Jimma University Plant Pathology MSc Program History

Jimma University
College of Agriculture & Veterinary Medicine
JUCAVM is a Pioneer Agricultural Higher Learning Institute in Ethiopia. Slide 1
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Plant Pathology MSc Program
Plant Bacteriology (PLPA 531)

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Plant Bacteriology

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INTRODUCTION
History of Plant Pathogenic Bacteria
Progress of microbiology depended on invention of new:
– Instruments
– Methods
– Techniques

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JUCAVM is a Pioneer Agricultural Higher Learning Institute in Ethiopia.
INTRO….
Visualization of bacteria (microbes)
 Human eye has resolution power of 150 µm &
 every thing under the size of 150 µm is said “micro”
 e.g. micro meter (µm) and microorganims

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Bacteria:
 0.1 to 1 µm in thickness
 0.3 to 2 µm in diameter and
 several µm in length
To discover/observe bacteria:
 Human eye should be augmented

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Thus the invention of microscope by
 Hans and Zaccharias Janssen (1655)
– With low resolution power of 20-10 µm
– This does not help for bacteria but fungal hypahe
(Mucor) were discovered by Robert Hook in 1665

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 To visualize still smaller organisms like bacteria improved
microscope lenses were necessary
 Antonie van Leeuwenhoek built the first lens for this
purpose and discovered protozoa in 1674
 The highest resolution of this microscope was 2 µm

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 Modern microscopes have a resolution power of 0.2
µm
 with contrasting techniques such as the dark fields
objects of diameter down to 20 nm, like bacterial
flagella can be distinguished

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Sterilization methods and sterile techniques:
Spallanzani (1765)
 boiling infusions for 45 minutes demonstrated that
liquids treated by heat and sealed did not decay
 No microorganisms could be found inside the vessels
after incubation

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Almost after 70 years:
Schultze (1836):
 proved that air passed through sulphuric acid could not produce
putrefaction
 demonstrated that organisms living in air were responsible for the
process
Schroder and Dusch (1853):
 Untreated air would not cause decay in boiled meat extracts if the
bottles were stoppered with cotton plugs

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Louis Pasteur (1861):
 Developed sterilization by heat, but his experiments were
not always reproducible
(cause of the failures were resistance of bacterial spores to
boiling point temperature)
A break through in sterilization was made by Robert Koch
who utilized pressurized steam in an autoclave and thus
reaching temperatures above 100°C (121°C)
This is another pillar of microbiology

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In 1876 also Robert Koch succeeded in proving the cause
of ‘anthracnose’ is the bacterium baccillus anthracis
This was by applying Koch’ postulates

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Robert Koch (1881) was the first to solidify culture
media by adding gelatin and was able to isolate and
count bacteria on plate
Wife of Hesse in 1882 proposed exchange of gelatin
with agar
Chamberland (1884):
 found that procelain filters could withhold bacteria
 This was the starting point of sterilizing liquids
without heat by sterile filtration

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General Characteristics of
Phytopathogenic Bacteria
 One bacterial cell is an
independent organism
– Feeds and respires
– Multiplies, inhabits certain
environment

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Morphology
 Most plant pathogenic bacteria are
rod shaped exception being
Streptomyces, which is
filamentous
 Most have delicate, threadlike
flagella, considerably longer than
the cells on which they are
produced
(A) Agrobacterium, (B) Erwinia, (C)
Pseudomonas, and (D) Xanthomonas.

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Branched filaments (hyphae) of
Streptomyces scabiei, causing
potato scab, in Gram stain
14-day-old colonies of S. scabiei on
yeast-malt agar. Active spore formation
in aerial hyphae that turn into a dusty
grey mass

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Position of flagella:
 One flagellum
 tuft of flagella at one end
 distributed over the entire
surface of the cell

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Streptomyces species,
 cells consist of branched threads, which usually have a
spiral formation and produce conidia in chains on aerial
hyphaebranched threads, which usually have a spiral
formation and produce conidia in chains on aerial hyphae

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Structure of bacterial cell
Genome - carrier of hereditary
information – DNA
Genome of bacteria- single round
(ring) molecule of double-
stranded DNA
- Nucleoid - DNA + proteins
- Plasmid - separate small
DNA ring, additional
information, facultative

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Components of bacterial cell
Capsule or slime: cell walls of bacteria of most species
are enveloped by a viscous, gummy material, which, if
thin and diffuse, is called a slime layer, but if thick,
forming a definitive mass around the cell, is called a
capsule.
 Missing in some bacteria

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Cytoplasm membrane:
 50%lipids, 50% proteins
 Phospholipid double layer
 Pheripheric and integral proteins
 Assistant-lipids
Cell wall:
 Different in Gram(+) and Gram(-) bacteria

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Functions of cell membrane
 Selective permeability
 Secretion of enzymes
– Gram(+) bacteria: to environment
– Gram(-) bacteria: to periplasmatic space

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Cell wall
 Between cell membrane and capsule
 Rigid
 Different in Gram(+) and Gram(-) bacteria
– Gram(+) thick, one layer
– Gram(-) thin but several layers

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Capsule -
• extracellular mucous polymere-polysaccharide,
tightly surrounds the cell
• Tasks:
. defends
. helps to attach

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Other components of bacterial cell:
 Pili, fimbria ø 3-8 nm, pilin
 Flagella ø 15-20 nm, flagellin
 Spores

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Pili
-for attachment
- For changing of DNA b/n
bacteria (sex of bacteria)

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Flagella
- Long spiral protein
chains-flagellin
- For motility

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Spores:
- For survival of bacteria in
bad environmental
condotions
- Very strong and
dehydrated
Only two genus:
- Bacillus sp.
- Clostridium sp.

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Colonies of different species may vary from
 a fraction of a millimeter to several centimeters in
diameter and
 may be circular, oval, or irregular
 edges may be smooth, wavy, or angular, and their
elevation may be flat, raised, or wrinkled.
 Colonies of most species are whitish or grayish, but
some are yellow
 Some produce diffusible pigments into the agar that may
be fluorescent with ultraviolet light

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Reproduction
Bacteria multiply by dividing
 DNA replication
 inward growth of the cytoplasmic membrane
toward the center of the cell, forming a trans-
verse membranous partition
 two layers of cell wall material, continuous
with the outer cell wall, are then synthesized
between the two layers of the membrane.
 Separation of the two layers splitting the two
cells apart
NB. Bacteria reproduce at an astonishingly rapid
rate

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Fig.

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Multiplication of bacteria

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 Under favorable conditions,
– they divide every 20 to 50 minutes, one bacterium
becoming 2, two becoming 4, four becoming 8, and so
on. At this rate, one bacterium could produce 1 million
progeny bacteria in less than a day.
 However, because of the diminution of the food supply,
accumulation of metabolic wastes, and other limiting
factors,
– reproduction slows and may finally come to a stop.

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Ecology and spread
Almost all plant pathogenic bacteria develop mostly:
 in the host plant as parasites,
 on the plant surface, especially buds as epiphytes,
 partly in plant debris or in the soil as saprophytes.

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There are great differences among species in the degree
of their development in one or the other environment.
 Some bacterial pathogens, such as Erwinia amylovora
(fire blight of pear) produce their populations in the
plant host, while in the soil their numbers decline
rapidly and usually do not contribute to the
propagation of the disease from season to season.

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 These pathogens have developed sustained plant-to
plant infection cycles, often via insect vectors, and, either
because of the perennial nature of the host or the
association of the bacteria with its vegetative propagating
organs or seed, they have lost the ability to survive in the
soil.

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Some other bacterial pathogens, such as:
 Agrobacterium tumefaciens - crown gall,
 Ralstonia solanacearum - bacterial wilt of solanaceous
crops, and particularly
 Streptomyces scabies -the common scab of potato, are
rather typical soil inhabitants.

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Soil inhabitants
 build up their populations within the host plants, but these
populations only gradually decline when they are
released into the soil
 If susceptible hosts are grown in such soil in successive
years, sufficiently high numbers of bacteria could be
present to cause a net increase of bacterial populations
in the soil from season to season.

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 Most plant pathogenic bacteria, however, can be
considered soil invaders.
 Such bacteria enter the soil in host tissue and, because
they have poor ability to compete as saprophytes, persist
in the soil either as long as the host tissue resists
decomposition by saprophytes or for varying durations
afterward, depending on the bacterial species and on the
soil temperature and moisture conditions.

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 When in soil, bacteria live mostly on plant material. Less
often they live freely or saprophytically, or in their natural
bacterial ooze, which protects them from various adverse
factors
 Bacteria may also survive in or on seeds, other plant
parts, or insects found in the soil.
 On plants, bacteria often survive epiphytically, in buds,
on wounds, in their exudate, or inside the various tissues
or organs that they infect

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The dissemination of plant pathogenic bacteria from one
plant to another or to other parts of the same plant is
carried out primarily by:
 water,
 insects,
 other animals,
 and humans
Even bacteria possessing flagella can move only very short
distances on their own power.

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TAXONOMY OF PLANT PATHOGENIC
BACTERIA
Taxonomy (= systematics) is the process of:
 classification,
 the application of names (nomenclature),
 and the practice of identification.

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1. Classification
 systematic arrangement of organisms into groups based
on shared characteristics or evolutionary relatedness
 Ordering of organisms into groups (taxa)
 Systematic grouping of microorganisms by certain
features
 It changes by time

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Classification can be:
– Phenotypic
– Genotypic
– analytical

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Phenotypic classification
 is based upon over all similarity
 Morphological, physiological, biochemical, serological
features are compared
 Developed in 1960’s, and was prevalent up to 1980’s

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Features or attributes for phenotypic classification:
 Quantitative – measured or counted
– Morphology in microscope
– Morphology of colony
 Qualitative – shape, color, etc. some feature present or
missing
– Biochemical activity
– Serotyping, etc

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Genotypic classification - is
based up on evolutionary
relationship
 Most precise method
 Started with the development of
molecular methods and genetic
studies
 Genetic taxonomy/nucleotide
composition are used to
determine similarity
 GC index
 DNA hyberdization
 Nucleic acid sequence
analysis
 Analysis of chromosomal DNA
fragements
 Plasmid analysis

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Analytical classification –
based upon detection of
structural components and
metabolic products using
chemical methods.
– Cell wall fatty acids
– Whole cell lipids
– Whole cell proteins
– enzymes
 Chromatography
 Mass spectrometry
 Enzyme
electrophoresis

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Bergey’s Manual of
Systematic Bacteriology
= the standard taxonomic reference on bacteria;
most widely accepted and used system
 Manual of Determinative Bacteriology
. 1923, . , 1994
. not based on phylogeny! (does not require molecular
testing)
. based on cell wall type (differential staining), oxygen
requirements, morphology, motility, biochemical
testing
 Manual of Systematic Bacteriology
. 1984, 2001, 2005
. based on phylogeny - requires molecular testing

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2. Nomenclature
 branch of taxonomy concerned with assignment of
names to taxonomic groups (the naming of
(micro)organisms)
 Binomial nomenclature
– Genus name + species name
– Captalize genus
– Underline or itlicize both the genus and species
e.g. Ralstonia solanacearum
 as the taxonomy of bacteria evolves, changes in
nomenclature will reflect this evolution

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 Rules for the assignment of names to bacteria are
established by the International Code of
Nemonclature of Bacteria
 When exact naming has to be mentioned:
– Author(s) giving the first description with prefix, and those who
made the last taxonomic classification following together with
the year of publication
e.g. Xanthomonas axonopoids pv. citri ( Hasse 1915) Vauterin,
Kersters and Swings 1995

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3. Identification of bacteria
 Process of determining the specific identity of an
isolate
 Placement of new strain into a previously described
group
It is a routine work in microbiology lab;
– Usually starts from getting pure culture of new microorganism
– Investigation of properties follows
 Morphological (shape, size, color, arrangement of flagella)
 Biochemical, and/or
 Serological,
 Sometimes molecular

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 Colonies of bacteria on
solid medium in Petri
dish

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 Investigation of the
biochemical properties
of bacteria using
different test tubes

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Chemical composition
 The chemical compositions of certain substances
in bacterial cells can be detected with specific
staining techniques.
 Information about the presence or absence of
such substances is used for the identification of
bacteria.
 Gram’s staining reaction differentiates bacteria
into gram-positive and gram-negative types.

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In this reaction,
 bacteria fixed on a glass slide
 are treated with a crystal violet solution for 30 seconds,
 rinsed gently,
 treated with iodine solution,
 and rinsed again with water and then alcohol.
Gram-positive bacteria retain the violet-iodine stain combination
because it forms a complex with certain components of their cell wall
and cytoplasm.
Gram-negative bacteria have no affinity for the stain combination, which
is therefore removed by the alcohol rinse, and bacteria remain as
nearly invisible as before.

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Ability to use nutrients
 Bacteria are also distinguished by the substances that
they can or cannot use for food and by the kinds of
enzymes produced when the bacteria are grown on
certain media.
 Over a hundred characteristics of a bacterium can be
determined by these tests, and the profiles for each
bacterium are often used in numerical taxonomy of
bacteria.

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Pathogenicity test
 Phytopathogenic bacteria are also tested for their
pathogenicity on various species and varieties
of host plants. This test, for practical purposes,
may be sufficient for tentative identification of the
bacterium.

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Using selective media
 An excellent method of isolation and identification of bacteria
obtained from plant tissues or soil is through the use of selective
nutrient media.
 Selective media contain nutrients that promote the growth of a
particular type of bacterium while at the same time contain
substances that inhibit the growth of other types of bacteria.
 Positive identification usually requires more than one subculturing on
selective media because seldom does only one bacterium grow on a
selective medium.

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 Positive identification usually requires more than one
sub-culturing on selective media because seldom does
only one bacterium grow on a selective medium
 The available selective media for plant pathogenic
bacteria are helpful for routine isolation and sometimes
identification of bacterial genera and of several species
and even pathovars.

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Fatty acid profile analysis
 fairly quick distinction and identification of bacterial
genera, species, and, in some cases, lower
subdivisions have been made by extraction and
comparison of the fatty acids present in the bacterial
cell membranes (fatty acid profile analysis).
 The same bacteria grown under identical conditions
also produce identical membrane proteins and
identical enzymes and isoenzymes.
 Isolation and comparison of such structural proteins or
enzymes are also used to identify bacteria

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DNA based methods
Polymerase chain reaction (PCR)
 A specific part of nucleic acid of a bacterium is artificially multiplied
by repeated cycles of
– Denaturation – melting of nucleic acid,ususally at 95C
– Annealing – of short strands of nucleic acids (primers) with known
sequence
– Extension – of nucleic acid in the presence of polymerase (Taq
polymerase)

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Other DNA based identification methods:
 Real-Time PCR
 Restriction fragment length polymorphism (RFLP)
analysis
 Random amplified polymorphic DNA (RAPD)
 Amplified fragment length polymorphisms (AFLP)

Jimma University Plant Pathology MSc Program History

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Jimma University Plant Pathology MSc Program History