generation (S.G.).

29. The germ theory of disease (Koch's Postulates) The Germ Theory also called kotch’s postulate Formulated by Robert Koch, a German scientist. He established that germs were the causes of diseases not the end product of diseases He discovered bacilli in the blood of cattle that had died of anthrax He grew these bacilli in the culture and examined under microscope. Then injected in to health animals. The infected animals developed symptoms of anthrax. He then re- isolated similar bacilli. These experiments lead to the formulation of Koch’s postulates.

30. There are four Kotch’s postulate on disease Animals such as; 1. A specific disease is caused by a specific organism. A specific organism can always be found in association with a given disease 2. The organism should be isolated and grown in the lab into a pure culture. 3. When the artificially cultured organism inoculated into health but susceptible animal it should produce symptoms of the same diseases 4. The organism should be re-isolated from artificially infected animal and grown into pure culture in the lab.

31. There are four Kotch’s postulate on diseased plant To demonstrate Koch’s Postulates, you must do the following: (i)  Describe and record the symptoms shown . (ii)  Isolate the suspected pathogen from the infected plant material and establish a pure culture. (iii) Inoculation of new healthy plant material. (iv) Re-describe and record the symptoms shown by the new plant. Check that these are the same as your original observations. (v) Re-isolate the organism. Check that this is the same as that isolated previously.

32. Kocth’s postulate on plant

33. Drawbacks (Limitations) of Kotch’s postulate 1. Not every microorganism associated with diseases. ex. Normal flora do not cause diseases 2. Many health people carry pathogens but do not exhibit symptoms of the disease (carriers) 3. Certain disease develops only when an opportunistic pathogen invades a weakened host. Opportunistic microorganisms can cause diseases only in immune compromised individuals. 4. Not all diseases are caused by microorganisms. Ex. Diabetes, Asthma, hypertension etc. 5. Some microbes are very difficult or impossible to grow in the laboratory in artificial media, such as most viruses and some bacteria ex. Treponema pallidivm and M.leprae are uncultivable organisms

34. Contribution of Kotch’s postulate 1. Emphasizes the importance of lab cultivation of microorganism in artificial growth media 2. Showed that a specific microbe has specific activity 3. Contributed to the development of pure culture

35. Lecture 2 Culture media and development of pure culture Culture Media- is a liquid or solid nutrient medium prepared in the laboratory for the growth of microorganisms. They can be available commercially as powdered media. used for identification and isolation of microorgnasim . it ranges from general purpose growth media to specifically differential media . it supplies carbon, and nitrogen in a variety of forms. Microorganisms that grow in size and number on a culture medium are referred to as a culture .

36. The most popular and widely used medium used in microbiology laboratories is the solidifying agent . Agar is a complex polysaccharide derived from red algae . The test tubes are held at a slant and are allowed to solidify on an angle, called a slant . When they solidify in a vertical tube it is called a deep . The shallow dishes with lids to prevent contamination are called Petri dishes .

37. A. Liquid Media (Broth): Do not contain any solidifying agent. Used for the propagation of large numbers of organisms, and fermentation studies. Ex: nutrient broth, citrate broth, glucose broth, litmus milk, etc. B. Solid Media : Contain solidifying agent such agar, silica gel or gelatine . Used for developing surface colony growth of bacteria and molds. Ex . Nutrient agars, blood agar

38. C. Selective Media: A culture media design t o suppress the growth of unwanted microorganisms and encourage the growth of the desired microorganisms. The goal of a selective medium is to isolate only the target organism . Such media may contain antibiotics , fungicides, or other compounds that inhibit the growth of unwanted organisms (exclusion). E.g. Bismuth sulfite agar inhibits all gram positive bacteria and gram negative bacteria except salmonella. D.Enrichment Media: A media used for preliminarily isolation that favours the growth of a particular microorganism. Unlike the above types of media, this medium may involve chemical, physiological, nutritional and environmental factors. It is used to enrich the required microbe.

39. Development of pure culture A pure culture is refers to the growth of single type of microbe in an environment free of any other kind of living thing. used to study several characteristics that identify and classify microbes one from one an other. When you attempt to isolate, several different organisms may grown in your plates. Mostly it is not easy to tell which organisms is the cause of the disease under study. You have to make pure cultures of these organisms for any further investigation that could lead to the identification of the causative organism.

40. Solidifying agent: Agar is used to solidify nutrient media for growing bacteria and fungi, Agar powder will only dissolve in boiling water, once dissolved; the solution will remain liquid. E.g. Sabouraud dextrose agar (SDA) and Malt extract agar (MEA). N.B. Ready mixed agar products are generally more expensive than purchasing the pure agar powder and the media components separately.

41. Chapter 2. Basic concepts in microscopy (2Hrs) Microscope is material which is used to study microbes. A microscope is an instrument used to see objects that are very small for the naked eye. The science of examining small objects using such a tool is called microscopy. Microscopic means invisible to the eye except aided by a microscope.

42. The basic principle of microscope is 1. Microscopes are responsible for formation of image and play a central role in determining the quality of images that the microscope is capable of producing. 2. To magnify particular specimen under which fine specimen detail can be observed in the microscope.

43. 2.1. Types of Microscopies Microscopes used in clinical practice are light microscopes . They are called light microscopes because they use a beam of light to view specimens. A. Compound light microscope : - is the most common microscope used in microbiology. It consists of two lens systems (combination of lenses) to magnify the image. Each lens has a different magnifying power. A compound light microscope with a single eye-piece is called monocular ; one with two eye-pieces is said to be binocular.

44. B. Electron microscopes: - is a beam of electrons (instead of a beam of light) and electromagnets (instead of glass lenses) for focusing are called electron microscopes . it provide a higher magnification and are used for observing extremely small microorganisms such as viruses . Sub classification of microscope is based on nature of field i. Light microscopy ( bright field microscopy) This is the commonly used type of microscope. In bright field microscopy the field of view is brightly lit so that organisms and other structures are visible against it because of their different densities. Differential staining may be used .

45. ii. Dark field microscopy the field of view is dark and the organisms are illuminated. A special condenser is used which causes light to reflect from the specimen at an angle. It is used for observing bacteria.

46. iii. Phase-contrast microscopy is allowing the examination of live unstained organisms . S pecial condensers and objectives are used. These alter the phase relationships of the light passing through the object and that passing around it.

47. iv. Fluorescence microscopy: - In this case specimens are stained with fluorochromes/ fluorochrome complexes. fluorochrome is any of a group of fluorescent dyes used to stain biological specimens. Light of high energy or short wavelengths is then used to excite molecules within the specimen or dye molecules attached to it. These excited molecules emit light of different wavelengths, often of brilliant colours. Auramine differential staining for acid-fast bacilli is one application of the technique; rapid diagnostic kits have been developed using fluorescent antibodies for identifying many pathogens.

48. Parts of compound microscope and its function The simplest optical microscope is the magnifying glass and is good to about ten times (10X) magnification.   it has two systems of lenses for greater magnification, 1)the ocular or eyepiece lens that one looks into and 2)the objective lens, or the lens closest to the object.  Eyepiece Lens :  the lens at the top that you look through.  They are usually 10X or 15X power.  Tube:   Connects the eyepiece to the objective lenses. Arm:   Supports the tube and connects it to the base. Base:  The bottom of the microscope, used for support. Illuminator:   A steady light source used in place of a mirror.  If your microscope has a mirror, it is used to reflect light from an external light source up through the bottom of the stage.

49. Stage:  The flat platform where you place your slides.  Stage clips hold the slides in place.  Revolving Nosepiece or Turret:  This is the part that holds two or more objective lenses and can be rotated to easily change power. Objective Lenses:   Usually you will find 3 or 4 objective lenses on a microscope.  They almost always consist of 4X, 10X, 40X and 100X powers.   When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. The shortest lens is the lowest power; the longest one is the lens with the greatest power. 

50. Condenser Lens:  The purpose of the condenser lens is to focus the light onto the specimen.  Are most useful at the highest powers (400X and above) .  Microscopes with in stage condenser lenses render a sharper image than those with no lens (at 400X).  Diaphragm or Iris:   Many microscopes have a rotating disk under the stage.  Used to project light upward to theslide.

51. 2.2. Preparation and Fixation for Light Microscopic Examination Living microorganisms can be directly examined with the light microscope , they often must be fixed and stained to increase visibility. Microscopic Techniques: Dyes and Staining Successful microscopy requires a proper specimen preparation (wet mount, smearing) and staining (various). Resolution and magnification are important in microscopy, the degree of contrast between structures to be observed and their backgrounds is equally important. Magnification is the enlargement of an image; Resolution is the ability to tell two objects apart.

52. Dyes or Stains:- are colouring agents used in the biological specimen.The presence of colour gives the cells significant contrast so are much more visible as a consequence. What is a Stain? A stain is a mixture of dyes that enhance the contrast of the microscopic image. A stain is a substance that adheres to a cell giving the cell colour. What is a Dye? A dye is a single chemical component contained in a stain. Microbiological stains are called dyes . used to differentiate different types of organisms or to view specific parts of organisms.

53. Basic dyes , which carry a positive charge, are more commonly used for staining than are negatively charged acidic dyes. Because microbes are known to possess a negative electric charge on them and get attracted to the positively charged ions of the stain. The most commonly used basic dyes are methylene blue, crystal violet, safrinine, and malachite green. Role Stain: To give a contrast to the tissue Dye: To highlight a specific component within a tissue Examples Stain: H&E, toluidine blue, Masson’s trichrome stain, Wright’s stain Dye: Methyl green, pyronin G, Aniline Blue, orange G Acidic dyes are sometimes used to stain backgrounds , against which colourless cells can be seen, a technique called negative staining.

54. Wet mount: is a non-dried specimen, typically a drop of specimen-containing medium. Wet mounts do not provide good contrast (i.e. it is difficult to see the microorganism) when using bright-field microscopy. Smears: It is a small volume of specimen containing medium that is spread (smeared) onto a microscope slide. A smear is the film obtained by placing a drop of a liquid containing a microbe on a glass microscope slide and allowing it to air dry. Fixing: A method of attaching the smeared organism to the slide is called fixing. Heat fixing is the most common form used.

55. Staining Techniques A)Simple staining Simple staining is a staining procedure that uses only a single dye to stain the cell to increase the contrast between colourless cells and a transparent background. It typically is only a single staining step and everything that stains is stained the same colour. It does not differentiate between different types of organisms. B)Differential staining Most stains used in microbiology are differential stains. is a staining procedure that uses more than one dye to distinguish one group of organisms from another or to distinguish differences in the chemical composition of the cell of a microorganism. The two most frequently used differential staining techniques are the Gram stain and the acid fast stain.

56. Gram stain Named after the Danish bacteriologist who originally devised it in 1844, Hans Christian Gram , is one of the most important staining techniques in microbiology. Gram staining procedure is usually the first to be performed to separate the bacteria into two major groups, the Gram-positive and Gram-negative . The Gram stain procedure involves four basic steps: 1.The smear is first flooded with the primary stain ( crystal viole t). 2. The Gram’s iodine is subsequently added as a mordant (a chemical used to fix the staining reaction) to form the crystal violet-iodine complex, thereby decreasing the solubility of the dye within the cell. This step is commonly referred to as fixing the dye . 3. 95% alcohol or a mixture of alcohol and acetone is added as a solvent to act as a decolourizing agent.

57. 4. Finally, a counter stain is applied to impart a contrasting colour to the now colourless Gram-negative bacteria. The applied counter stain (either basic fuchsin or safrinin) then colourized the Gram-negative bacteria to pink . Note that The Gram-positive bacteria retain the stain (crystal violet-iodine complex ) while the Gram-negative cannot retain this stain . The microorganisms that do not retain the crystal violet-iodine complex appear purple brown under microscopic examination. Bacteria that are stained by the Gram's method are commonly classified as 1. Gram-positive or Gram non-negative. Others that are not stained by crystal violet-iodine complex are referred to as 2. Gram-negative .

58. Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. Bacteria stained by Gram’s staining method fall into two groups – Gram positive, (which appear deep violet in color) and Gram negative (which appear red in color). Gram staining is generally not applicable to other microorganisms. However, yeasts consistently stain gram positive.

59. Chapter 3. Taxonomy of microbes What is Taxonomy? Taxonomy is the science of classification of organisms based on a presumed natural relationship. Organisms have traits that are similar to and different from other organisms. Having this inmind, Scientists organize organisms into groups by developing taxonomy. Organisms that have similar characteristics are presumed to have a natural relationship and placed in the same group.

60. Taxonomy has three components: 1. Classification -the arrangement of organisms into groups based on, similar characteristics, evolutionary similarity or common ancestry. These groups are also called taxa . 2. Nomenclature- the act of given names to each organism. Each name must be unique and should depict the dominant characteristic of the organism. 3. Identification- the process of observing and classifying organisms into a standard group that is recognized throughout the biological community.

61. There are two basic types of cells , 1. Eukaryotic and 2. Prokaryoti c.

62. Difference B/t

63. 3.1. Eukaryotic microbes (algae, fungi and protozoa) Eukaryotes have the nucleus that enclosed by two concentric membranes and cytoplasm contains several membrane bound structures to perform specialized functions like energy generation and electron transport in and across the cells . The eukaryotic cells are about 10X the diameter of a typical prokaryote and can be as much as 1000X greater in volume .

64. Eukaryotic cells contain membrane bound compartments in which specific metabolic activities take place. Have a “ well defined nucleus or "true nucleus." ”, having a membrane-delineated compartment that houses the eukaryotic cell's DNA. are multi-cellular Including plants, animals, algae, fungi and protists (protozoa).

65. Eukaryotic Microorganisms Algae are relatively simple plants; their size varies from 1 μm to several feet, they contain the green pigment chlorophyll, so can carry out photosynthesis (autotrophic) and are found most commonly in aquatic environments or in damp soil . They cause problems by clogging (block) water pipes, releasing toxic chemicals into water bodies, or growing in swimming pools But extracts of some species have commercial uses : as emulsifiers for foods such as ice-creams; as a source of agar used as solidifying agent in microbial medias and as anti-inflammatory drugs for ulcer treatment.

66. Typical structure of Fungi cell

67. Fungi Eucaryotic lower plants devoid of chlorophyll . usually multicellular but are not differentiated into roots, stems and leaves. They range in size and shape from single celled microscopic yeast to giant multicellular mushrooms and puff balls . are either saprophytes or parasites. They have eukaryotic cell structures which, like algae, have rigid cell walls . Size range of molds is 2.0-10 μm and yeast has size varying in the range of 5-10 μm.

68. They reproduce by: fission budding or spores – e.g. molds, mildews, yeasts and rusts belong to this group. They form fruiting structures called conidia or exospores and endospores . Spores of fungi are always present in air, dust and soil. Fungal Nutrition Heterotrophs that acquire nutrients by absorption Digests food outside their body by secreting enzymes into the food Fungi then absorb decomposed molecules

69. Structure of Fungi Fungi can be unicellular (e.g., yeasts). Most fungi are multicellular in structure. The thallus (body) of most fungi is a mycelium. True fungi are composed of filaments and masses of cells, which make up the body of the organism called mycelium . Mycelium: - the vegetative part (body) of a fungus made of a collection of thread like structure . Hyphae is the individual branches or filaments of the mycelium . They form characteristic hyphae called mycelium which may be septate, non septate or coenocytic. It could be Aseptate :- mycelium which do not have cross wall. Eg. Oomycetes, P. infestans Septate: - mycelium which do not have cross wall -Eg. Fusarium

70. Hyphae are filaments that provide a large surface area and aid absorption of nutrients . When a fungus reproduces, a portion of the mycelium becomes reproductive structures. Fungal cells lack chloroplasts and have a cell wall made of chitin, not cellulose. The energy reserve of fungi is glycogen as in animals, and not starch. are nonmotile ; their cells lack basal bodies and do not have flagella at any stage in their life. Fungi move to a food source by growing toward it; hyphae can grow up to a kilometer a day ! Nonseptate hyphae lack septa or cross walls; hyphae are multinucleated. Septate fungi have cross walls in their hyphae; pores allow cytoplasm and organelles to pass freely.

71. Taxonomy of Fungi The classification is based on the following:-  The morphology of hyphea Absence or presence of sexual cycle The type of spores Class Zygomycetes  Have aseptated hyphea The hyphea is coenocytic (contain more than one nuclei) Produce no motile cell Produce sporangia Sexually reproduced by conjugating of mating hyphea Eg. Rhizopus stolonifa ( black bread mold)

72. Class Ascomycetes Possess asci (a sac like structure) – fruiting body consists of 8 ascospores Hyphea produce partially Septated (Yeasts are unicellular, but most ascomycetes are composed of septate hyphea ) Sexual reproduction involves production of eight ascospores within ascus contained within sac like ascocarp Asexual reproduction, which is the norm, involves the production of conidiospores . There are no sporangia in ascomycetes Conidiospores (conidia) develop directly on tips of conidiophores, modified aerial hyphae, and are windblown when released Eg. yeast - Saccharomyces cerevisiae

73. Class Basidomycetes Have completely Septated hyphea Sexual reproduction involves production of basidiospores within clublike basidia contained within a basidiocarp; Asexual reproduction is rare and involves the production of conidiospores. Eg. Smuts and Rusts

74. Class Deutromycetes They reproduce asexually by forming conidiospores ; They are “ imperfect ” because no sexual stage is known and may not exist; thus it cannot be easily classified. Cell morphology and biochemistry indicate some are sac fungi that lost ability to reproduce sexually. Several species of imperfect fungi are of great economic importance Some species of Penicillium mold provide antibiotic penicillin ; others give a characteristic aroma and flavor to certain cheeses (e.g., Roquefort and Camembert). Aspergillus is used in the production of citric acid and gallic acids Some imperfect fungi cause human diseases. Aspergillosis is a respiratory infection caused by inhaling spores . An aspergillus that grows on moist seeds secretes aflatoxin , a potent natural carcinogen.

75. Protozoa are unicellular eukaryotic organisms (animals), are motile having cilia, flagella Pseudopodia , they are either saprophytic or parasitic. They are generally present in soil, water and marshy ( muddy ) place s their size varies from 5-200 μm . They are differentiated on the basis of morphological, nutritional and physiological characteristics. The best known protozoa are the few that cause disease in human beings and animals. Some protozoa are beneficial, such as those found in stomach of cattle, sheep and termites help digest food.

76. Other features include: Eukaryotes are hypothesized to be more complex than prokaryotes. Prokaryotes have evolved a multitude of metabolic strategies and are found in a wide range of habitats, including conditions where most other organisms (Eukaryotes) fail to survive. The plasma membrane resembles that of prokaryotes in function, with minor differences in the set up. Cell walls may or may not be present. The eukaryotic DNA is organized in one or more linear molecules, called chromosomes , which are associated with histone proteins. All chromosomal DNA is stored in the cell nucleus, separated from the cytoplasm by a membrane. Some eukaryotic organelles also contain some DNA. Eukaryotes can move using cilia or flagella.

77. Part 2 3.2 . Prokaryotic microbes (Bacteria, archae and virus) Prokaryotes Are organisms that lack membrane-bound organelles, have no true necuelus Are unicellular, Their cells are usually single. Prokaryotes are one-celled and often live in clusters or colonies. lack most of the intracellular organelles and structures that are characteristic of eukaryotic cells (an important exception is the ribosomes, which are present in both prokaryotic and eukaryotic cells). Most of the functions performed by the plasma membrane . Prokaryotes consist of two domains – Bacteria and Archaea and viruses. .

78. In contrast to most eukaryotes, prokaryotes reproduce asexually and reproduce their clones. While sexual reproduction in eukaryotes results in offspring with genetic material . During reproduction, eukaryotes generate genetic variation by sexual reproduction whereas genetic variation mechanisms of prokaryotes are not tied to reproduction. are generally smaller than eukaryotes. have higher growth rates and shorter generation times. Because of the asexual reproduction and short generation time relative to larger organisms, prokaryotes pass the genome rapidly on to subsequent generations.

79. Other features include:

80. The plasma membrane (a phospholipid bilayer) separates the interior of the cell from its environment. Most prokaryotes have a cell wall (some exceptions are Mycoplasma , a bacterium , and Thermoplasma , an archaea ). It consists of peptidoglycan in bacteria, and acts as an additional barrier against exterior forces. It also prevents the cell from "exploding" (cytolysis) from osmotic pressure against a hypotonic environment. A cell wall is also present in some eukaryotes like fungi, but has a different chemical composition. A prokaryotic chromosome is usually a circular molecule (an exception is that of the bacterium Borrelia burgdorferi, which causes Lyme disease). Even without a real nucleus, the DNA is condensed in a nucleoid. Prokaryotes can carry extrachromosomal DNA elements called plasmids, which are usually circular.

81. Ch-4.The structure and functions of prokaryotic cells 4.1. General Characteristics of Bacteria Why bacterial study so important in this course? Because: Some cause disease, some perform important role in natural cycling of elements which contributes to soil fertility, some useful in industry for manufacture of valuable compounds, some spoil food and some make foods.

82. Bacteria Bacteria (singular: bacterium) are a large domain of prokaryotic microorganisms. Bacterial Morphology Size, shape and arrangement Size : Their size varies from 0.5-1 μm are prokaryotic, unicellular microorganisms. It can be grown on artificial media in laboratory, reproduce asexually by simple cell division i.e. Binary fission and Budding . They have rigid cell wall, cells are rod, spherical (cocci), bacilli (cylindrical rods), spiral and vibrios shape and some motile with flagella. They have high surface area to volume ratio, so have efficient rate of material exchange, They can be anaerobic, aerobic and facultative or obligate parasite.

83. On the basis of chemical composition of cell wall the bacteria are divided in (1) Gram Positive and (2) Gram Negative. Shape: The shape of bacterial cell is governed by rigid cell wall. The bacterial shapes directly affect biological functions, including mode of nutrition, motility, dispersion, stress resistance and interactions with other organisms. Although, bacterial shape is genetically determined, but physical or environmental forces (may be internal and/or external) exerted on cells are increasingly recognized as responsible players in deciding bacterial shapes.

84. Based on their Shape bacteria are grouped as

85. Some bacteria having mycelia morphology are known as Actinomycetes Actinomycetes are very important in production of antibiotics . The Actinomycetes or Streptomycetes are the bacteria which form branched filamentous hyphae having resemblance with fungal hyphae. Actinomycetes are so called because of a fancied resemblance to the radiating rays of the sun when seen in tissue lesions (from actis meaning ray and mykes meaning fungus ). Mycoplasma is another example of structural variant. Mycoplasmas are the bacteria which are cell wall deficient and hence do not possess a stable morphology . They grow as round or oval bodies as interlacing filaments.

86. Binary fission is a form of asexual reproduction in which an organism divides into two, each part carrying one copy of genetic material.” Budding is the term used to describe the asexual reproduction in which progeny develops from, the generative tissue or cell of the parent organism. Difference b/n Budding and Binary fission Binary fission , parent organism is divided into two daughter organisms by evenly separating the cytoplasm whereas, during budding, a new organism is formed from the existing organism by sprouting out. Bacterial Reproduction

87. Binary fission in bacteria

88. Based on Cell Arrangement: The Cocci are further grouped into Diplococci, Streptococci, Tetracocci and Staphylococci based on the characteristic arrangement of the cells. Bacilli are mostly singular or in pairs (Diplococci). But some species may be Streptobacilli (Ex: Bacillus subtilis) or trichomes (Ex: Beggiatoa) or may have palisade arrangement (Corynebacterium diphtheria). Some other bacilli may form long, branched multinucleated filaments called hyphae , which collectively form mycelium (Ex: Streptomyces).

89. Cell arrangement

90. 4.2. Structure of Bacterial Cell The cell wall is common to all bacteria. The structures that are present external and internal to the cell wall are not common to all bacteria. Components of bacterial cell wall

91. Stracture of bacterial cell

92. a. Capsule- The capsules are the outmost structures of bacterial cells. These are the gelatinous (Jellylike) secretion of some bacteria which provides cell with additional protection helps them in preventing phagocytosis of bacteria. Phagocytosis is a type of endocytosis in which any cells uses their plasma membrane to swallow up a large external particle. These capsules are secreted by the cell into the external environment and are highly i mpermeable (water-resistant).

93. However, the capsules are considered to be a major virulence factor of bacteria. Note: Virulence is described as an ability of an organism to infect the host and cause a disease. Virulence factors are the molecules that assist the bacterium colonize the host at the cellular level. That means almost all the bacterial pathogens including Streptococcus pneumoniae, Klebsiella pneumonia, Neisseria meningitidis, Haemophilus influenza and Escherichia coli etc. have polysaccharide capsules on their surface.

94. b. Flagella- These are long (about 20 nm) hair or whip like helical filaments extending from cytoplasmic membrane to exterior of the cell. Bacterial flagella are hair like helical appendages that protrude through the cell wall and are responsible for swimming motility. It grows at the tip unlike hair, which grows at the bottom. It help bacteria to move towards nutrients and other stimuli. The position of the flagella varies with the bacterial species. Functionally and structurally, it is divided into three parts, the filament, hook and the basal body. Filament is connected to the hook at cell surface, the hook and basal body are bordered in the cell envelope.

95. The arrangement of flagella is described as follow (figure) (i) Monotrichous – single flagella on one side (ii) Lophotrichous – tuft of flagella on one side (iii) Amphitrichous – single or tuft on both sides (iv) Peritrichous – surrounded by lateral flagella along the periphery

96. Arrangement of flagella in bacterial cell

97. C. Pili / Fimbriae - It is hair-like proteinaceous appendage used for adherence to a host (in case of a pathogen), or for transferring DNA when bacteria conjugate. As compared to flagella, fimbriae is thinner, smaller and more in number (as many as 1,000 fimbriae in one bacterial cell). Also, they do not have role in cell motility. Bacteria use fimbriae to adhere to other bacteria or animal cells. The fimbriae is comprised of a protein subunit called pilin . D. Slime (extracellular polysaccharide) - This is an extracellular material, loosely associated with some bacterial species. Slime facilitates colonization of smooth, prosthetic surfaces such as intravascular catheters.

98. 4.3. The cell envelop (cell wall and cell membrane composition) Cell wall: In bacteria the cell wall is very rigid and gives the shape to the cell. In most bacteria a cell wall is present on the outside of the cytoplasmic membrane . The cytoplasm of all the bacteria is enclosed within cell membrane. A common bacterial cell wall material is peptidoglycan called murein in older sources, which is made from polysaccharide chains cross-linked by peptides containing D-amino acids. Bacterial cell walls are different from the cell walls of plants and fungi, which are made of cellulose and chitin, respectively. T

99. The cell wall is essential to the survival of many bacteria, and the antibiotic penicillin is able to kill bacteria by inhibiting a step in the synthesis of peptidoglycan. There are broadly speaking two different types of cell wall in bacteria, call ed Gram-positive and Gram-negative.

100. Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids. In contrast, Gram-negative bacteria have a relatively have thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and n lipoproteins. Most bacteria have the Gram-negative cell wall. In many bacteria an S-layer of rigidly arrayed protein molecules covers the outside of the cell. This layer provides chemical and physical protection for the cell surface and can act as a macromolecular diffusion barrier. S-layers have diverse but mostly poorly understood functions, but are known to act as virulence factors in Campylobacter and contain surface enzymes in Bacillus stearothermophilus.

101. The major functions of the bacterial cell wall are as follow: (i) Protection from osmotic lysis: the cell wall prevents the cell from expanding and eventually bursting due to water uptake (the pressure inside the cell = 300 lbs/in2) (ii) Virulence factor: cell wall can be responsible for causing virulence in host organisms. (iv) Defence against host immune response (v) Protection from some toxic substances

102. 4.4. Internal structures (cytoplasm, nucleus, etc ) Cell (plasma) membrane – is the cellular structure which separates (limits), the cyctoplasmic contents from the external environment. is the site of many biological functions. It is specialized to perform such important functions as: selective transport of molecules into and out of cells respiration and photosynthesis secretion of extracellular enzymes regulation and replication

103. Ribosome – densely packed organelles found throughout cytoplasm, protein factory. Cytoplasmic inclusions. are the platform of protein synthesis whereby they receive the genetic commands and translate these in the form of specific proteins. Ribosomes are composed of ribosomal RNA and protein. Nucleoid - The nucleus is not distinct in prokaryotes and hence called nucleoid . It doesn’t have a uniform shape and size as there is no nuclear membrane around it. The nucleoid is principally composed of several copies of DNA which exist in the form of closed, continuous and coiled thread which carries hereditary information.

104. i.The bacterial chromosome and plasmids Most bacterial chromosomes are circular although some examples of linear chromosomes exist (e.g. Borrelia burgdorferi). Along with chromosomal DNA, most bacteria also contain small independent pieces of DNA called plasmids that not essential for growth and often encode for traits that are advantageous but not essential to their bacterial host. Plasmids can be easily gained or lost by a bacterium and can be transferred between bacteria as a form of horizontal gene transfer.

105. i ii.Intracellular membranes Groups of phototrophs, nitrifying and methane-oxidizing bacteria e tc all have intracellular membranes. iv. Cytoskeleton play essential roles in cell division, protection, shape determination, and polarity determination in various prokaryotes.

106. i v.Nutrient storage structures In order to accommodate excess (transient) levels of nutrients, bacteria contain several different methods of nutrient storage. Many bacteria store excess; Carbon in the form of polyhydroxyalkanoates or glycogen. Nitrate in vacuoles. Sulfur is most often stored as elemental (S0) granules which can be deposited either intra- or extracellularly. Sulfur granules are especially common in bacteria that use hydrogen sulfide as an electron source.

107. Cytoplasmic membrane- It is present just below the cell wall and present in both Gram positive as well as Gram negative bacteria. It is a thin but semi-permeable layer that encloses the cytoplasmic contents of the bacterial cell and is made up of a phospholipid bilayer and proteins. Being hydrophobic in nature, it acts as a barrier and prevents the outflow of the cytoplasmic constituents which is hydrophilic. Cytoplasm - Similar to the eukaryotes, bacterial cytoplasm is also a colloidal system consisting of a variety of organic and inorganic constituents such as 80% Water and 20% Salts, Proteins. They are rich in ribosomes, DNA and fluid. Apart from chromosomal DNA, the extra choromosomal DNA is characteristically closed and circular. These extra chromosomal DNA is called Plasmids. They are highly coiled and complexed with polyamines and other support proteins

108. Mesosomes- They are vesicular structure produced by localized and inward folding of plasma membrane into the cytoplasm. are rich in respiratory enzymes and other enzymes responsible for DNA replication and cell division. Spore- Some bacteria form highly resistant resting stage called spores, which helps them to sustain in adverse environmental conditions. They are neither a reproductive form nor a storage granule. These spores enable bacteria to be resistant against the adverse environmental conditions and bactericidal agents as well as. There exists three layers in the spore namely core, cortex and spore coat. Endospores Perhaps the most well-known bacterial adaptation to survival stress is highly resistant to many different types of chemical and an environmental stress is the formation of endospores.

109. Exospores are formed external to the vegetative cell by budding at one end of the cell in the methane oxidizing genus Methylosinus. They are desiccation and heat resistant. Conidiospores and Sporangiospores: The bacteria, actinomycetes form branching hyphae. From the tips of these hyphae spores develop singly or in chains. If the spores are contained in an enclosing sac (sporangium), they are termed sporangiospores , if not they are called conidiospores . The spores can survive long periods of drying but they do not have high heat resistance.

110. Cysts : are thick walled, desiccation resistant, dormant forms that develop by differentiation of vegetative cells. Azotobacter and some other genera produce cysts.

111. Chap-5. Microbial growth and nutrition 5.1. Microbial growth 5.1.1. Bacterial growth and reproduction Although widely varying in morphology, bacteria share one major characteristic: they divide by Simple binary fission . This means that one cell grows to about double its original size and then splits into two genetically identical cells. Since DNA replication occurs before the cells divide, each new cell, called a daughter cell, gets a complete genome (a full set of genes). Part 4

112. The two genetically identical daughter cells are called clones . All the progeny of a single original cell form a mass of cells on a solid surface such as agar is called a colony . Bacterial Colony - means a mass of bacterial cells grown on culture media. If the original form was not a single cell, for example, it was a chain of cocci, that entire chain of cells and all its progeny will form a single colony. So a colony forming unit (CFU) may include the progeny of a single cell, or it may include the progeny of several cells that were originally connected to each other.

113. Bacterial conony on agar media

114. Steps involved for bacteria reproduction

115. Steps involved for bacteria reproduction

116. Transverse (Cross) binary fission is the most common and important in the growth cycle of bacterial population, which is an asexual reproductive process. Fragmntation : Some bacteria produce extensive filamentous (threadlike) growth, which is followed by the fragmentation of these filaments into small bacillary or coccoid cells, each of which give rise to new growth. Eg. Nocardia species. Spore Production : Some genera of bacteria produce reproductive spores called conidiospores or sporangiospores at the tip of filamentous growth, each of these spores give rise to a new organism. Eg : Streptomyces D. Budding : A few bacteria also reproduce by a process known as budding where in the parent cell remains intact while a new cell buds off which again grows into a new organism. Eg: Rhodopseudomonas, Hyphomicrobium

117. image of bacterial reproduction

118. The growth curve Bacterial growth over time can be graphed as cell number versus time . This is called a growth curve . The cell number is plotted as the log of the cell number, since it is an exponential function. Regardless of the generation time, in a growing culture the plot of the log of cell number versus time gives a characteristic curve. Generation Time is the amount of time needed for a cell to divide. This varies among organisms and depends upon the environment they are in and the temperature of their environment. Some bacteria have a generation time of 24 hours, although the generation time of most bacteria is between 1 to 3 hours .

119. Bacterial cells grow at an enormous rate. For example, with binary fission , bacteria can double every 20 minutes . In 30 generations of bacteria (10 hours), the number could reach one billion. It is difficult to graph population changes of this magnitude using arithmetic numbers, so logarithmic scales are used to graph bacterial growth. Growth phase: There are four basic phases of growth: 1. Lag phase, 2. Exponential (log) phase, 4. Stationary phase, and 5. Death phase.

120. Example 2 In one micriobiological experiment the research calculate the 20 cells in original numbers of bactrial populations (No) that having 8 number of division (n) within 6 hours assuming 20 minute generation time , then calculate number of cell in the bacterial population (N t )? Given Required (N t )? No= 20 n= 8 Soln: N t= No *2 n = 20*2 8 =

121. 1. THE LAG PHASE : In the lag phase there is little or no cell division . This phase can last from one hour to several days. Here the microbial population is involved in intense metabolic activity involving DNA and enzyme synthesis. This is like a factory “shutting down ” for two weeks in the summer for renovations. New equipment is replacing old and employees are working, but no product is being turned out. 2. THE LOG PHASE: In the log phase, cells begin to divide and enter a period of growth or logarithmic increase. This is the time when cells are the most active metabolically. This is the time when the product of the factory must be produced in an efficient matter. Growth rate is high or fast growth rate phase which means the number of dead microorganisms much lower than the number of new microorganisms’ In this phase, however, microorganisms are very sensitive to adverse conditions of their environment.

122. 3. THE STATIONARY PHASE: This phase is one of equilibrium phase . The growth rate slows , the number of dead microorganisms equals the number of new microorganisms , and the population stabilizes. The metabolic activities of individual cells that survive will slow down. The reason why the growth of the organisms stops? is possibly that the nutrients have been used up, waste products have accumulated, and drastic harmful changes in the pH of the organisms environment have occurred. There is a device called a chemostat that drains off old, used medium and adds fresh medium. In this way a population can be kept in the growth phase indefinitely.

123. 4. THE DEATH PHASE: Here the number of dead cells exceeds the number of new cells. This phase continues until the population is diminished or dies out entirely. 5. THE PROLONGED (Chronic) DECLINE PHASE : This phase is marked by a very gradual decrease in the number of viable cells in the population lasting for months to years.

124. The growth curve

125. Summary of bacterial growth phase

126. GROWTH RATE Growth rate is the change in cell number or mass per unit time. It is expressed as ‘R’ which is the reciprocal (Inverse) of generation time ‘g’. It can be defined as the slope of the line when log of cells versus time is plotted (R = 1/g). Microbes generally respond linearly to a limiting nutrient concentration in the medium, which forms the principle for microbiological assays.

127. GROWTH YIELD Balanced growth is a condition where all biochemical constituents are being synthesized at the same relative rates. Growth yield is the mass of cells produced per unit of a limiting nutrient concentration. It is denoted by Y = X-X0/C, where X0 = mass of initial population immediately after inoculation, X = mass of final population after cells enter stationary phase , C = concentration of the limiting chemical constituent in the medium. This is the basis used in microbiological assays of various vitamins and amino acids by auxotrophic mutants of bacteria.

128. Excercise Given Xo= mass of initial population immediately after inoculation= 50 X= mass of final population after cells enter stationary phase= 350 at the C = Glucose concentration of the limiting chemical constituent in the medium (20 g/l) Required= Growth yield = Y? Solution, Y = X-X0/C Y= (350-50)/20= 15 cells produced

129. 5.1.3. Measurements of microbial growth (Bacterial counts) Bacterial growth can be measured by both Direct and Indirect methods . It generally considered at two levels, that is growth in size and growth in numbers . Growth in numbers can be measured by bacterial counts. There are two types of bacterial counts . 1. Total count 2. Viable count

130. Total count : this gives the total number of cells in the sample which includes the living and dead cells . It can be obtained by following methods. Direct counting under microscope By using culture counter Direct counting using stained smears prepared by spreading a known volume of culture over a measured area on the slide. B . Viable count: it measures the number of living cells only, that is, the cells capable of multiplication. Viable counts are obtained by dilution or plating methods. Dilution method : the suspension is diluted to a point where there is growth when inoculated in to suitable liquid media. Several tubes are inoculated with varying dilutions and the viable count is measured statistically from the number of tubes showing growth.

131. Plating method : appropriate dilutions are inoculated on solid media. The numbers of colonies develop after incubation gives the estimate of viable count.

132. 5.2. Nutrient Requirement The growth of any microbe depends on the physical environment and the available source of chemicals to use as nutrition . Therefore, every microbe has its own minimum physical environment and nutritional requirement. Each microbe can grow only if presented with the right nutrients/conditions. Major Element Trace Element

133. A. Major elements: are elements that are required in large amounts to make up the cell constituents. They are the essential components of proteins, carbohydrates, lipids and nucleic acids. It includes carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorous, potassium, magnesium, calcium, and iron.

134. B.Trace elements : are elements that are required in small amount by all cells . These elements form parts of enzymes or may be required for enzyme function. It includes cobalt, zinc, copper, molybdenum and manganese.

135. Growth factors: Some bacteria cannot synthesize some of their cell constituents, such as amino acids, vitamins, purines and pyrimidines, from the major elements. These organisms can only grow in environments where such compounds are available. Those low molecular weight compounds (amino acids, vitamins, purines and pyrimidines) that must be provided to a particular bacterium are called growth factors.

136. Based on the source of carbon prokaryotes groups as autotrophs and heterotrophs . Autotrophs are microbes that use inorganic carbon in the form of carbon dioxide as a carbon source . eg . Cyanobacteria , purple bacteria, green sulfur bacteria. carbon dioxide is generally the sole source of cellular carbon . These autotrophs also use hydrogen sulphide (H2S), ammonia (NH3) or hydrogen gas to convert carbon into sugars required for energy. Nitrifying bacteria, which oxidize and convert ammonia to nitrites and nitrates are specifically categorized as chemoautotrophic nutrition.

137. Heterotrophs are microbes that use organic carbon as a carbon source . require organic molecules such as sugars, fats and amino acids for example Saprophytic bacteria . They manage their nutrition from dead and decaying organic matters . These bacteria will break down complex compounds with the aid of enzymes to release energy . Saprophytic bacteria are decomposers and play an important role in ecosystem by releasing simpler products which plants and animals can use

138. B. Based on the way of harvesting energy, prokaryotes are classified into two major groups ; phototrophs and chemotrophs . Phototrophs : is organisms that harvest the energy of sunlight . Chemotrophs : is organisms that obtain energy by metabolizing chemical compounds.

139. 5.3. Environmental Requirement Factors affecting the growth of bacteria Nutrition, Temperature, Oxygen, Carbon dioxide, Light, pH, Moisture and salt concentration.

140. (a) Nutrition - Water, proteins, polysaccharides, lipids, nucleic acid and mucopeptides are the principal constituents (Parts) of the bacterial cells. In addition, for adequate growth and multiplication , bacteria require a source of energy, carbon, nitrogen and some inorganic salts. Therefore, bacteria can be classified on the basis of their nutritional requirements energy requirement and ability to synthesise essential metabolites.

141. (b)Temperature- The optimum temperature requirement for bacterial growth varies with the species. Bacteria which grow best at temperatures of 25-40°C are called mesophilic bacteria . Psychrophilic bacteria : are those that growth best favoured at temperatures below 20°C. are known as cold-loving bacteria Thermophiles bacteria: that grow adequately at higher temperatures that are at 55-80°C . This group is non pathogenic bacteria. Thermal Death Point (TDP) is  the lowest temperature that is required to kill a population of bacteria when applied for a specific time .

142. (c) Oxygen - Based on their dependence on oxygen b acteria divided into aerobes and anaerobes , Bacteria that necessitate oxygen for their growth are called aerobic . Aerobic bacteria may be obligate or facultative aerobes . Obligate aerobes : is bacteria which well grow strictly in the presence of oxygen Facultative anaerobes are the bacteria which are normally aerobic but can grow even in the absence of oxygen . E.g. clostridia grow in the absence of oxygen are classified under anaerobic bacteria . They are also the obligate anaerobes and Microaerophilic anaerobes . The obligate anaerobes do not survive on exposure to oxygen , whereas, microaerophilic bacteria grow adequately in the presence of low oxygen tension.

143. (d) Carbon Dioxide - Small amounts of carbon dioxide are also required by all bacteria for their growth. The atmospheric carbon dioxide fulfils this demand. However, some bacteria like Brucella abortus need much higher levels of carbon dioxide for its growth. (e) Light - Bacteria that use light as food- Phototrophic bacteria are autotrophs that absorb light energy, to create cellular energy through photosynthesis. Bacteria are generally negatively phototropic , they die if exposed to light and on the contrary, grow well in the dark. Bacteria are sensitive to ultraviolet light and other radiations too. There are two types of phototrophs . Anaerobic phototrophs are the bacteria that do not produce oxygen as a byproduct, while aerobic phototrophs are those which produce oxygen. For example, Cyanobacteria . Both autotrophs and heterotrophs can be phototrophs . Heterotrophic phototrophs use organic compound, besides producing organic molecules through photosynthesis.

144. (f) pH - The requirement for pH also varies with the bacterial species. Each bacterial species has a specific pH range, above or below which it cannot grow and survive. Similarly, every species has an optimum pH at which it grows well. Most of pathogenic bacteria grow best at either neutral or slightly alkaline pH (7.2 – 7.6) .

145. (g) Moisture - Water is a vital constituent of bacterial protoplasm. Lack of moisture or drying is lethal to the bacterial cells; however, the effect of moisture or drying varies in different species. (h) Salt concentration of the culture media - Salt concentration of the culture media is also an important consideration for bacterial growth. However, bacteria are more tolerant to osmotic variation as compared to other cells because of good mechanical strength of their cell wall. Plasmolysis (or shrinkage of cell) may occur on abrupt exposure of bacterial cells to a hypertonic solution because of osmotic withdrawal of water from the protoplasm.

146. Chap-6: Microbiology of Plant Pathogenic Microorganisms Definition of terms related to plant diseases what is disease? what is pathogen? Plant pathology : is the study of plant pathogens, the diseases they cause and their control. Disease : Any deviation in the general health, or physiology or function of plant or plant parts, is recognized as a disease Pathogen : An entity, usually a micro-organism that can cause the disease Pathogenicity : The relative capability of a pathogen to cause disease.

147. Pathogenesis : It is a process caused by an infectious agent (pathogen) when it comes in contact with a susceptible host. Sign : manifestation (Physical appreance ) of the pathogen or the pathogen by itself or its parts or products seen on the affected parts of a host plant is called sign . e.g. some structural parts of the pathogen Symptoms : A visible or detectable abnormality expressed on the plant as a result of disease or disorder is called symptom . Syndrome: The totality of symptoms is collectively called as syndrome. Or Syndrome is also defined as a group of signs and symptoms that occur together and characterize a particular abnormality or condition.

148. Sign of yellow rust fungi spore Symptoms of leaf spot

149. 6.1. Causes of Plant Disease Plant diseases caused by pathogens . Hence a pathogen is always associated with a disease. In other way, disease is a symptom caused by the invasion of a pathogen that is able to survive, perpetuate and spread. Further, the word ‘ pathogen ’ can be broadly defined as any agent or factor that incites ‘pathos or disease in an organism or host.

150. 6.2. Fungi Most plant pathogenic fungi form hyphae i.e. filamentous cells which extend by apical growth and an ordered system of branching. The network of hyphae which results from such growth is called a mycelium , and the interconnected hyphal network derived from one propagule is termed a colony . The apical mode of growth of most fungi is the key to the success of these organisms both as saprotrophs and parasites . Unlike unicellular organisms, filamentous fungi are able to extend through soil, plant litter or living tissues . . As the nutrients become exhausted the hypha simply grows on to explore a new area.

151. Assignment 10% Self learning: write note on different fungal disease that affect crops. Gruop 1:- write a note on 1 major fungal disease that affect Faba bean crop (Name of the disease, syptom , causal pathogen (Scientific name of the pathogen, common name of the pathegens ), favoarable condition for the Pathogen and the host, Host range, Geographic distribution of the causal organism ). From those pathogens, please select one disease and write its artificial inoculation methods by following koch’s postulate. Gruop 2. write a note on 1major fungal disease that affect Wheat crop (Name of the disease, syptom , causal pathogen (Scientific name of the pathogen, common name of the pathegens ), favoarable condition for the Pathogen and the host, Host range, Geographic distribution of the causal organism ).From those pathogens, please select one disease and write its artificial inoculation by following koch’s postulate.

152. Gruop 3. write a note on 1 major fungal disease that affect Mango fruit (Name of the disease, syptoms , causal pathogen (Scientific name of the pathogen, common name of the pathegens ), favoarable condition for the Pathogen and the host, Host range, Geographic distribution of the causal organism ).From those pathogens, please select one disease and write its artificial inoculation by following koch’s postulate. Gruop 4. write a note on 1 major fungal disease that affect Banana fruit (Name of the disease, syptoms , causal pathogen (Scientific name of the pathogen, common name of the pathegens ), favoarable condition for the Pathogen and the host, Host range, Geographic distribution of the causal organism ).From those pathogens, please select one disease and write its artificial inoculation by following koch’s postulate.

153. Gruop 5 :- Referee and make a note on 1 major Viral diseases that affect field crop that affect Tomato (Name of the disease, syptoms of the disease, syptom , causal pathogen (Scientific name of the pathogen, common name of the pathegens ), favoarable condition for the Pathogen and the host, Host range, Geographic distribution of the causal organism ).From those pathogens, please select one disease and write its artificial inoculation by following koch’s postulate. Gruop 6: - Referee and make a note on 1 major Viral diseases that affect field crop that affect Hotpepper (Name of the disease, syptoms of the disease,causal pathogen (Scientific name of the pathogen, common name of the pathegens ), favoarable condition for the Pathogen and the host, Host range, Geographic distribution of the causal organism ).From those pathogens, please select one disease and write its artificial inoculation by following koch’s postulate.

154. 6.3. Bacteria The majority of plant-pathogenic bacteria are unicellular, cell division/ multiplication by binary fission. Many plant pathogenic bacteria possess flagella Therefore, motile and capable of moving along nutrient gradients and towards host signal molecules . Most plant pathogenic bacteria are rod shaped (bacilli) .

155. Bacterial diseases Crown gall disease caused by Agrobacterium tumefaciens Most bacteria that are associated with plants are actually saprotrophic , and do no harm to the plant itself. Bacterial diseases are much more prevalent in sub-tropical and tropical regions of the world . had rod shaped (bacilli) . In order to be able to colonize the plant they have specific pathogenicity factors.

156. Five main types of bacterial pathogenicity factors are known: 1. Cell wall degrading enzymes used to break down the plant cell wall in order to release the nutrients inside. Used by pathogens such as Erwinia to cause soft rot. 2. Toxins: these can be non-host specific, and damage all plants , or host specific and only cause damage on a host plant. 3. Effector proteins - these can be secreted into the extracellular environment or directly into the host cell . Some effectors are known to suppress host defense processes . 4. Phytohormones – for example Agrobacterium changes the level of auxins to cause tumours . 5. Exopolysaccharides – these are produced by bacteria and block xylem vessels, often leading to the death of the plant.

157. Bacterial Leaf Diseases of Foliage Plants Many foliage plants are susceptible to bacterial diseases, especially during gloomy winter months. Common symptoms include leaf spots, blights, and wilting.  Bacterial diseases restricted to the leaves can often be controlled. Bacterial Leaf Spot Bacterial Leaf Blight ( Xantomonas spp.) Bacterial Blight ( Erwinia chrysanthemi ) Bacterial Leaf Spot and Stem Canker ( Xanthomonas campestris pv . hederae )

158. Bacterial leaf spot Bacterial leaf blight Bacterial wilt

159. 6.4. Viruses As first sight viruses might appear ill-equipped to act as pathogens, due to their extreme dependence on living cells. Virus parasitism is unique, in that the parasite is incorporated into the metabolism of the host cell. After gaining entry into a living cell, the nucleic acid component of the virus is released from its protein coat. The viral genome is then translated and replicated, and numerous new virus particles are assembled from the newly synthesized nucleic acid and protein. A virus can thus be visualized as a set of instructions for making more viruses, packaged in a protective coat. In contrast to fungi and bacteria, viruses do not attack the structural integrity of their host tissues. Example: Tobacco mosaic virus (TMV).

160. 6.5. Nematode Nematodes are small, multicellular worm-like creatures. Many live freely in the soil, but there are some species which parasitize plant roots. They are a problem in tropical and subtropical regions of the world , where they may infect crops. Root knot nematodes have quite a large host range, whereas cyst nematodes tend to only be able to infect a few species. Nematodes are able to cause radical changes in root cells in order to facilitate their lifestyle.

161. Chap-7. SOIL MICROORGANISMS What Is a soil? Soil is the loose material of the earth’s surface, which supports the growth of plants, bacteria, fungi, algae and protozoa, which make up for the living organisms of soil. It consists of five major components. a) Living organisms, b) Organic matter, c) Air , d) Water and e) Minerals .

162. What Is a Microbiome ? a microbiome is a community of microbes — eukaryotes, archaea , fungi, viruses, bacteria — that act together both with and within a specific soil environment. It some times defined as the “ set of resident micro-organisms that inhabit a given host/environment”. Do you know that in a spoonful of agricultural soil contains  30,000 taxonomic varieties of microbes . Among them are several fungal filaments that-  convert dead matter to biomass, or attach to plant roots to boost their nutrient uptake; up to a billion bacteria that - convert nitrogen gas into compounds that “feed” those plants and other organisms; a few dozen nematodes and a few thousand protozoa that- keep bacterial populations in check, mineralize nutrients and protect plants from pathogens.

163. Rhizosphere microflora Although, a number of microorganisms ( Microbiome ) are found in the rhizosphere include bacteria, fungi, algae, viruses, nematodes, protozoa, and arthropods, but bacteria are the dominant and have direct prominent roles in plant growth.

164. The type and quantity of microorganisms present in a soil vary depending upon the ; a. Physical characteristics of soil b. Agricultural practices c. Amount and type of nutrients, d. Available moisture, e. Degree of aeration, f. Temperature and pH.

165. In the case of plants, the microbiota associated acts in the acquisition of soil nutrients, tolerance to abiotic stresses, and disease control the consequent increase of ecological fitness under the natural environment or food, fiber, and energy production under agricultural systems  .

166. Therefore, when the soil microbiome is healthy and in balance , it directly affects the health of the plants that grow in it and protects them from drought or pests, It can shove out (squeeze) pathogens trying to attack plants, produce toxins to kill them off and also trigger (activate) the plants to defend themselves. It also has other critical ecosystem functions; most notably, it acts as a carbon sink , helping keep atmospheric carbon in check for a critical climate benefit . They have significant contribution on soil fertility

167. 7.2. Influence of Plants on Soil Microorganisms Rhizosphere environment The rhizosphere is the narrow region of soil that is directly influenced by root secretions and associated soil microorganisms. The rhizosphere contains many Bacteria and microorganisms that feed on sloughed-off plant cells, termed rhizodeposition , and the proteins and sugars released by roots. Much of the nutrient cycling and disease suppression needed by plants occurs immediately adjacent to roots due to root exudates and communities of microorganisms. The rhizosphere differs from the bulk soil because of the activities of plant roots and their effect on soil organisms.

168. A major characteristic of the rhizosphere is the release of organic compounds into the soil by plant roots. These compounds called root exudates. How plant influence soil microorganism? The Root exudates : Increase the availability of nutrients in the rhizosphere & also provide a carbon source for heterotrophic microorganisms . Can cause the no. of microbes to be far greater (500 times higher) in the rhizosphere than in the bulk soil.

169. How soil microorganism influence plant? Organisms in the rhizosphere can affect the plant roots by altering the movement of carbon compounds from roots to shoots . Many are Plant growth promiting rhizobacteria (PGPR). Symbiosis - can increase nutrient uptake by plants in nutrient poor environment ( eg . Mycorrhizal or Rhizobia ) Associative N2 fixing bacteria with grasses etc – eg . Azospirillum . produce hormones that stimulate plant growth are antagonistic to plant pathogens . But some are pathogenic & attack living plant roots.

170. Importance of Rhizosphere Rhizosphere plays a very important role through microorganisms in regulating the decomposition of soil organic matter and nutrient cycling . in the nutrients absorption and water uptake. Many soil habitat microorganisms are beneficial for plant growth and development, some plant pathogenic microorganisms such as Agrobacterium bacteria that colonize the soil around plant roots and cause diseases. some facultative human pathogenic bacteria are also found in the rhizosphere and can be carried on in plant tissues. Knowledge of rhizosphere is vital for adaptive or curative measures from these pathogenic microorganisms . As far as the beneficial effects are concerned, the rhizosphere represents a congenial soil habitat where introduction of beneficial microorganisms as inoculants such as biofertilizers , phytostimulators , and biopesticides can result significant improvements in crop yield and/or crop quality.

171. Rhizoplane :- is the surface of the plant roots in the soil . is the site of the water & nutrient uptake & the release of exudates in to the soil. Through chemotaxis (movement to ward chemical agent) processes, followed by microbial adhesion mechanisms, bacteria, and fungi attach epidermal-cell wall root surface. These epiphytic interactions in the  rhizoplane   may involve the participation of anchoring structures and adhesive proteins that result in the firm adhesion of micro-organisms to the plant cell wall, with subsequent formation of aggregates and bio-films . Epiphytic interactions in the  phylloplane  can result from systemic spreading through the xylem vessel elements by rising water flux in the transpiratory processes of the plant or by direct access of micro-organisms to the surface of the stem and leaves.

172. Phyllosphere: The region on the leaf surface where microorganisms are present abundantly. Because of leaf and stem surfaces facilitate a favourable environment to allow growth of microbial communities. This above-ground habitat of microorganisms is termed as the phyllosphere The inhabiting microorganisms are called epiphytes . An epiphyte is an organism that grows on the surface of a plant and derives its moisture and nutrients from the air, rain, water or from debris accumulating around it.

173. Phyllosphere microflora The above-ground parts (leaf and stem) of plants are also colonized by a variety of microorganisms The epiphytes of phyllosphere are very wide and cover many different genera of bacteria, filamentous fungi, yeasts, algae, and, less frequently, protozoa and nematodes.

174. Importance of Phyllosphere Phyllosphere plays key role; in affecting the plant-microbe interactions on plant growth In suppression of plant disease. Eg . Bacteria,( actinomycetes ) and fungi are directly control the growth and development of plants as well as regulate plant pathogens . involved in biogeochemical cycles such as carbon and nitrogen cycles. In carbon cycle , epiphytes intercept the carbon compounds released from plants or removed by sucking arthropods, while in nitrogen cycles , epiphytes are involved in nitrification of ammonium pollutants and nitrogen fixation .

175. External and internal factors directly influence the growth of epiphytes Nutrient availability, Moisture, Temperature, Topography, Leaf architecture, Presence of enzymes and growth inhibitors etc.

176. Role of plant microbiome in protection from pathogens and host immunity The rhizosphere antagonistic microorganisms ward off pathogens by producing Antibiotics or Hydrolytic enzymes Competing for nutrients and Space Bacteria and fungi are two major groups of the plant microbiome , and their interactions via antibiosis, modulation of the physiochemical environment, cooperative metabolism, protein secretion, or even gene transfer can lead to either antagonism or cooperation.

177. Pseudomonas fluorescens   suppresses soilborne pathogens like  Meloidogyne incognita  and  Fusarium oxysporum  by production of the antibiotic 2,4-diacetylphloroglucinol (DAPG) . Bacteria are also known to parasitize and degrade spores of fungal plant pathogens through the production of extracellular cell wall-degrading enzymes such as chitinase and β-1,3 glucanase . Most microbial biocontrol strains produce more than one antibiotic compound with varying degrees of antimicrobial activity.  Agrobacterium radiobacter  produces agrocin 84, which is antibiotic to closely related strains, and polyketide antibiotics which are broad-spectrum in nature. Siderophores produced by  Bacillus subtilis  significantly managed the  Fusarium  wilt of pepper caused by  Fusarium   oxysporum  . Siderophores produced by  Aspergillus niger ,  Penicillium citrinum , and  Trichoderma harzianum  were found to be effective biocontrol agents and enhanced the growth of chickpeas.

178. Role of plant microbiome in tolerance to abiotic stresses Rhizosphere microorganisms contribute to alleviate abiotic stresses in plants. Eg Pseudomonas ,  Bacillus ,  Achromobacter ,  Burkholderia ,  Enterobacter ,  Azotobacter ,  Methylobacterium , and  Trichoderma  have been widely studied in plant growth promotion by mitigation of multiple kinds of abiotic stresses .

179. Role of plant microbiome in phytohormone production Plant growth-promoting rhizobacteria and fungi are known to improve plant growth by the production of phytohormones ( Gibberellins, Auxin and cytokinin ) . Gibberellins can stimulate plant growth and regulate developmental processes like seed germination, stem elongation, sex expression, and fruit formation. Some bacterial genera contribute in gibberellin production-including  Azospirillum  sp.,   Rhizobium  sp.,  Acetobacter diazotrophicus ,  Herbaspirillum seropedicae ,  Bacillus  sp., and  Fusarium moniliforme .

180. Auxin and cytokinin production are thought to be involved in root growth stimulation by beneficial bacteria and in associative symbiosis. Auxin biosynthesis by  Pseudomonas ,  Agrobacterium ,  Rhizobium ,  Bradyrhizobium ,  Azospirillum ,  Botrytis ,  Aspergillus , and  Rhizopus  . Therefore, microorganism-mediated phytohormone production is a potent mechanism to alter plant physiology , leading to diverse outcomes from pathogenesis to promotion of plant growth .

181. Role of microbiome in impairing plant health and productivity The root microbiome also consists of plant growth-promoting microorganisms, rhizosphere microorganisms which are detrimental to plants, competing for nutrients and space. Plant pathogenic microorganisms that cause various plant diseases resulting in substantial economic damage to crops. Eg . Agrobacterium tumefaciens ,  Ralstonia solanacearum ,  Pectobacterium carotovorum ,  Pythium  sp.,  Phytopthora  sp.,  Fusarium oxysporum ,  Rhizoctonia  sp.   Colletotrichum  sp., and  Magnaporthe oryzae  are a few of the major plant pathogenic microorganisms prevalent in soils.

182. Effect of agricultural practices (fertilizers, green manure, compost, vermicompost , agrochemicals, crop rotation, monoculture) Agricultural practices are often related to changes in the plant microbiome due to changes in soil properties, mainly nutritional and can affect the microbiome directly, stimulating or inhibiting its activity according to their nutritional preferences, or indirectly, interfering in the way plants select their micro-organisms. The use of green manure and  agroforestry system  alters the composition of the  yerba mate   bacterial microbiome , and its cultivation in  monoculture  favors the development of an abundant  fungal microbiome . Where the use of organic compost in soil with crop rotation increased the phylogenetic richness, diversity and bacterial heterogeneity of the soil. In the potato rhizosphere , the use of mulch increased the diversity of fungi, with  Ascomycota  being the dominant phylum. On the other hand, this practice inhibited the reproduction of the  Fusarium  pathogen.

183. Chapter 8. MICROBIAL INTERACTION IN SOIL SYSTEM Interaction of organisms with each other and with their physical environment contributes to the functioning of ecosystems . The reasons for microbial interactions are t he competition for nutrients (including oxygen ) and space in an ecological niche. On the basis of relative advantage to each partner i.e. hosts and microorganisms, the relationships (interactions) are basically of three types.

184. Hosts and microorganisms, the relationships (interactions) are basically of three types .  Neutralism: when the host remains unaffected by the microbes. Mutualism: when both partners get benefits from the association. Eg . Mycorrhizas ; the fungus is involved in phosphate acquisition and the plant supplies carbon .  Parasitism: when one partner gets benefits and the second partner suffers from damages.

185. Aboveground microbe-plant interaction ( Phyllosphere or Phylloplane ) Microbial interactions on aboveground parts of plants occur in several ways where the foliage especially leaf surface ( phyllosphere and phylloplane ) acts as microbial niche. The association may be destructive (diseased) which is caused by pathogenic microorganisms or beneficial (symbiosis). Some examples of microbial pathogens causing disease on phyllosphere include Pseudomonas pisi (stem blight of pea), Alternaria solani (early blight of potato), Puccinia tritici (rust of wheat), etc. The excellent example of beneficial association is the development of stem nodules by some legumes species like Neptumia when interact with Azorhizobium bacteria.

186. Below ground microbe-plant interaction ( Rhizosphere or Rhizoplane ) The rhizosphere is the zone of soil immediately adjacent to plant roots in which the kinds, numbers, or activities of microorganisms differ greatly from that of the bulk soil. The rhizosphere is today regarded as the zone of microbial proliferation in and around roots. A variety of light and electron microscopic techniques have been used to observe bacteria and fungi around roots (the ecto rhizosphere ), on the root surface (the rhizo plane ) and within the root (the endo rhizosphere ). Some examples of microbial pathogens causing disease on the rhizosphere include Streptomyces scabies ( scab of potato ), Rhizoctonia solani ( root rot of potato and many plants), Meloidogyne incognita ( root knots of tomato and many plants), etc. The beneficial associations (Symbiosis) as a result of interaction of microorganisms with plant roots may or may not develop apparent symbiotic structure.

187. Symbioses between two organisms vary based on; 1. Degree of intimacy: the degree of closeness, proximity of the association between interacting organisms. The degree of intimacy could be:  Ectosymbiosis : in which one organism remains outside the other organism i.e. external to the cells and tissues of the partner.  Endosymbiosis : in which one organism is found within the other organism . Ecto / endosymbiosis : in which one organism lives both on the inside and the outside of the other organism. 2. Relative advantage each partner gets from the association

188. Microorganism form symbiotic relationship for either of the following purposes: Protection against: extreme environment: (e.g. desiccation, temperature, pH, etc.), invasion by disease causing microorganisms, toxic products (e.g. breakdown and removal of toxic substance like urea and uric acid by bacteria in the excretory organs of insects). Direct provision of nutrients (for instance, fungi in roots of plants increase water absorption capacity of roots and increase absorption of nutrients. Indirect provision of one or more nutrients to its partner (e.g. protozoa and bacteria in cellulose digestion in ruminants). Many microbes (bacteria, fungi, etc) have important symbioses with plant roots. The rhizosphre , which is a thin layer soil immediately adjacent to root hairs of plants typically, contains 10 9 microbes/g of soil. Many rhizosphere organisms are ectosymbionts and others are endosymbionts .

189. Chapter 9. Role of Microorganisms in Soil Fertility and Crop Production Cycles Benefits of the soil microbiome : Soil microbes underpin key benefits that soils provide, such as Movement and exchange of key plant growth limiting nutrients such as nitrogen and phosphorus; Protection of plants from stress, pests and pathogens; Decontamination of soils through bioremediation; Helping to maintain the physical structure of soil; Decomposition of organic wastes while storing carbon; Regulating the flow of greenhouse gases, such as carbon dioxide and methane; and, A repository of undiscovered biochemicals , including antibiotics, that can be used to address antibiotic resistance.

190. The major roles of soil microbes in increasing and maintaining the soil fertility and thereby promoting the crop production are as follow: Physical support : Microbial products play very important roles for to soil aggregation and improved soil structure. Raw materials : Soil microbes also produce antimicrobial agents and enzymes used for physiological as well as reproductive growth of the plants. Growth medium for plants : Soil microbes mobilize nutrients from insoluble minerals to support plant growth. Buffering water flows : Soil macropores are formed by plant roots, earthworms and other soil biota, which may depend on soil microbes as food or for nutrients. Nutrient cycling: The activities of soil bacteria, archaea and fungi drive nutrient cycling. Recycling of wastes and detoxification: Microbial processes like mineralization and immobilization are responsible for recycling of wastes and detoxification of certain toxic elements in the soil. Biological control of pests, weeds and pathogens: Carbon storage and regulation of greenhouse gas emissions: By mineralizing soil carbon and nutrients, microbes are major determinants of the carbon storage capacity of soils. For example, denitrifying bacteria and fungi regulate nitrous oxide (N2O) while methanogenic bacteria regulate methane (CH4) emissions from the soils.

191. Biogeochemical Cycling: Microorganisms, in the course of their growth and metabolism, interact with each other in the cycling of nutrients, including carbon, nitrogen, phosphorus, sulfur, iron, and manganese. This nutrient cycling, called biogeochemical cycling when applied to the environment, involves both biological and chemical processes.

192. 1. Carbon cycle Carbon is the building block of life. Carbon is the basic constituent of all organic compounds. Plants obtain carbon from atmospheric carbon dioxide (CO2) through photosynthesis, during which the chloroplasts in the plant cells convert CO2 to carbohydrates. It is the cycling of carbon from the atmosphere through plants and algae, to animals and micro-organisms and back to the atmosphere that maintains earth's atmosphere and climate in its current balance. The greenhouse effect, or warming of the planet, is a consequence of an excess of atmospheric CO2 caused by deforestation (reduced CO2 consumption) and compounded by excessive fossil fuel energy use (increased CO2 production).

193. Carbon is a critical element in the formation of stable humus. The carbon: nitrogen (C : N) ratio of the organic matter supplied to the soil is a controlling factor in this process. A ratio of about 20:1 is considered ideal. If greater amounts of carbon are present, decomposition slows as micro-organisms become nitrogen-starved and compete with the plants for available nitrogen. Nitrate nitrogen practically disappears from the soil because microbes need nitrogen to build their tissues. carbon fixation – is the conversion of CO2 into organic matter .

194. Figure. Carbon cycle

195. The return of carbon is brought about through the decomposition organic carbon in nature. Some carbon is also released to the atmosphere as CO2 in respiration and combustion of both plants and animals. The soil microorganisms play an important role in completing the cycle. They convert organic matter into body substances, liberate CO2 and water, increase and concentrate the nitrogen content, and bring down the ratio between carbon and nitrogen in the soil. This improves soil fertility. The released CO2 goes back to complete the cycle and this ensures that there is no major lock-up in organic tissues on earth.

196. 9.3. Significance of microbes to carbon cycling Role of microorganisms in carbon cycle Microorganisms degrading cellulose  Bacteria: Species of Cellulomonas , Cytophaga , Bacillus, Clostridium, Pseudomonas, Streptomyces etc .  Fungi: Species of Trichoderma , Chaetomonium , Aspergillus , Penicillium etc. Microorganisms degrading hemicellulose Bacteria: Species of Bacillus, Pseudomonas, Cytophaga , Streptomyces , Actinomyces etc.  Fungi: Species of Chaetomonium , Aspergillus , Penicillium etc. Microorganisms degrading lignin  Bacteria: Species of Pseudomonas, Micrococcus, Flavobacterium , Arthobacter ,  Fungi: Species of Polyporus , Poria , Trametes , Mycena , Clavaria , Aspergillus , Phanerochaete

197. 9.4. Organic Matter Decomposition and the Soil Food Web 9.4.1. Soil organic matter When plant residues are returned to the soil, various organic compounds undergo decomposition. Decomposition is a biological process that includes the physical breakdown and biochemical transformation of complex organic molecules of dead material into simpler organic and inorganic molecules. Its speed is determined by three major factors : Soil organisms, The physical environment and The quality of the organic matter. In the decomposition process, different products are released: Carbon dioxide (CO2), Energy, Water, Plant nutrients and Resynthesized organic carbon compounds. Successive decomposition of dead material and modified organic matter results in the formation of a more complex organic matter called humus . This process is called humification .

198. Humus affects soil properties. As it slowly decomposes, it colours the soil darker; Increases soil aggregation and aggregate stability; Increases the CEC (the ability to attract and retain nutrients); and Contributes N, P and other nutrients. Soil micro-organisms, use soil organic matter as food. As they break down the organic matter, any excess nutrients (N, P and S) are released into the soil in forms that plants can use. This release process is called mineralization. The waste products produced by micro-organisms are also soil organic matter. This waste material is less decomposable than the original plant and animal material, but it can be used by a large number of organisms.

199. Beneficial property of soil organic matter to soil  Improved fertilizer efficiency;  Long life N for example, urea performs 60-80 days longer;  Improved nutrient uptake, particularly of P and Ca;  Stimulation of beneficial soil life;  Provides magnified nutrition for reduced disease, insect and frost impact;  Salinity management - humates “buffer” plants from excess sodium;  Organic humates are a catalyst for increasing soil C levels.  

200. Nitrogen cycle In atmosphere, 78% nitrogen is present.  Less then 0.1% of this N is fixed.  Through rainwater trace N is dissolved and added in soil. In process of lightning N2 + O2 combine to form nitrous and nitric acids .

201. Nitrogen demand of plant is very high, so nitrogenous fertilizers are required to be added to soil.  Due to symbiotic N-fixation 10-500 kg N/ha/year is fixed in soil.  Due to non-symbiotic process N-fixed in soil ranges between 50-150 kg/year/ha. Steps of Nitrogen Cycle: 1. Proteolysis /decomposition of protein 2. Ammonification 3. Nitrification 4. Denitrification 5. Nitrogen fixation

202. Nitrogen cycle

203. Amino acids utilized by:  Soil Microorganisms  Plants with the help of mycorrhiza .  Converted into ammonia (NH3) gas. This process is called Ammonification 2) Ammonification  It is process by which Amino acids are converted into ammonia with the help of microorganisms.  Bacteria are relatively more active than other organisms.  Spore-forming bacteria Pseudomonads , Actinomycetes and fungi seem to readily attack amino acids in soil.  However, in acid soils, fungi are more important agents of ammonification than bacteria.  Amino acids are broken down by oxidative deamination by aerobic microorganisms.

204. 3). Nitrification  Microorganisms convert ammonia to nitrate, the process is called nitrification .  It occurs in two steps, each step preformed by a different group of bacteria .

205. 4. Denitrification  This is reverse process of nitrification i.e . nitrate is reduced to nitrites and then to nitrogen gas and ammonia.  This process is harmful at the view point of agriculture because it reduces soil fertility.  Some aerobic organisms, Pseudomonas denitrificans , also reduce nitrate under certain conditions.  The presence of denitrifying bacteria in the soil is sufficient.  The enzyme required is nitrate reductase or nitratase . This enzyme is found in bacteria viz. E. coli, P. aeruginosa , Micrococcus denitrificans .  Denitrification may result in the formation of gaseous nitrogen.

206. 5. Nitrogen fixation  In this biological process, atmospheric nitrogen utilized by nitrogen fixing bacteria with the help of nitrogenase enzyme and converts to ammonia , readily utilizable form of nitrogen by plants.  Symbiotic, non symbiotic and associative symbiotic nitrogen fixing bacteria plays an important role in this process.  Symbiotic nitrogen fixation: Carried out by Rhizobium with leguminous plants.  Non-symbiotic nitrogen fixation: by Azotobacter directly into soil.  Associative nitrogen fixation : by Azospirillum with roots of grass.

207. 9.8. Legume inoculation Legume inoculation : the inoculation of legume seeds with a specific culture of bacteria that multiply in the roots of a legume plant forming nodules where the bacteria fix atmospheric nitrogen for the nutrition of the plant. Legumes growing together with soil bacteria called rhizobia work together to take atmospheric nitrogen (N 2 ) found in soil air spaces and transform—or fix—it into a plant-available form through the process called Biological Nitrogen Fixation (BNF) (Fig. 1). Even though the atmosphere is almost 80% N, the N 2  gas is such that plants can't use it for their own growth and development unless it is fixed. However, neither legumes nor the rhizobia can do the job alone. The process must occur as part of a mutually beneficial—or symbiotic—relationship with soil-dwelling rhizobia bacteria. Rhizobia form root nodules on the host legume, thereby providing the plant with transformed N in exchange for a portion of the carbohydrates made by the plant.

208. Figure 1. Biological Nitrogen Fixation provides nitrogen fertility in legume-based cropping systems.

209. What Is Inoculation? The application of the recommended type of bacteria to the seed or soil prior to planting is called inoculation . Inoculation simply means bringing the appropriate rhizobia into contact with legume seeds or roots. Inoculation defined as the  process of adding effective bacteria to the host plant seed before planting . Inoculation is a farming practice that helps growers give their crops a productive start to the season, improving plant vigor and return-per-acre potential each year. 

210. Need of inoculation Inoculation is needed if any of the following situations exist: Cool, wet soils (no-till and conservation tillage) Early planting Low pH (<5.8) and high pH (>8.5) soils Sustained use of some soil-applied pesticides Previous flooding or ponding (even for a short period) Low organic matter (<1%) Legume crop not grown in previous years Topsoil temperature exceeding 80° F Eroded soils

211. Methods of inoculation SEED INOCULATION: Farmers should coat their seed with inoculant just before planting 2. SOIL INOCULATION : Some inoculants are designed to be placed directly into the soil. This practice is recommended under the following conditions: When seeds are precoated with pesticides or herbicides. When planting in hot, dry soil. When seed inoculation has failed. When very large numbers of rhizobia are needed.

212. Phosphorus cycle Phosphorus is never found in the free state in nature, because contact with air causes combination with oxygen. It is found abundantly as calcium, phosphate in many minerals.  The residue of man, animals, plants, birds etc contain several phosphates. When they reach the soil, they are acted upon by several microorganisms, break-down the phosphorus containing compounds with the liberation of mineral elements like Ca, Fe, Na and this process is known as Mineralization .

213. Fig.: P-CYCLE

214. Sulfur cycle in soil Sulphur is present in different forms: Trace in air In industrial area, atmosphere contains high amount of sulfur, due to burning of coal. Sulfur in air, reaches the soil through rainwater. Sulfur and H2S abundantly coming out from volcanoes. Sulfur is present in some springs. Plants utilize Sulfur in the dissolved from as sulfate.

215. S-cycle

216. Some of the Biochemical changes by micro-organisms involved in this cycle are as follows: Elemental sulfur cannot be utilized by plants or animal. Bacteria, Thiobacillus thiooxidans is capable of oxidizing sulphur to sulphates : The reaction is: 2S + 2H2O + 3O2 2H2SO4 Sulfate is assimilated by plants and is incorporated into sulfur-containing amino acids and then into proteins. Degradation of protein (Proteolysis) librate amino acids, some of which contain sulfur is released from the amino acids by enzymatic activity of many bacteria. Sulfates may also be reduced to H2S by soil microorganisms. The bacterium involved is Disulfotomaculum .

217. H2S resulting from sulfate reduction and amino acid decomposition is oxidized to elemental sulfur. This reaction is characteristics of some pigmented sulfur bacteria: The H2S may be photosynthetically utilized and elemental sulfur may be released. The sulfates and sulphuric acid, when dissolved in water, are made available for plant growth. The plants utilize sulfates to form various amino acids, hormones, growth factors, etc.

218. Microorganisms in agriculture Followings are the few major application of soil beneficial microorganisms: - Microbes break down the complex organic matter Microbes help in recycling of nutrients Microbes help in maintaining the soil moisture Microbes create soil structure Microbes fix nitrogen Microbes promote plant growth Microbes control pests and diseases

219. 9.11. Composting of organic wastes BIODEGRADATION Microbial conversion of waste materials such as agricultural, industrial and domestic wastes, by microorganisms into inorganic compounds is called biodegradation . For successful survival of mankind on earth, recycling of organic and inorganic materials is essential. Microorganisms degrade various organic wastes by various biochemical processes and purify them to a stage of reutilization.

220. Chapter 10. Bioremediation of contaminated soil Bioremediation is defined as use of biological processes to degrade, break down, transform, and/or essentially remove contaminants or impairments of quality from soil. Bioremediation is a natural process which relies on bacteria, fungi, and plants to alter contaminants as these organisms carry out their normal life functions. Metabolic processes of these organisms are capable of using chemical contaminants as an energy source, rendering the contaminants harmless or less toxic products in most cases.

221. 10.1. Approaches to Bioremediation Soil type is an important consideration when determining the best suited bioremediation approach to a particular Situation. In situ bioremediation : - refers to treatment of soil in place. In situ biostimulation treatments usually involve bioventing , in which oxygen and/or nutrients are pumped through injection wells into the soil. It is imperative that oxygen and nutrients are distributed evenly throughout the contaminated soil. 2. Ex situ bioremediation , in which contaminated soil is excavated and treated elsewhere, is an alternative. Ex situ bioremediation approaches include use of bioreactors, land farming, and biopiles . In the use of a bioreactor, contaminated soil is mixed with water and nutrients and the mixture is agitated by a mechanical bioreactor to stimulate action of microorganisms.

222. 10.2. Advantages of Bioremediation Advantages of bioremediation include: 1. It is possible to completely breakdown organic contaminants into other nontoxic chemicals. 2. Equipment requirements are minimal compared to other remediation technologies. 3. Can be implemented as an in-situ or ex-situ method depending on conditions. 4. Low cost of treatment per unit volume of soil or groundwater compared to other remediation technologies. 5. Low-technology equipment is required i.e. readily available equipment e.g. pumps, well drilling equipment etc. 6. It is perceived positively by the public because it is a natural process. 7. Complete breakdown of pollutants in to non-toxic compounds is possible because the process does not involve transferring of contaminants to other another environmental medium.

223. 10.3. Disadvantages of Bioremediation Disadvantages of bioremediation include: 1. If the process is not controlled it is possible the organic contaminants may not be broken down fully resulting in toxic by-products that could be more mobile than the initial contamination. 2. The process is sensitive to the level of toxicity and environmental conditions in the ground 3. Field monitoring to track the rate of biodegradation of the organic contaminants is advised. 4. If an ex-situ process is used, controlling volatile organic compounds (VOCs) may be difficult. 5. Treatment time is typically longer than that of other remediation technologies. 6. Range of contaminants that can be effectively treated is limited to compounds that are biodegradable. 7. Leaves residual levels that can be too high (not meeting regulatory requirements), persistent, and/or toxic. 8. Performance evaluations are difficult because there is not a defined level of a "clean" site and therefore performance criteria regulations are uncertain

224. CHAPTER 11. BIOPESTICIDES Agriculture has had to face the destructive activities of numerous pests like fungi, weeds and insects from time immemorial, leading to radical decrease in yields. With the advent of chemical pesticides, this crisis was resolved to a great extent. But the over dependence on chemical pesticides and eventual uninhibited use of them has necessitated for alternatives mainly for environmental concerns. Degraded soils and groundwater pollution has resulted in nutritionally imbalanced and unproductive lands.

225. Biopesticides or biological pesticides based on pathogenic microorganisms specific to a target pest offer an ecologically sound and effective solution to pest problems. They pose less threat to the environment and to human health. The most commonly used bio-pesticides are living organisms, which are pathogenic for the pest of interest. These include bio-fungicides ( Trichoderma ), bio-herbicides ( Phytopthora ) and bio-insecticides (Bacillus thuringiensis ).

226. Biopesticides fall into three major categories: Microbial pesticides: Contain a microorganism (bacterium, fungus, virus, protozoan or alga) as the active ingredient. Plant-pesticides: are pesticidal substances that plants produce from genetic material that has been added to the plant. Biochemical pesticides : are naturally occurring substances that control pests by non-toxic mechanisms .

227. 10 U for attention