ACP601: Plant Mycology and Bacteriology Plant Bacteriology · 2020. 11. 25. · Bacterial streaming...
Transcript of ACP601: Plant Mycology and Bacteriology Plant Bacteriology · 2020. 11. 25. · Bacterial streaming...
James W. Muthomi
Department of Plant Science and Crop Protection
University of Nairobi
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
ACP601: Plant Mycology and Bacteriology
Plant Bacteriology
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Bacterial blight of cotton
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Bacterial blight of cassava (Xanthomonas axonopodis pv. manihotis)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Bacterial soft rot on pepper and carrot (Erwinia carotovora subsp. carotovora)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Angular leaf spot on cucumber (Pseudomonas lacrymans)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Banana Xanthomonas wilt (Xanthomonas campestris pv musacearum)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Halo blight of bean (Pseudomonas savastanoi pv phaseolicola)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Common bacterial blight of bean (Xanthomonas axonopodis pv phaseoli)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Black rot of cabbage: http://www.extension.umn.edu/distribution/horticulture/DG1169.html;
http://www.infonet-biovision.org/default/images/105/pests
Bacterial wilt of potato: http://www.infonet-biovision.org/default/ct/79/pests
https://research.cip.cgiar.org/confluence/display/SET/Bacterial+Wilt+Protocol Bacterial wilt of potato and tomato (Ralstonia solanacearum)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Black rot of cabbage: http://www.extension.umn.edu/distribution/horticulture/DG1169.html;
http://www.infonet-biovision.org/default/images/105/pests
Bacterial wilt of potato: http://www.infonet-biovision.org/default/ct/79/pests
https://research.cip.cgiar.org/confluence/display/SET/Bacterial+Wilt+Protocol
(Xanthomonas campestris pv campestris)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Bacterial canker of tomato (Clavibacter michiganense subsp. michiganense)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Bacterial spot on tomato and pepper (Xanthomonas campestris pv. vesicatoria)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Crown gall of roses (Agrobacterium tumefasciens)
Citrus canker
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Bacterial Diseases of Plant in East Africa
Citrus greening disease (Candidatus liberobacter)
Morphology of Bacteria
Cellular structure s
Arrangement of cells
Types flagellation
Differences between Gram positive and Gram
negative bacteria
Examples of plant pathogenic bacteria
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Morphology of Bacteria
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Morphology of Bacteria
Gram positive genera Gram negative genera
Clavibacter Acidovorax
Curtobacterium Agrobacterium
Rathayibacter Burkholderia
Streptomyces Enterobacter
Erwinia
Pantoea
Pseudomonas
Ralstonia
Xanthomonas
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Plant Pathogenic Bacteria
Bacterial growth curve and calculation of generation
time
Measurement of bacterial growth
o Use of haemocytometer
o Plate counting techniques (use of serial dilution)
o Measurement of turbidity (spectrophotometer)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Growth and Reproduction in Bacteria
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Isolation of Bacteria
Sources of bacterial disease inoculum
o Primary sources – seed, planting materials, water, soil, crop debris, volunteer crops, weeds, insect vectors
o Secondary sources – human activities, rain splash
Dissemination of bacterial diseases
Entry into host plants
o Natural openings – stomata, lenticels, nectaries, hydathodes
o Wounds
Survival
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Epidemiology of Bacterial diseases
Extracellular polysaccharides
Pectic enzymes
Toxins
Plant growth regulators
Others
o Necrosis
o Ammonia accumulation
o Ice-nucleating proteins
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Mechanisms of infection in Bacterial diseases
Bacterial spots and blights
Bacterial soft rots
Bacterial vascular wilts
Bacterial cankers
Bacterial galls
Bacterial scabs
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Types of diseases caused by phytopathogenic bacteria
Preliminary diagnosis o Symptoms o Signs of bacteria – streaming, ooze
Isolation and cultural characteristics Gram staining Flagellation and mortility Physiological and biochemical tests
o Oxidative/ fermentative tests o Levan production o Utilization of nitrogen compounds o Catalyse test o Starch hydrolysis o Salt tolerance
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
Water soaking
bacterial "ooze" or exudate seen coming out of water soaked lesions (see arrows). The "ooze" forms in the readily seen droplets. These droplets are a sign of the pathogen, being composed mostly of bacterial cells
Bacterial ooze
Black rot of cabbage: http://www.extension.umn.edu/distribution/horticulture/DG1169.html;
http://www.infonet-biovision.org/default/images/105/pests
Bacterial wilt of potato: http://www.infonet-biovision.org/default/ct/79/pests
https://research.cip.cgiar.org/confluence/display/SET/Bacterial+Wilt+Protocol
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
Bacterial streaming test – diagnosis of bacterial wilt
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
To properly diagnose 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. This only occurs with bacterial wilt and not with any other type of pathogen or abiotic cause
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
Isolation of bacteria
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
Cultural characteristics
Colony morphology
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
Colony morphology
KOH solubility test for Gram stain
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
Gram stain test
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
Cell walls of Gram positive and Gram negative bacteria
Biochemical Tests
Biochemical tests are used to identify bacteria to genus level
i. Oxidation Test
ii. Gas Production
iii. Starch hydrolysis
iv. Salt tolerance
v. Catalyse test
vi. Nitrate reduction test
vii. Urease Activity
viii. Gelatin hydrolysis
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
Oxidation / fermentation Test
o In presence of oxygen, glucose is oxidized; in absence of
oxygen glucose is fermented.
o Oxidative (aerobic) bacteria metabolize glucose only in
presence of oxygen to produce acid, resulting in pH change
o Fermentative (anaerobic) bacteria metabolize glucose both in
presence or absence of oxygen.
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – Biochemical tests
Gas Production
oBacteria metabolize carbohydrates to produce either acid alone, or acid together with gas.
oGas production is tested by growing bacteria in a liquid medium to which a carbohydrate (glucose, sucrose, fructose, xylose, arabinose, mannital, sorbitol) has been added. A small tube is inverted into the medium.
oIf the bacterium utilizes the carbohydrate & produces a gas, the gas will collect in the small inverted tube
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – Biochemical tests
Tetrazolium salts tolerance test
o certain bacteria are sensitive to tetrazolium salts.
o To carry out the test, bacteria is spot inoculated onto a medium containing 0.02% and 0.1% triphenyl tetrazolium chloride (TTC) and incubated.
o Tolerance to tetrazolium salt will be indicated by presence of growth.
Salt tolerance test
o bacteria is inoculated into nutrient broth containing required sodium chloride concentration.
o Bacterial growth, seen as turbidity, which signifies sodium chloride tolerance.
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – Biochemical tests
Gelatin hydrolysis o The bacterium is grown by making a
single streak in the middle of a solid medium containing gelatin.
o Gelatin hydrolysis may be detected by a clear halo around colonies after addition of 10% potassium dichromate in hydrochloric acid.
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – Biochemical tests
Nitrate reduction test o Some oxidative bacteria have the ability
to grow in anaerobic conditions rich in nitrate ions.
o Nitrate medium is stab inoculated with bacteria and incubated fro 3-5 days until good growth has been obtained.
o An equal volume of Follet and Ratcliff’s reagent is added and shaken well.
o Development of an orange or orange–brown colour is a positive reaction, indicating the presence of nitrite.
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – Biochemical tests
Starch hydrolysis o A single streak of test bacterium is inoculated
at the middle of starch-containing medium.
o Incubate for 7 days, after which the plate is flooded with lugoli’s iodine.
o Starch hydrolysis is indicated by the presence of clear zone in the black-stained medium around or under the colonies.
o Reddish zones indicate that starch has been partially hydrolyzed to dextrins, and is usually considered a negative reaction.
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – Biochemical tests
Urea test
Utilization of urea o Bacterium is inoculated into urea-
containing medium and incubated for up to 7 days.
o Urease activity is indicated by the development of a pink-red colouration.
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – Biochemical tests
Catalyse test oMany oxidative (aerobic) bacteria
produce enzyme catalyse that helps to protect them from toxic oxygen compounds oCatalyze regulates release of molecular
oxygen from peroxides e.g. hydrogen peroxide.
Test oA few drops of 3% hydrogen peroxide
solution are placed onto a bacterial culture. oA positive result is indicated by the
generation of oxygen bubbles (effervescence)
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – Biochemical tests
Serology – ELISA Fatty acid analysis PCR-based analysis (molecular markers) Determination of pathogenicity
o Inoculation of detached fruits o Stem inoculation for canker and gall inducing
pathogens o Leaf inoculation for leaf spot pathogens o Cotyledon inoculation o Root inoculation o Inoculation of storage organs for soft rot bacteria
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases
Fatty acid analysis o Bacterial cell membranes contain many different fatty acids o Types and relative proportions of these are unique to a particular
species. o Fatty acids are extracted & analyzed by gas chromatography and the
fatty acid profiles are used to identify the bacteria genus, species, and, in some cases, to strain level
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants
Gas Chromatogram fatty acid profiles
DNA – based analysis
o Methods are based on gene sequences in the DNA of the
bacterial chromosome.
o Gene sequence is unique to a particular species or strain.
o The steps in DNA analysis include:
i. Bacterial DNA is first extracted from the cell
ii. The extracted DNA is amplified by polymerase chain reaction
(PCR),
iii. The amplified DNA fragments are separated by
electrophoresis on agarose gel to produce finger prints (DNA
profiles).
iv. Based on the banding pattern produced on the DNA profiles,
the bacteria are identified by comparison to profiles of known
DNA
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants
Nucleic acid-based analysis PCR analysis of bacteria
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – PCR analysis
o A specific antibody is conjugated to polystyrene wells and a
suspension of the test bacterium is placed in the well and
allowed to react with the antibody.
o A positive reaction is indicated by colour change, which can be
detected by eye or measured by spectrophotometer (ELISA
reader).
Antigen-antibody reaction ELISA plate
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of bacterial diseases of plants – Serology (ELISA)
Pathogenicity test
Isolated bacteria
Inoculate on germinated bean cotyledons
Water soaking sympom
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Diagnosis of Bacterial Diseases of Plants – Host inoculation tests
Sanitation
Adjusting fertilization and watering
Crop rotation
Use of resistant varieties
Soil sterilization
use of clean seed and planting materials
Use of chemicals
Use of biological control agents
ACP601: Plant Bacteriology Dr. J. W. Muthomi Dept of Plant Science & Crop Protection, University of Nairobi
Management of bacterial diseases