ULTRA STRUCTURE OF FUNGAL CELL AND GROWTH

Ultra structure of fungi K R.pptx

1. ULTRA STRUCTURE OF FUNGAL CELL AND GROWTH K R MICRO NOTES 1

2. INTRODUCTION FUNGI is the plural for the word FUNGUS which is derived from the latin word FUNGOUR, which means to flourish. Fungi can be defined as eukaryotic, spore bearing, achlorophyllous organisms and Thallophytic plants that may reproduce sexually or asexually and whose filamentous, branched and somatic structures are typically surrounded by cell walls containing chitin, cellulose, or combination of both. The branch of biology which deals with the study of fungi is called mycology ( Mykes = Mushroom or fungi, logos = Discourse) K R MICRO NOTES 2

3. They are ubiquitous in terrestrial and fresh water habitats, less in the marine environment. They are cosmopolitan in distribution Characteristically, they are saprophytic, parasitic, symbiotic or hyper parasitic in nature. They are also used for preparation of different industrial substances including antibiotics, enzymes and acids etc.. K R MICRO NOTES 3

4. ULTRASTUCTURE OF FUNGAL CELL K R MICRO NOTES 4

5. Cross-section view of a typical fungal cell K R MICRO NOTES 5

6. CELL WALL CELL WALL The main identifying characteristics of fungi is the make up of their cell walls. The composition of the fungal cell wall is rather variable. It gives strength and shape to fungi. Iit provides protection for the protoplasm from UV RAYS (presence of melanin) Glucan and Chitin are components of the primary wall, proteins are the components on the secondary cell. Ability to resist Lysis by organic solvents such as enzymes, toxins and osmotic integrity. They have ability to bind with metal ions. Except slime moulds ( Myxomycetes ) ,the fungal cell consist of a rigid cell wall and cell organelles. 6

7. The cell wall of Ascomycotina and Basidiomicotina contains chitin (β 1,4 N- acetylglucosamine ). In the Zygomycotina , the chitin fibres are modified to produce poly-β-(1,4)glucosamine, which is called chitosan. The cell walls of oomycetes contain cellulose and lack chitin. K R MICRO NOTES 7

8. CELL MEMBRANE In fungal cells, the living protoplast is enclosed in a cell membrane,also called the plasma membrane or plasmalemma . It is delicate, extremely thin semipermeable membrane. The principal components of plasmalemma are proteins and lipids. At the surface of plasmalemma ,some membrane structures known as lomasomes and plasmalomosome s have been reported. Glucose residues, Glucosamine, sterols( er gosterol ), Mannose are present. K R MICRO NOTES 8

9. CYTOPLASM Within the plasma membrane ,is the colourless cytoplasm in which sap-filled vacuoles may occur. Immersed in the cytoplasm are structures known as the organelles and inclusions. The organelles are living structures,each with a specific function.The inclusions are dead,have no specific function and thus are not essential to cell survival. Among the cell organelles are included the E.R,mitochondria,ribosomes,Golgi apparatus and vacuoles. Examples of inclusions are the stored foods( glycogen,oil drops) pigments and secretory granules. K R MICRO NOTES 9

10. NUCLEUS The cytoplasm contains one, two or more globose or spherical nuclei It measures up to 1-3 µm in diameter. Structurally the nucleus consists of : A central dense body(nucleolus). Chromatin strands. The whole structures surrounded by a definite nuclear membrane. Under the electron microscope, nuclear membrane is seen to consist of 2 unit membranes - inner and outer layers of electron dense material. It has pores, at certain points and the membrane is continuous with ER. K R MICRO NOTES 10

11. ENDOPLASMIC RETICULUM Presence of endoplasmic reticulum in fungal cytoplasm is observed through electron microscope. It is made up of flattened sacs of membrane- cisternae. It is composed of a system of membranes or micro tubular structures with small granules (ribosomes) In many fungi, the E.R is highly vesicular. It is loose and irregular as compared with cells of green plants. K R MICRO NOTES 11

12. RIBOSOMES Ribosomes is found on the ER , others free floating in the cytoplasm. They are proteinaceous bodies with high RNA content. They are concerned with protein synthesis . They aggregate to form polyribosomes (or polysomes ) K R MICRO NOTES 12

13. GOLGI BODIES( DICTYOSOMES) Moore and Muhlethaler in 1963 reported 3 flattened sacs in Saccharomyces cells. Golgi Bodies or dictyosomes are found comparatively rarely in fungi (except oomycetes ). Not organized as stacks of flattened cisternae . Instead, the Golgi bodies of fungi appear as single tubular, cisternae that vary in shape from cup-like to planar bodies. functionally equivalent to the stacked Golgi bodies.  Major function is to process and package macromolecules (proteins) and transportation of lipids around the cell. K R MICRO NOTES 13

14. K R MICRO NOTES 14

15. MITOCHONDRIA Richard Altmann , in 1890, established cell organelles and called them “ bioblasts ”. Carl Benda coined the term “mitochondria” in 1898. The cytoplasm contains small, usually spherical bodies known as the mitochondria. Each mitochondrion is enveloped by a double membrane. The inner membrane is infolded to form the cristae which are in the form of parallel flat plates. The mitochondria function as the power house of the cell. Mitochondria has its own machinery for transcription and translation of organelle specific DNA. K R MICRO NOTES 15

16. VACUOLES Vacuoles are essential for cell function in fungi. Fungi are characterized by the presence of spherical to tubular vacuoles. Vacuoles are found in the old cells of hyphae. The end of hyphal tip of young hyphae lacks vacuole. Vacuoles are surrounded by a membrane known as tonoplast . The function of the vacuoles is to provide the turgor needed for cell growth and maintenance of cell shape. Besides osmotic function,it stores reserve materials( Volutin - polymetsphosphate ; in yeasts), pigments , amino acids and hydrolases. K R MICRO NOTES 16

17. LYSOSOMES Thornton (1968) described them as “ Autophagic vesicles”. They have been described only in Botrytis cinerea and Phycomyces .   They are thought to be derived from Golgi cisternae. Simple lysosomes have a diameter of 400nm and are bound by a unit membrane.    K R MICRO NOTES 17

18. VESICLES The term vesicle is used for any cell or organ that is inflated by the stuff that is used to store. (swollen end cells) This are generally formed by Endomycorrhizal fungi either between root cells or within the cell wall. Vesicles are common in fungi, especially in the apical regions and where ever wall synthesis is in progress. The apex of hyphae contains a large number of vesicles and is termed Apical Vesicular Complex (AVC) . They transport the products formed by the secretary action of Golgi apparatus to the site where these products are to be utilized. K R MICRO NOTES 18

19. MICROTUBUL E S Discovered by transmission electron microscopy in the late 1950s. In filamentous fungi,microtubule is an essential component of tip growth machinery that enables continuous and rapid growth. Composed of the protein tubulin which consist of a dimer composed of two protein subunit. Microtubules are long, hollow cylinder -25nm in diameter Involved in the movement of organelles, nuclei and Golgi vesicles containing cell wall precursor. Assist in the movement of chromosomes during mitosis and meiosis The destruction of cytoplasmic microtubules interferes with the transport of secretory material to the cell periphery, which may inhibit the cell wall synthesis. K R MICRO NOTES 19

20. Micro Bodies It was first described by Frederick and commerce workers in 1975. These organelles are round, oval, 1.5-2.0 I'm in diameter surrounded by single unit membrane. Their origin is unknown. They may be identical to or may be the precursors of peroxisomes or lysosomes, which contains either catalase or histolytic enzymes. " Woronin bodies " named after M. S woronin , are generally spherical and highly refractive bodies and are bound by unit membrane. These are found associated with septal pores of discomycetes and many deuteromycetous fungi K R MICRO NOTES 20

21. GROWTH Growth is defined as the irreversible constant increase in the dry mass of an organism. It is brought about by an increase in cell size or number. It is the fundamental characteristics of living bodies accompanied by various metabolic processes. (anabolic or catabolic) Factors effecting growth : External factors - light, temperature, water, nutrients; Internal factors - hormones. K R MICRO NOTES 21

22. OPTIMUM CONDITION FOR GROWTH Presence of water : 80–90% of the fungi is composed of water by mass, and requires excess water for absorption due to the evaporation of internally retent water. Presence of oxygen Neutral-acidic pH : Optimum pH 5.0 Low-medium temperature : ranges between 1°C and 35°C, with optimum growth at 25 °C. K R MICRO NOTES 22

23. Growth in fungi can be seen by unicellular organization in form of hyphae K R MICRO NOTES 23

24. UNICELLULAR ORGANIZATION Unicellular organization occur in some fungal groups. The most widely known are the yeast. Budding takes place during favourable condition. The proto­plasm of vegetative cell swells up at one side in the form of a bud. The nucleus undergoes mitotic division. Out of two nuclei formed by mitosis, one goes to the bud and other one remains in the mother. Bud enlarges and even­tually cuts off from the mother by parti­tion wall and grows individually. The size of the bud is always smaller than the mother cell. K R MICRO NOTES 24

25. Sometimes due to rapid division, large num­ber of buds develop without being detached from one another and persist in the form of branched or unbranched chain, called pseudo- mycelium. K R MICRO NOTES 25

26. IN FORM OF HYPHAE Under favourable environmental conditions, fungal spores germinate and form hyphae. The hypha may be roughly divided into three regions: (1) the apical zone about 5–10 micrometres in length, (2) the  subapical region , extending about 40 micrometres back of the apical zone, which is rich in cytoplasmic components, such as nuclei, Golgi apparatus, ribosomes, Mitochondria, the Endoplasmic Reticulum, and vesicles, but is devoid of Vaculoes . (3) the zone of vacuolation , which is characterized by the presence of many vacuoles and the accumulation of Lipids. K R MICRO NOTES 26

27. Fungal hyphae extend continuously at their extreme tips. The rate of tip extension can be extremely rapid - up to 40 micrometres per minute. It is continuous movement of materials into the tip from older regions of the hyphae. This unique mode of growth -  apical growth  - is the hallmark of fungi, and it accounts for much of their environmental and economic significance. K R MICRO NOTES 27

28. MECHANISM OF APICAL GROWTH K R MICRO NOTES 28

29. SPITZENKÖRPER The mature vegetative hyphae elongate by means of tip growth. The extreme tip of a growing hypha has very few organelles; instead, it contains a body termed the Spitzenkörper . This consists of a cluster of small, membrane-bound vesicles embedded in a meshwork of actin microfilaments. The Spitzenkörper is always present in growing tips, disappears when growth stops, reappears when growth restarts. K R MICRO NOTES 29

30. A region within the centre of the Spitzenkörper (the ‘core’) is largely devoid of nuclei. Spitzenkörper is generally regarded as a ‘ vesicle supply centre ’, which regulates the initiation, maintenance, and direction of hyphal growth. K R MICRO NOTES 30

31. CYTOSKELETON There are two main cytoskeletal elements in fungi: microtubules, actin microfilaments . Microtubules and actin microfilaments play a variety of roles: formation of spindles. allowing chromosome segregation during nuclear division. nuclear positioning. providing long distance transport of secretory vesicles to hyphal tips. Intracellular movement of organelles and protein complexes. K R MICRO NOTES 31

32. The rate of hyphal extension might be controlled, and bursting prevented, by the actin/spectrin cap The transport of secretory vesicles and other organelles along cytoskeletal elements is driven by motor proteins Depolymerization of microtubules results in a disappearance of the Spitzenkorper, termination or reduction of apical growth and enzyme secretion. Actin depolymerization leads to uncontrolled tip extension to form giant spheres. The integrity of the Spitzenkorper is maintained by an interplay between actin and microtubule. K R MICRO NOTES 32

33. SECRETORY PROCESS OF HYPHAL TIP GROWTH K R MICRO NOTES 33

34. SYNTHESIS OF CELL WALL K R MICRO NOTES 34

35. FUNGAL GROWTH PHASES From the time a spore or a hyphal fragment germinates to form a colony to the time the fungus dies, there are a number of growth phases. Although these phases have been determined under laboratory conditions, it is possible that the same occur in nature. The unicellular organisms (Yeast) may present different phases :- 1.Lag phase 2. Log phase or exponential phase 3.Retardation phase 4.stationary phase . K R MICRO NOTES 35

36. K R MICRO NOTES 36

37. Lag phase Once the growth conditions become favourable for the fungi to germinate, new transport systems must be induced before growth starts. Thus growth starts slowly. This phase is referred to as the lag phase. Exponential or log phase Hyphae branches are initiated The new hypha extends at a linear . The biomass of the growing fungus doubles per unit time. As long as the nutrients are in excess growth remains constant during the exponential phase. K R MICRO NOTES 37

38. Retardation Phase : After the end of the exponential phase the growth of the culture ceases long before the nutrients in the medium where exhausted. Stationary phase As soon as the nutrients are depleted or toxic metabolites are produced growth slows down or is completely stopped. During the stationary phase, hyphal growth stops and, in some molds , cell differentiation occurs, resulting in spore formation. During this process nutrients are transferred from the vegetative mycelium to the developing spores. The spores are dispersed by air movement to other areas of the building where they can start new mold growth once the conditions for growth are favourable. K R MICRO NOTES 38

39. Different Phases of Growth In filamentous Fungi is due to linear phase of growth Here Usually exponential Phase is replaced by linear phase of growth. In linear Phase growth of fungi is limited to terminal portion of the hyphae. This phenomenon of terminal growth was studied in Fusarium , Aspergillus , Pencillium , Rhizopus . Three dimension growth can also be seen in standing liquid cultures , Fungi usually grow as floating mats. K R MICRO NOTES 39

40. GENERTION TIME Generation time is the average time between two consecutive generations in the lineages of a population. When growing exponentially by binary fission, the increase in a fungal population is by geometric progression. If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The generation time is the time interval required for the cells (or population) to divide Calculation of Generation Time G (generation time) = (time, in minutes or hours)/n (number of generations) G = t/n t = time interval in hours or minutes B = number of cell at the beginning of a time interval b = number of cell at the end of the time interval n = number of generations (number of times the cell population doubles during the time interval) . K R MICRO NOTES 40

41. b = B x 2n Solve for n: Log b = log B + n log 2 n = log b – log B log 2 n = log b – log B 0.301 n = 3.3 log b/B G = t/n Solve for G G = t . 3.3 log b/B K R MICRO NOTES 41

42. MEASUREMENT OF FUNGAL GROWTH The following two methods used for measuring the growth in fungi. The methods are: Linear Method (Agar Plate) Mycelial Dry Weight (Biomass) K R MICRO NOTES 42

43. Linear Method Linear Growth means that the fungus grows by the same amount in each time. To measure the diameter of the colony at two places - at right angles to each other An average of the cross diameter gives the growth of a fungus On solid medium the fungal growth can be measured. K R MICRO NOTES 43

44. Procedure Pour the media in sterile petriplate . Aseptically inoculate the culture Incubate the plates at 25°C (room temperature) for a week or 10 days. Measure the diameter of each colony at 2-3 places and take the average. This measurement can be done after 24, 48, 72, 96, 120 and 144 hours. Record the measurements. plot a graph with time against diam­eter of colony. A growth curve is obtained. K R MICRO NOTES 44

45. Mycelial Dry Weight (Biomass) On liquid medium the mycelial growth can be measured. Determined by inoculating cultures into broth, followed by filtering of mycelium, drying at 105°C  for 48 hours to a constant weight and weighing the mycelium. K R MICRO NOTES 45

46. Procedure Pour the media in sterile petriplate. Aseptically inoculate the culture Incubate the plates at 25°C (room temperature) for a week or 10 days. Filtered the content through a pre weighed filter paper. Dry the filter paper in an oven for 48 hours at 105°C. Reweigh the filter papers with dry myc­elium, subtract the weight of filter paper . Prepare growth curve with time (hours) against weight of mycelium. K R MICRO NOTES 46

47. REFERENCE An Introduction to Mycology – --R.S Meherotra & K.R. Aneja. Introduction to Fungi - Webster. Introductory Mycology – D P Tripathi K R MICRO NOTES 47

48. K R MICRO NOTES 48