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Unit 4 Diagnosis of Plant Diseases Caused by Bacteria Prepared by: Mellprie B. Marin A schematic representation of the basic functions in a plant (left) and of the interference with these functions (right) caused by some common types of plant diseases. (Modified f...

Unit 4 Diagnosis of Plant Diseases Caused by Bacteria Prepared by: Mellprie B. Marin A schematic representation of the basic functions in a plant (left) and of the interference with these functions (right) caused by some common types of plant diseases. (Modified from Agrios, G.N. 1997. Plant Pathology (4th ed.). Academic Press, NY, NY.) What is Disease Diagnosis? Ø the art or act of identifying a diseased plant from a healthy plant through its signs and symptoms Ø Disease diagnosis is a critical first step for successful disease management. Rationale for Diagnosis v Plant abnormally cannot be diagnosed solely on symptoms v Similar pathological conditions may be caused by quite different agents e. g. soft rots may be caused by fungi and bacteria Ø Bacterial soft rot may be primarily associated with species of Pseudomonas, Dickeya, Bacillus, Pectobacterium or Erwinia vSeveral diseases and/or plant health problems can cause similar symptoms e.g. leaf and fruit spot of tomato Major Types of Bacterial Diseases: Ø Vascular Diseases – Wilt Ø Necrotic Diseases – Necrotic leaf spots Ø Soft Rot Diseases – Soft rots Ø Tumor Diseases – Gall/hyperplasia Ø Signs are actual physical evidence of the occurrence of the pathogen in association with the unhealthy plant material. Ø Bacterial ooze and specific odors: associated with tissue macerations by certain pathogens Ø Bacterial ooze and specific odors: associated with tissue macerations by certain pathogens B acterial "ooze" or exudate seen coming out of water-soaked lesions (see ). The "ooze" forms in the readily seen droplets. These droplets are a sign of the pathogen, being composed mostly of bacterial cells.  From wilted tomato plant: To p r o p e r l y d i a g n o s e bacterial wilt, a horizontal cut was made in the lower stem and the cut stem immersed partway in water. Within a few minutes, copious amounts of bacterial exudate emerged from the cut end, forming the white streamers you see in the water. bacterial streaming Test for Rapid Diagnosis  watersoaking - the spots look like drops of water and are caused by water accumulation in tissues as a direct result of bacterial attack; occurs early in the development of bacterial diseases Watersoaking (at the underside of the leaf)  lesions "scabby“ (bacterial spot of pepper) Fruit spot of pepper- Xanthomonas campestris pv. vesicatoria Black leg of tobacco- Pectobacterium carotovorum subsp. atrosepticum  Methods Used in Diagnosis of Bacterial Diseases 1. Symptomatology Ø Direct examination of infected tissues (visual and microscopic) Ø Staining Techniques - performed when it is difficult to differentiate the bacterial ooze from cellular plant materials under low magnification A. Negative Staining - for leaf spots, streaks, cankers, blights, pustules, galls, etc. on fleshy plant tissues  Methods Used in Diagnosis of Bacterial Diseases 1. Symptomatology Staining Techniques A. Negative Staining solutions and reagents: 1. Congo Red 2 g Congo red (80% dye) 100 ml distilled water 2. Acid Alcohol 3 drops concentrated HCl 30 ml 95% ethyl alcohol RESULT: Bacteria appear as colorless rods in dark background  Methods Used in Diagnosis of Bacterial Diseases 1. Symptomatology Staining Techniques A. Negative Staining solutions and reagents: RESULT: Bacteria appear as colorless rods in dark background  Methods Used in Diagnosis of Bacterial Diseases 1. Symptomatology Ø Staining Techniques B. Gram Staining - for any pathogenic bacteria associated with any bacterial disease, esp. soft rot and wilt; the test is essential for differentiating plant bacteria into two broad groups: gram-positive and gram-negative  Methods Used in Diagnosis of Bacterial Diseases 1. Symptomatology Staining Techniques B. Gram Staining solutions and reagents: 1. Ammonium Oxalate - Crystal Violet solution Sol'n A: 2 g crystal violet (90% dye) 80 ml distilled water Sol'n B: 0.8 g ammonium oxalate 80 ml distilled water  Methods Used in Diagnosis of Bacterial Diseases 1. Symptomatology Staining Techniques B. Gram Staining solutions and reagents: 2. Gram's modification of Legel's solution 1 g Iodine 2 g potassium iodide 300 ml of distilled water 3. Counterstain 10 ml Safranin (2.5% sol'n in 95% ethanol) 100 ml distilled water  Methods Used in Diagnosis of Bacterial Diseases 1. Symptomatology 3. KOH Test - can be used as rapid test for presumptive identification Mix loopful of bacteria with 2 drops of 3% KOH. RESULTS: Gram Negative bacteria will become gummy upon mixing with a loopful while gram positive bacteria will not.  Methods Used in Diagnosis of Bacterial Diseases 1. Symptomatology B. KOH Test Major points in successful staining: 1. Use reagent less than one year old (particularly iodine). 2. Iodine (if not put in brown bottle or kept in the dark) will decolorize and become ineffective 3. Use freshly grown bacteria (exponential phase). Older stationary phase cells may give a gram-variable reaction 4. Use a known positive and known negative for control C. Flagella Staining C. Flagella Staining D. Spore Staining D. Spore Staining 2. Isolation a. Streaking b. Dilution-pour Plate c. Selective Media Common bacterial diseases and crops affected: Factors Crops Bacterial conducive affected Symptoms disease to spread Light-brown to yellow V-shaped lesions on Black rot the leaf, which (Xanthomonas Warm, wet become brittle and dry Brassicas campestris pv. conditions with age. Vein campestris) blackening with the necrotic area.  Seedlings may die and older plants may wilt Bacterial and die eventually. canker Moderate Older plants have Tomato; (Clavibacter temperatures leaves that turn yellow capsicum; michiganensis and high and wilt only on one chilli pv. humidity side. Cankers on michiganensi stems and fruit. Tissue s) inside stems becomes discoloured.  Wide range of Wet, slimy, soft rot vegetables, Bacterial that affects any part including soft rot of vegetable crops Warm, wet lettuce; brassicas; (Pseudo- including heads, conditions. cucurbits; tomato; monas spp., curds, edible roots, capsicum; potato; Erwinia stems and leaves. sweet potato; spp.) May have a carrots;herbs. disagreeable odour.  Lettuce – Large brown to black circular areas that start as small translucent spots; usually on outer leaves. Tomatoes and capsicums – Greasy Bacterial leaf Range of spots on leaves and spot/Bacterial vegetables stems that go from tan to Overhead spot including black; fruit may have irrigation (Xanthomonas lettuce; circular spots with central and windy campestris - cucurbits; scab. Cucurbits – Begin conditions. various tomato; as small water- strains) capsicum. soaked/greasy spots on underside of leaves with corresponding yellowing on upper side; fruit may produce light-brown ooze from water-soaked  High Wilting, yellowing and temperatures, stunting of plants but high soil they may wilt rapidly moisture and and die without any poor drainage. spotting or yellowing; Bacterial Once infection Potato; vascular tissue appears wilt has occurred, tomato; brown and water- (Ralstonia severity of capsicum; soaked; a white ooze solana- symptoms is eggplant. appears when pressure cearum) increased with is applied to affected hot and dry tubers or stems. conditions, which facilitate wilting.  Bacterial leaf Long Beet; Beet – irregular, spot/Bacterial periods of onions; round leaf spots with blight leaf leeks; a grey centre (Pseudomonas wetness. rocket; surrounded by a syringae - coriander. purple margin. various strains) Spring onions/shallots – pale yellow to light- brown lesions with a water-soaked appearance around the margins; outer leaves wither and die and youngest leaf turns lemon to light- green. Leeks – brown streaking on  Bacterial blight Cool, wet, Water-soaked spots (Pseudomonas windy on leaves and syringae pv. pisi) conditions. stipules which become dark-brown and papery in warm weather or black in Peas cool weather. Water- soaked spots on pods that become sunken and dark- brown.  Small dark spots Bacterial speck Humidity and surrounded by a (Pseudomonas overhead yellow halo on leaves; Tomato. syringae pv. irrigation. dark raised specks on tomato) fruit.  Tan to reddish-brown Bacterial brown spots on leaves. spot Cool, wet, Water-soaked spots (Pseudomonas windy on pods which Beans. syringae pv. conditions. enlarge and become syringae) sunken and tan with distinctive reddish- brown margins. Other bacterial diseases of vegetables include: Corky root – Rhizomonas suberifaciens (lettuce) Other bacterial diseases of vegetables include: Angular leaf spot – P. syringae pv. lachrymans (cucurbits); Other bacterial diseases of vegetables include: Common bacterial blight – Xanthomonas campestris pv. phaseoli (beans) Other bacterial diseases of vegetables include: Halo blight – Pseudomonas syringae pv. phaseolicola (beans) Other bacterial diseases of vegetables include: Black leg – Erwinia carotovora pv. atroseptica (potatoes)  MANAGEMENT: Disease management strategies aim to favor the host plant’s growth and development while attacking vulnerable stages in the life cycle of the pathogen to prevent or restrict its development.  MANAGEMENT: 1. Exclusion or eradication of the pathogen (quarantine and use of pathogen-tested seeds and propagation materials) MANAGEMENT: Use of clean transplants Monitor crops regularly and use predictive models Reduce the pathogen levels by crop rotation Remove weeds and incorporate crop residues that can host the disease Protect the host plant by using resistant plant varieties Minimize mechanical damage to crops and damage by insect pests Avoid working in crops when they are wet Spray with a registered bactericide when weather conditions favor disease development to prevent infection Understand chemical resistance and rotation of chemical groups If the plants are already infected, isolate and destroy them and pr une infected leaves, but avoid excessive handling of diseased plants; if the disease is systemic and has spread throughout the p l a n t , t h e p l a n t c a n n o t r e c ove r a n d should be destroyed (burning or burying) Use correct temperatures and packing conditions during transport and storage. Thank you! Aim high! CULTIVATION AND GROWTH OF BACTERIA   Chocolate agar (CHOC) or chocolate blood agar (CBA), is a nonselective, enriched growth medium used for isolation of pathogenic bacteria. It is a variant of the blood agar plate, containing red blood cells that have been lysed by slowly heating to 80°C. "blood agar" is usually prepared from Tryptic Soy Agar or Columbia Agar base with 5% Sheep blood. Rabbit or horse blood may be used for growth of NAD (nicotinamide adenine dinucleotide) - requiring organisms (electron acceptor organisms), such as Haemophilus species, but the hemolytic patterns may be inconsistent with those on sheep blood  Eosin – Methylene Blue (EMB) agar contains peptone, lactose, sucrose, and the dyes eosin Y and methylene blue; it is commonly used as both a selective and a differential medium. EMB agar is selective for gram- negative bacteria  Xylose Lysine Deoxycholate agar (XLD agar) is a selective growth medium used in the isolation of Salmonella and Shigella species  XLD agar is composed of yeast extract, sodium chloride, xylose, lactose, sucrose, l-lysine hydrochloride, sodium thiosulfate, iron (III) ammonium citrate, phenol red, sodium deoxycholate, agar, and distilled or deionized water.  MacConkey agar (MAC) is a bacterial culture medium named after bacteriologist Alfred T. MacConkey (1861-1931). MacConkey agar is a selective and differentiating agar that only grows gram-negative bacterial species; it can further differentiate the gram-negative organisms based on their lactose metabolism  Nutrientagar consists of peptone, beef extract and agar.It is a simple formulation but provides the nutrients necessary for the replication of a large number of microorganisms that are not too demanding. Beef extract contains water-soluble substances (carbohydrates, vitamins, nitrogen compounds and salts). Methods for Studying Microorganisms culture - the process of growing a bacterial or other biological entity in an artificial medium.  fastidious - microorganisms that are difficult to culture since they need specific nutrients in their medium to grow. Methods for Studying Microorganisms  agar - gelatinous material obtained from the marine algae, used as a bacterial culture medium, in electrophoresis and as a food additive. sterilization - any process that eliminates or kills all forms of microbial life present on a surface, solution, or solid compound. Methods for Studying Microorganisms  How can growth of the microbes be attained? - by providing the microbes the necessary nutrition needed for increase in size and number. Culture media may contain the desired element to favor the growth of wanted organism and may exclude all others. Methods for Studying Microorganisms  Culture Medium – the nutrient solution used to grow microorganisms in the laboratory; - any nutrient material prepared for the growth and cultivation of microorganisms Methods for Studying Microorganisms Components/ingredients of culture media: 1. Commercial digests – ex. agar, beef extract, peptone, dextrose 2. Local ingredients – ex. potato, sweet potato, sugar, gulaman, coconut water, other plant materials Methods for Studying Microorganisms Classification of Culture Media:  Based on Chemical Composition 1.Chemically-defined/Synthetic – prepared by adding precise amounts of highly purified inorganic or organic chemicals to distilled water; the exact composition of this medium is known. Methods for Studying Microorganisms Classification of Culture Media:  Based on Chemical Composition 2.Undefined (complex)/Non-synthetic – the exact composition of this medium is not clear (at least 1 ingredient is not chemically definable); employs digest of casein (milk protein), beef, soybeans, yeast cells, blood, and other highly nutritious yet chemically-undefined substances Classification of Culture Media:  Based on Physical State (Medium’s Normal Consistency) 1.Liquid Media – water-based solutions that do not solidify at temperatures above freezing and that tend to flow freely when the container is tilted; termed as broth, milks or infusions (ex. Nutrient Broth, tomato juice) Classification of Culture Media:  Basedon Physical State (Medium’s Normal Consistency) 1.Liquid Media – these are available for use in test tubes, bottles or flasks. Certain aerobic bacteria (ex. Bacillus anthracis) are known to grow as a thin film called ‘surface pellicle’ on the surface of undisturbed broth Classification of Culture Media:  Based on Physical State (Medium’s Normal Consistency) 2.Semi-solid Media – exhibit a clot-like consistency because they contain an amount of solidifying agent (agar or gelatin) that thickens them but does not produce a firm substrate; useful in demonstrating bacterial motility and separating motile from non-motile strains (ex. Stuart’s and Amie’s Media)  Based on Physical State 3. Solid Media – provides a firm surface on which cells can form discreet colonies. Liquefiable media are reversible solid media; contains a solidifying agent which melts at boiling temperature and solidifies at room temperature. Non-liquefiable media have less versatile application; they start out solid and remain solid after heat sterilization.  ex. cooked meat, potato slices Classification of Culture Media:  Based on Chemical Contents of the Media o Synthetic Media – compositions are chemically defined; contains pure organic and inorganic compounds and have a molecular content specified by means of an exact formula; most useful in research and cell culture when the exact nutritional needs of the test organisms are known. Classification of Culture Media:  Based on Chemical Contents of the Media o Non-synthetic Media – contain at least one ingredient that is not chemically definable; not a simple, pure compound and not representable by an exact chemical formula; most of these substances are extracts of animals, plants or yeasts. ex. blood serum, meat extracts or infusions Blood Serum Agar Water Agar Classification of Culture Media:  Media Based on Purpose/Function o General-purpose Media – designed to grow a broad spectrum of microbes as possible; contain a mixture of nutrients that could support the growth of pathogens and non-pathogens alike ex. Nutrient Agar (NA) Classification of Culture Media:  Media Based on Purpose/Function o Enriched Medium – contains complex organic substances such as blood, serum, haemoglobin or special growth factors (vitamins) for certain microbes to grow. ex. fastidious bacteria which requires growth factors and complex nutrients Streptococci - blood agar Neisseria gonorrheae - chocolate agar Classification of Culture Media:  Media Based on Purpose/Function o Selective Media – contains one or more agents that inhibit growth of certain microbe but not others and thereby encourages or selects and allow it to grow. o useful in the isolation of a specific type of microorganism from samples containing dozens of different species – feces, saliva, skin, water, soil o ex. Mannitol Salt Agar (MSA) contains NaCl (75%) that is nhibitory to most human pathogens except Staphylococcus. Classification of Culture Media:  Media Based on Purpose/Function o Differential Media – grow several types of microorganisms; designed to display visible differences among microbes. These variations come from the type of chemicals these media contain and the ways by which the microbes react to them. ex. Mac Conkey Agar contains neutral red, a dye that is yellow when neutral and pink or red when acidic. E. coli which produces acid when it metabolize lactose in the medium develops red to pink colonies.  Classification of Culture Media:  Media Based on Purpose/Function o Reducing Media – contains substances that absorbs oxygen thus reducing its availability; important in growing anaerobic bacteria; for growth of Clostridium spp. o ex. Robertson cooked meat – contains heart meat and nutrient broth o  Classification of Culture Media:  Media Based on Purpose/Function o Transport Media – used to maintain and preserve specimens that are to be held for a long time before clinical analysis or to sustain delicate species that die rapidly if not held under stable conditions o Cary o Blair o Medium o o Venkatraman Ramakrishnan Classification of Culture Media:  Media Based on Purpose/Function o Assay Media – used by technologists to test the effectiveness of antimicrobial drugs and by drug manufacturers to assess the effect of disinfectants, antiseptics, cosmetics and preservatives on the growth of microorganisms.  Classification of Culture Media:  Media Based on Purpose/Function o Enumeration Media – used by industrial and environmental microbiologists to count the number of microorganisms in milk, water, food, soil and other samples. o for detecting and enumerating yeasts and molds o Ex. Dichloran rose bengal chloramphenicol agar o Oxytetracycline glucose yeast extract agar Nutritional Requirements of Bacteria: o Minimum Nutritional Requirements: H2O, C, N and some inorganic compounds Water - 80% of the total weight of the cell Protein, polysaccharides, lipids, nucleic acid, mucopeptides and low molecular weight compounds - 20% Nutritional Requirements of Bacteria: o Monoelements - required in large amount, 95% of the dry wt. of the bacterial cell a. C – sources (amino acids, lipids, nucleic acid, sugar) b. N- source (amino acids and nucleic acid) - NH4+2 - source of N c. O - source (sugars, etc.) d. P - source (PO4-3) e. S - source (amino acids) f. Minerals - K, Ca, Mg and Fe - found as ions Nutritional Requirements of Bacteria: o Microelements - Mn, Zn, Co, Mo, Ni, Cu – required in trace amounts as part of enzymes and co-factors. o Minerals can also be provided in tap water (Mn, Mg, Co, Cu & Zn) Cell Division and Multiplication of Bacteria:  Growth - an increase in all cell components which ends in multiplication of cell leading to increase in population - involves an increase in the size of cell and increase in the number of individual cells Cell Division and Multiplication of Bacteria:  Growth Cell Division and Multiplication of Bacteria:  Growth 1. Cell elongates and DNA is replicated 2. Cell wall and plasma membranes begin to constrict 3. Cross-wall forms, completely; the two DNA copies 4. Cell separate Cell Division and Multiplication of Bacteria:  Growth Cell Division and Multiplication of Bacteria:  Generation Time - interval of time between two cell divisions - time required for a bacterium to give rise to 2 daughter cells under optimum conditions - population doubling time Cell Division and Multiplication of Bacteria:  Growth  INTRODUCTION AND HISTORY OF PLANT BACTERIOLOGY Prepared by: Mellprie B. Marin, PhD INTRODUCTION AND HISTORY OF PLANT BACTERIOLOGY Learning Outcome: Ø Discuss the concepts of Plant Pathology and importance of Plant Bacteriology in sustainable agriculture Bacteria (singular: bacterium)- constitute a large domain of prokaryotic microorganisms that cause plant diseases.  Important Bacterial Diseases in the Philippines: 1. Bacterial wilt of solanaceous crops- Ralstonia solanacearum 2. Bacterial wilt of banana - Ralstonia solanacearum Xanthomonas vasicola pv. musacearum 3. Soft rot of vegetable – Pectobacterium carotovorum subsp. carotovorum 4. Crown gall of roses – Agrobacterium tumefaciens 5. Black leg of tobacco – Pectobacterium sp. 6. Bacterial spot - Xanthomonas axonopodis pv. vesicatoria Bacterial wilt of tomato Bacterial wilt of banana Fusarium wilt of banana (just for comparison)  Wilting and stunted growth Wilting on one side of the tobacco plant Bacterial wilt on pepper  Squash Potato Ginger Bacterial Soft Rot of Vegetables Cabbage Onion  Crown Gall of Roses Crown Gall of Chrysanthemum Black leg of tobacco (Pectobacterium sp. ) Witches Broom of Cassava- Phytoplasma Witches Broom of Cassava- Phytoplasma Bacterial Spot of Pepper- Xanthomonas axonopodis pv. vesicatoria Hypersensitive Reaction (Test)  Historical Background 1863- J. C. Davine Showed that rod-shaped elements were 1868 present in animals infected with anthrax. The disease can be transmitted to healthy animals thru blood containing rod-shaped elements Bacillus anthracis Louis 1865 Pasteur Discovered the pathogen affecting silk worms; his works led to the development of vaccines for rabies and anthrax. Pasteurization involves heating liquids at high temperatures for short amounts of time. Pasteurization kills harmful microbes in milk without affecting the taste or nutritional value (sterilization= all bacteria are destroyed). 1876 Robert Koch A German scientist who provided the first conclusive demonstration on the etiology of anthrax. 1. He conducted a transmission experiment (successive inoculation on 20 mice; introduced affected blood to healthy mice and resulted to diseased mice); 2. He cultivated the bacterium from a piece of spleen in sterile serum and observed hourly the growth of rod-shaped elements. It changed into filaments which were ovoid, refractile bodies (spores) Observed the spores germinated (made a series of transfer (8x)  3. Inoculated the healthy mice – reisolated – re inoculated the bacterium to a healthy mice-which resulted to diseased mice His work is the milestone of Microbiology known as “Koch’s Postulates” a criterion in establishing the specific microorganisms in a specific disease Considered the Father of Microbiology (Bacteriological Technique) First to demonstrate the biological specificity of organisms (Doctrine of Specific Etiology) Etiology describes the cause or causes of a disease. 1878- Thomas Jonathan Discovered that bacteria cause disease in 1884 Burril plants (pear blight and apple twig disease)- Micrococcus amylovorus now known as Erwinia amylovora; First report of the association of bacteria in plant disease came 2 years after Robert Koch’s demonstration of bacterial etiology of anthrax in animals 1883 Walker Reported that bacteria caused yellow disease of hyacinth- Bacterium hyacinthi now known as X. campestris pv. hyacinthi 1887 Savastoni Olive knot disease, Bacillus oleae tuberculoses now known as Pseudomonas syringae pv. savastoni 1893- Erwin First paper mainly on crown gall and bacterial wilt of 1894 Smith solanaceous crops 1895 Published his work on bacterial wilt of cucurbits 1905 Published his first book on Phytobacteriology entitled “Bacteria in Relation to Plant Diseases” 1920 Published his 2 nd book entitled “Introduction to Bacterial Diseases in Plants” 1930 Charlotte Published the first edition of “Manual of Bacterial Plant Elliot Pathogens. A Comprehensive Compilation of Bacterial disease” 1943 Delbruck Discovered mutation in bacteria which provided the and Luria technical conceptual basis for genetic work on bacteria (Molecular Genetics) 1944 Avery Discovered the process of bacteria genetic transfer known Mcleod and as “Transformation by free DNA” McCarty 1967 Doi, First report on Mycoplasma diseases in plants Teranaka, (mycoplasma-like bodies in mulberry, potato witches Yora, broom and aster yellows) Asuyama 1971 Diener Determined that the potato-spindle tuber disease was caused by small molecule of infectious naked RNA called “Viroid” 1972 Davis/Whorle Observed a motile organisms on corn stunt disease- y/Withcomb/ caused by Spiroplasma (walless bacteria) Isiyama/Steer 1973 Gotten, Discovered Rickettsia-like organisms (RLO) in Pierce Nyland, Lowe, disease of grape known as Xylella fastidiosa (wavy Hopkins walls) 1974 Van Lareveke, Discovered a large specific Plasmid in virulent strains of Engler, Agrobacterium tumefaciens. The plasmid is essential for Holster, virulence and codes for TIP (Tumor-Inducing Principle or Vande, Ti) of the bacterium. Elsacker, Salmen, Scelperoort, Shell Thank you for your time! Prepared by: Mellprie B. Marin, PhD Learning Outcome: Ø Familiarize the different parts and functions of plant pathogenic bacteria  BACTERIA GENERAL CHARACTERISTICS  Typically one-celled  Possess a unit membrane and a rigid cell wall  Reproduce by binary fission  Nuclear material consists of DNA which may appear in the cell as: circular or ellipsoidal dumbbell-shaped BACTERIA BACTERIA  BACTERIA GENERAL CHARACTERISTICS  Gene transfer in bacteria may occur through: transformation transduction conjugation lysogenization  During transformation, DNA fragments (usually about 10 genes long) are released from a dead degraded bacterium and bind to DNA binding proteins on the surface of a competent living recipient BACTERIA bacterium. Transduction involves the transfer of a DNA fragment from one bacterium to another by a bacteriophage.. BACTERIA BACTERIA  BACTERIA GENERAL CHARACTERISTICS  Shape: spherical or ellipsoidal (cocci) ex. Staphylococcus, Streptococcus, rod-shape or cylindrical (bacilli) ex. Lactobacillus, E. coli, Virgibacillus spiral-shape or helicoidal (spirilla) ex. Treponema, Borellia BACTERIA Treponema  BACTERIA GENERAL CHARACTERISTICS  Occurrence: single couple chain cluster  BACTERIA GENERAL CHARACTERISTICS  Position of Flagella: Lophotrichous – 2 or more flagella are located at one end of the bacterial cell  BACTERIA GENERAL CHARACTERISTICS  Position of Flagella: Amphitrichous – 1or more flagella are at each end of the bacterial cell  BACTERIA GENERAL CHARACTERISTICS  Position of Flagella: Peritrichous – flagella are scattered all over the cell surface  BACTERIA GENERAL CHARACTERISTICS  Position of Flagella: Monotrichous – a flagellum is attached at one end of the cell  BACTERIA GENERAL CHARACTERISTICS Position of Flagella: Atrichous –bacterial cell with no flagellum  BACTERIA GENERAL CHARACTERISTICS  Position of Flagella:  BACTERIA COLONY MORPHOLOGY  Colony Size: Punctiform –very small dots Small – colonies with a diameter of 1-2 mm Large – colonies ranging over 8mm  BACTERIA COLONY MORPHOLOGY  Borders (the outer edge of the colony): circular - round colonies with even borders filamentous - fibrous looking, with stringy extensions irregular - extending unevenly from the center with asymmetrical borders loboid - border of colony appears to extend out in lobes rhizoid - border of the colony appears to extend out like tree roots spindle - colony is shaped long and thin like a spindle spreading or swarming - edges of the colony appear wavy  BACTERIA COLONY MORPHOLOGY  Borders (the outer edge of the colony):  BACTERIA COLONY MORPHOLOGY  Elevation (the height or depth of the colony compared to the plane of the agar): flat – colony seems even with the surface of the agar raised - colony appears as a mound on the agar umbonate - slightly raised in the center pitted or concave - depressed into the agar  BACTERIA COLONY MORPHOLOGY  Elevation (the height or depth of the colony compared to the plane of the agar):  BACTERIA COLONY MORPHOLOGY  Texture (the moistness or dryness): shiny or wet - colony appears shiny and moist mucoid - colony appears covered with mucous dry - colony appears dry and sometimes folded opaque - colony is solid, an object behind the colony can not be seen  BACTERIA COLONY MORPHOLOGY  Texture (the moistness or dryness):  BACTERIA COLONY MORPHOLOGY  Color: colored - orange, yellow, white, pink, black, red translucent - colony would appear clear, like glass, or a drop of water  BACTERIA COLONY MORPHOLOGY  Color:  BACTERIA COLONY MORPHOLOGY colored translucent  BACTERIA large, rhizoid, raised, opaque, white BACTERIA  UNIT 5 Description and Characterization of the Different Genera of Plant Pathogenic Bacteria KINGDOM PROKARYOTAE SUB-KINGDOM EUBACTERIA ARCHEABACTERIA 1. GRACILICUTES 1. MENDOSICUTES DIVISIONS 2. FIRMICUTES 3. TERINICUTES GRACILICUTES 1. OXYGENIX PHOTO BACTERA 2. ANOXYGENIC PHOTO BACTERIA 3. SCOTOBACTERIA CLASS FIRMICUTES 1. FIRMIBACTERIA 2. THALOBACTERIA TENERICUTES 1. MOLLICUTES  1. GRACILICUTES Gram-negative type cell wall (contains Gram - bacteria)]; thinned cell wall photobacteria 2. FIRMICUTES Firmacutes [Gram positive type cell wall (Gram + bacteria and actinomycetes)]; non-photosynthetic DIVISIONS 3. TERINICUTES Mollicutes (No cell wall; Gram Variable) 4. MENDOSICUTES Mendosicutes (No peptidoglycan on cell wall; contains class Archaeobacteria; uneven Gram stain)  Prokaryotic and smaller than bacteria Unlike bacteria, mollicutes have no cell walls but have a unit plasma membrane that is 9-12 nm thick which makes them pleomorphic and very sensitive to osmotic change. They contain both RNA and DNA. Pathogenic to plants, arthropods and other animals including man. Mollicutes are resistant to penicillin but are sensitive to tetracycline and chloramphenicol. Causes disease by blocking translocation in the phloem and interferes with plant’s hormonal balance. DIVISION GRACILICUTES CLASS CHARACTERISTICS Example Scotobacteria Non Phototrophic Pseudomonadaceae metabolism (cannot derive Enterobacteriaceae energy from light) Oxygenic Photo These are photosynthetic Cyanobacteria Bacteria and produce oxygen Anoxygenic These are photosynthetic Photobacteria but cannot produce oxygen  Archaebacteria Kingdom F I G. 1. T h e G r a n d P r i s m a t i c S p r i n g i n Yellowstone National Park. The brilliant colors observed in the spring are attributed to archaebacteria.  Archaebacteria Kingdom ❖ Discover how bacteria evolved to survive in Earth's most-extreme habitats, such as seafloor volcanic vents (300°C) ❖ Extremely thermophilic archaebacteria can live with carbon dioxide as their sole carbon source, obtaining energy from the oxidation of hydrogen by sulfur, producing hydrogen sulfide (H2S). Interesting facts about archaea: No archaean species can do photosynthesis. Archaea only reproduce asexually. Archaea show high levels of horizontal gene transfer between lineages. Many archaea live in extreme environments. Unlike bacteria, no archaea produce spores. Bacteria are susceptible to antibiotics; archaeans are not. Bacteria can cause illnesses; archaeans do not. Bacterial cell walls have peptidoglycan; archaean cell walls do not. Bacteria engage in both glycolysis and the Calvin cycle; archaea do not. According to the 8th Edition of Bergy’s Manual of Phytopathogenic Bacteria, bacteria are classified as: Division II: Scotobacteria (indifferent to light) Class I: The Bacteria Gram Negative, Aerobic rods and cocci. Family: Pseudomonaceae Genus: 1. Pseudomonas Genus: 2. Xanthomonas Family: Rhizobiaceae Genus: Agrobacterium Gram Negative, Facultative Anaerobic Rods Family: Enterobacteriaceae Genus: Erwinia According to the 8th Edition of Bergy’s Manual of Phytopathogenic Bacteria: Division II: Scotobacteria (indifferent to light) According to the 8th Edition of Bergy’s Manual Phytopathogenic bacteria are classified as: Division II: Scotobacteria (indifferent to light) Division II: Scotobacteria (indifferent to light) Class I: The Bacteria Gram negative aerobic rods and cocci Family: Pseudomonaceae Genus 1. Pseudomonas Genus 2. Xanthomonas Division II: Scotobacteria (indifferent to light) Class I: The Bacteria Gram negative aerobic rods and cocci. Family: Pseudomonaceae Family: Rhizobiaceae Genus: Agrobacterium Division II: Scotobacteria (indifferent to light) Class I: The Bacteria Gram negative aerobic rods and cocci. Family: Enterobacteriaceae Genus: Erwinia Gram Positive, Irregular Rods and Filamentous Bacteria a) Irregular Rods: Corynebacterium (Plant Pathogenic) Genera: 1. Curtobacterium Gram Positive, Irregular Rods and Filamentous Bacteria a) Irregular Rods: Corynebacterium (Plant Pathogenic) Genera: 2. Clavibacter Gram Positive, Irregular Rods and Filamentous Bacteria b) Filamentous Bacteria: Order: Actinomycetales Family: Streptomycetaceae Genus: Streptomyces  Important Phytopathogenic Bacteria Genus Characters Symptoms Example produced on host Pseudomonas Rod straight to curved, leaf spot 1. Brown rot or size 0.5-1 x 1.5-4 µm, blights bacterial wilt of one to many polar flagella, vascular wilts potato. non-spore forming, gram soft rots 2. Bacterial wilt of negative; strict aerobes ; canker solanaceous crops chemoorganotrophs; metabolism respiratory, never fermentative. Colonies not yellow, do not produce acid from lactose Xanthomonas Straight rods, size 0.4-1 x leaf spot 1. Citrus canker 1.2-3 µm. Gram fruit spots blight 2. Black arm of –negative , motile by a canker cotton single polar flagellum; non- spore forming; 3.Blight of paddy copious extracellular rice slime produced; colonies yellow due to Xanthomonadin, produce acid from lactose, strict aerobes; metabolism respiratory, never fermentative; oxidase negative and catalase positive Agrobacterium Rod shaped, size 0.8 x 1.5-3 µm, Crown Crown Gall of 1 to 4 peritrichous flagella, Gall stone fruit; roses colonies mostly white, do not hydrolyze starch; when only one flagellum is present, it is more often lateral than polar. When growing on carbohydrate – containing media the bacteria produce abundant polysaccharide slime. The colonies are non-pigmented and usually smooth. These bacteria are rhizosphere and soil inhabitants. Erwinia Straight rods, size 0.5-1, 0-3.0 µm , Fire blight 1. Fire blight several peritrichous flagella; Wilt of apple cells predominantly single, Soft rot 2. Soft rot of straight rods, measuring 0.5 to 1.0 vegetables x 1.0 to 3.0 microns; motile (except E. stewartii and E. dissolves) by peritrichous flagella; gram negative ; grows well on artificial media aerobically as well as anaerobically and produce acid. The genus has three major groups of species. i) Pectolytic bacteria that cause soft rot. ii) Those do not cause soft rot but dry necrosis and wilt and iii) Epiphytes that cause neither soft rot nor necrosis. Clavibacter Straight to straightly curved Wilt Wilt of (Corynebacterium) rods, 0.5-0.9 x 1.5-4.0 µm, potato and non motile but some Tomato species are motile by one or two polar flagella. The cells are Gram positive; non ovoid, fast plemorphic rods, often arranged at an angle to give V-formation as a result of snapping or bending type of cells division, no coccoid cells are seen; non- endospore forming; non- motile; strict aerobes; nutritionally exacting; nitrate not reduced; cell wall peptidoglycan contains diaminobutyric acid; G+C content of the DNA is 70 +-5 moles per cent. Streptomyces Slender branched hyphae Scab Potato without cross walls scab (coenocytic) 0.5-2 µm in diameter; aerial mycelium at maturity forms chains of three to many spores; cell wall contain diaminopimelic acid; Gram Positive; aerobic; heterotrophs; generally reduce nitrates, on isolation colonies are small, 1 to 10 mm in diameter, discrete and lichenoid , leathery or butyrous, initially relatively smooth but later develop a weft of aerial mycelium that may appear granular, ‘Top 10’ Plant Pathogenic Bacteria based on scientific/economic importance: