ACP601: Plant Mycology and Bacteriology Plant Bacteriology · 2020. 11. 25. · Bacterial streaming test – diagnosis of bacterial wilt ACP601: Plant Bacteriology Dr. J. W. Muthomi

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


Bacterial Diseases of Plant in East Africa

Bacterial blight of cotton



Bacterial blight of cassava (Xanthomonas axonopodis pv. manihotis)



Bacterial soft rot on pepper and carrot (Erwinia carotovora subsp. carotovora)

http://www.omafra.gov.on.ca/IPM/images/peppers/diseases/bracterial_soft_rot/peppers_bacterial_soft_rot_green_zoom.jpg



Angular leaf spot on cucumber (Pseudomonas lacrymans)



Banana Xanthomonas wilt (Xanthomonas campestris pv musacearum)



Halo blight of bean (Pseudomonas savastanoi pv phaseolicola)



Common bacterial blight of bean (Xanthomonas axonopodis pv phaseoli)



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)






https://research.cip.cgiar.org/confluence/display/SET/Bacterial+Wilt+Protocol

(Xanthomonas campestris pv campestris)



Bacterial canker of tomato (Clavibacter michiganense subsp. michiganense)



Bacterial spot on tomato and pepper (Xanthomonas campestris pv. vesicatoria)



Crown gall of roses (Agrobacterium tumefasciens)

Citrus canker





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





Gram positive genera Gram negative genera

Clavibacter Acidovorax

Curtobacterium Agrobacterium

Rathayibacter Burkholderia

Streptomyces Enterobacter

Erwinia

Pantoea

Pseudomonas

Ralstonia

Xanthomonas


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)


Growth and Reproduction in Bacteria


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


Epidemiology of Bacterial diseases

Extracellular polysaccharides

Pectic enzymes

Toxins

Plant growth regulators

Others

o Necrosis

o Ammonia accumulation

o Ice-nucleating proteins


Mechanisms of infection in Bacterial diseases

Bacterial spots and blights

Bacterial soft rots

Bacterial vascular wilts

Bacterial cankers

Bacterial galls

Bacterial scabs


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


Diagnosis of bacterial diseases of plants


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




https://research.cip.cgiar.org/confluence/display/SET/Bacterial+Wilt+Protocol



Bacterial streaming test – diagnosis of bacterial wilt



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





Isolation of bacteria



Cultural characteristics

Colony morphology



Colony morphology

KOH solubility test for Gram stain



Gram stain test



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



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.


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



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.



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.



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.



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.



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.



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)



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


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





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



Nucleic acid-based analysis PCR analysis of bacteria


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


Diagnosis of bacterial diseases of plants – Serology (ELISA)

Pathogenicity test

Isolated bacteria

Inoculate on germinated bean cotyledons

Water soaking sympom


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


Management of bacterial diseases

Documents

ACP601: Plant Mycology and Bacteriology Plant Bacteriology · 2020. 11. 25. · Bacterial streaming test – diagnosis of bacterial wilt ACP601: Plant Bacteriology Dr. J. W. Muthomi