1.
Identification and classification of plant pathogenic
bacteria
Successful control of any plant pathogenic disease depend up on the
availability of quick accurate methods of diagnosing and identification

2.
Identification and classification of plant pathogenic bacteria
 A simple diagnosis may require only symptom recognition and a rapid test to
confirm identity of a known bacterial plant pathogen
 A knowledge of Symptom is a prerequisite for successful isolation and
identification
 Enable to understand disease development in susceptible and resistant plants

3.
Identification by Symptoms
 Excellent genetical or molecular biological research is mistakenly based on atypical
symptoms = No useful conclusion
 Therefore, it is important to stress some fundamental aspects of diversity of
symptoms
 Typical bacterial disease symptoms appear during early phase disease development
 Early phase of disease development = easy isolation of the pathogen
 General characteristics PPB = attacked tissues are water soaked=
Bacterial polysaccharides
 Later stage of symptoms similar to other fungi or virus

4.
Types of Symptoms caused by Bacteria: Cause various symptoms
= five groups
Types of diseases Invaded tissue Kind of symptoms Pathogen
Leaf spot Invasion of inercellualry space and
necrotization of parenchymatous tissues
Water soaked spots Bacteria exudates
or ooze Angular leaf spot halo
blight chlorosis of young leaves
leaf blight fruit spots
Pseudomonas, Xanthomonads
Canker and die back Invasion of bark and woody tissues through
wounds , buds and leaf scars, young
leaves and blossoms
Bark necrosis canker, gummosis, die
back (apopelexy) shoot blight
shot hole on leaves bud blast
bark necrosis canker, bacterial
ooze die of blossom and twig
blight
Pseudomonas syringae Pv syringae
group
Erwina amylovora
Vascular wilt Invasion of vascular elements Wilting dwarfing browing of vascular
tissue ring rot of potato Bird’s
eye spots on tomato fruits
Coryneform bacteria, R.
solanacerum a few
xanthomonads
Soft rot diseases Maceration of middle lamella and primary
cell wall
Soft rot of tubers and onions and
others, Black leg disease
Soft rotting erwinias afew
pseudomonas
Proliferation and
tumer
Stimulation of plant cells and tissues to grow
abnormal
Agrobacteria, R. fascians, P.
syringae pv savastanoi

5.
Identification and classification of plant pathogenic
bacteria
Diagnosis of truly unknown pathogens requires
 Field observation,
 Examination of plant tissues,
 Isolation of the pathogen,
 Characterization, and
 Proof of Koch’s postulates

6.
Identification and classification of plant pathogenic bacteria
Robert Koch (1843–1910). Based on his experiences, in 1887, Koch set out the four
steps or criteria that must be satisfied before a microorganism isolated from a
diseased human, animal, or plant can be considered as the cause of the
disease.
These four steps, rules, or criteria are known as “Koch’s postulates”
1. The suspected causal agent (bacterium or other microorganism)
must be present in every diseased organism (e.g., a plant)
examined.
2. The suspected causal agent (bacterium, etc.) must be isolated from
the diseased host organism (plant) and grown in pure culture.
3. When a pure culture of the suspected causal agent is inoculated into
a healthy susceptible host (plant), the host must reproduce the
specific disease.
4. The same causal agent must be recovered again from the
experimentally inoculated and infected host, i.e., the recovered
agent must have the same characteristics as the organism in step 2.

7.
Identification and classification of plant pathogenic bacteria
 The constant presence of large numbers of bacteria in the affected
area, and the absence of any other pathogens = the causative agent
 However, care must be taken to exclude the possibility that the
observed bacteria are secondary saprophytes, i.e., bacteria that are
growing in tissue killed by some other cause
 Selective media are available for the selective cultivation of almost
all plant pathogenic bacteria free of common saprophytes so that the
genus and even some species can be identified
 The easiest and surest way to prove that the observed bacterium is
the pathogen is through isolation and growth of the bacterium in
pure culture and, using a single colony for re-inoculation of a
susceptible host plant, reproducing the symptoms of the disease
and comparing them with those produced by known species of
bacteria

8.
Isolation of Bacterial Pathogen

9.
Schematic isolation and culturing Plant Pathogenic Bacteria

10.
Identification and classification of plant pathogenic bacteria
Recommended steps for identifying a disease
Identify the diseased plant
 Examine symptoms both macro and microscopically
Under take literature search for pathogens occurring in a particular country
Compare disease symptoms with symptoms described in the literature
Select isolation media
Isolate
Incubate at appropriate temperature
Examine agar plates with bacterial growth
Select suspect colonies
Purify selected colonies
Perform required test (s)
Perform pathogenicity test

11.
Identification and classification of plant pathogenic bacteria

12.
Identification and classification of plant pathogenic bacteria
 Many bacterial diseases can be diagnosed quickly and efficiently using
established methods and materials that are already widely available
Hence, Identification of Plant Pathogenic bacteria involves
 Observation and comparison of visible characteristics such as size,
shape, structure, and color
 Obscure properties as chemical composition,
 Serological reactions
 Ability to use certain nutrients = Physiological properties,
 Enzymatic action
 Pathogenicity to plants
 Growth on selective media: For isolation and culture of PPB there exist
different general purpose and semi selective media. For instance see
the followings lists.

13.
General Diagonistic ad selective media

14.
General Diagnostic ad selective media

15.
Serological Test
Agglutination
 Slide agglutination
 Tube agglutination
 Latex agglutination
Precipitation in Agar
 Piazzi test
 Oucherlony double diffusion
Immuno-dot blot
Nitrocellulose membrane
DAS ELISA

16.
Physiological criteria
 Acetion (Methyle Red Test)= Erwina
 Amino peptidase activitiy = All pathogens
 Arginine dihyrolase- Pseudomonase
 Carbon Source utilization= All Genera
 Catalase= All Genera
 Crystal Violet Pectate= Erwina and Pseudomonas
 Egg yolk agar
 Erythromycin senstivity = Erwina
 Esculin Hydrolsis= Xanthomonas
 Easrerase activity
 Flourescent=Pseudomonas
 Gelatine Hydrolysis Pseudomonas= Xanthomonas and Erwina
 Gram stain= All Genea
 Hyrogen sulphide production from from cysteine= Erwina and Xanthomonas
 Indigoidine (Blue)= Erwina and Clavibacter
 Indol production= Erwina
 2 keto Gluconate Production= pseudomonas and Erwina
 Kelactose Production= Agrobacterium tumefaciens

17.
Physiological criteria..continued
 Levan Formation= Pseudomonas and Agrobacterium
 Milk Proteolysis= Xanthomonas
 Miniatured biochemical procedures= Erwina
 Nitrate reducation= Pseudomonas
 Oxidase test= pseudomonas and Agro bacterium
 Oxidation/Fermentation Test= All genera
 Phenylalanine deaminase = Erwina
 Phosphotatase Activity=Erwina
 Poly BetaHydroxybuttrate inclusions= Pseudomonas and Agrobacerium
 Potassium hydroxide solubility test= All Genera
 Potato soft rot= Erwina and Pseudomonas
 Reducing Substances from sucrose=Erwina
 Sodium chloride tolerance= All Genera
 Temperature Requirement= All Genera
 TTC agar= Ralstonia solanacerum
 Tyrosinase= Streptomyces
 Urease Production =Erwina

18.
Identification and classification of plant pathogenic bacteria
 The chemical compositions of certain substances in bacterial cells can be
detected with specific staining techniques
 Gram’s staining reaction differentiates bacteria into gram-positive and
gram-negative types
 Gram staining reaction involves
1. Fixing bacteria cell on a glass slide with a crystal violet solution
2. Gentle rinsing gently and treatment with iodine solution,
3. Rinsing again with water and treating with alcohol.
 Gram-positive bacteria retain the violet-iodine stain combination
 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.

19.
Gram Negative bacteria

20.
Identification and classification of plant pathogenic bacteria
 KOK (Test)= is an alternative to Gram staining procedure to identify Gram
negative or positive bacteria.
 The procedure is
 Grow the test bacteria is pure culture
 Mix loopful of bacteria with 2 drops of 3% KOH
 Gram –Ve = Gummy uponmixing
 Gram +Ve = Non gummy
 If questionable use Gram stain

21.
Differentiation of Major Genera
Character
Erwina
Pantoea
Acidovorax
Pseudomonas
Ralstonia
Burkholderia
Xanthomonas
Xaylophilus
Agrobacterium
Clavibacter
Clostridum
Bacillus
Streptomyces
Gram positive - - - - - - - - - + + + +
Grows anaerobically + + - - - - - - - - + + -
Grows aerobically + + + + + + + + + + _ + +
Colonies yellow or Orange on YDC or NBY - +a - - - - +b +c - +d - - -
Colonies mucoid on YDC or at 30OC - - + - + - + - + + ND ND -
Fluoreescent pigment on KB - - - + - - - - - - - - -

22.
Differentiation of Major Genera
Character
Erwina
Pantoea
Acidovorax
Pseudomonas
Ralstonia
Burkholderia
Xanthomonas
Xaylophilus
Agrobacterium
Clavibacter
Clostridum
Bacillus
Streptomyces
Diffusiable non-fluorencent pigment
on KB
- - - - - + - - - - - - -
Urease -e - + - + v - + ND - ND ND ND
Oxidase - - + - + +f - - + - - v +
Growth at 40 OC - v + - - +g - - - - + + -
More than four Petrichous flagella + + - - - - - - - - v v -
Growth on DIM agar - - - - - - - - - - - - -
Spores formed - - - - - - - - - - + + -
Aerial mycelium - - - - - - - - - - - - +
+, 80% or more strains positive after five days, V, between 21-79% of strains positive,-, 80% of strains negative, ND Not
determined
a- Colonies of Pantoea citrea and some strain of P.agglommerans are generally white
b- Colonies of X.campestris pathovars manihotis and mangiferaeindicea are white
c- Xaylophilus grows very slowly on these media , but somewhat better on Difco nutrient agar
d- Colonies Clavibacter michiganesis subsp.sepedonicus are generally white
e- Erwina nigrifluens is positive
f- Burkholderia andropogodiis is oxidase negative
g- Burkholderia andropogodiis and B.glumae pv.agricola are negative

23.
Identification and classification of plant pathogenic bacteria
The concept of species in Bacteria
 In plants and animal, species is defined as a population of individuals in
which the fundamental characteristics are stable, and intraspecific crosses
do not cause sterility nor reduce reproduction
 In bacteria, the concept of species is less clear compared with that of
higher organisms because of their simple cell morphology and the
primitive form of sexual reproduction
 Bacterial species are the taxonomic groups of strains defined on the basis
of common phenotypic characteristics.

24.
Identification and classification of plant pathogenic bacteria
The concept of species in Bacteria
 In practice, the International Code of Nomenclature of Bacteria
requires a designation of the strain of the group as the type strain,
the representative holding the name of the species.
 These factors make the concept of the bacterial species indistinct
and classification unstable.
 A bacterial species is a group of bacterial strains that share certain
phenotypic and genotypic characteristics

25.
Identification and classification of plant pathogenic bacteria
The concept of species in Bacteria…continued
 Some groups of phytopathogenic bacteria can only be differentiated
by their pathogenecity on plants; an intraspecific division called
pathovar
 Bacterial strains serve as the type strain, with the other strains of the
species differing to a lesser or greater extent from the type strain
 When a strain or group of strains infect a host plant not infected by
the other strains of the species, that strain or group of strains
comprise a pathovar (pv.) of the species
 Pathovar in plant pathogenic bacteria is a subspecies (group of
strains) that can infect only plant within a certain genus or species.

26.
Identification and classification of plant pathogenic bacteria
The concept of species in Bacteria…continued
Nomenospecies
A nomenospecies is the species with binominal names given in accordance
with the rules of nomenclature, irrespective of its inappropriateness to the
rules of nomenclature,
 The names may be claimed for another taxa on the basis of some criteria.
For example, X.campestris is a nomenospecies superseded by
X.campestris pv. Phaseoli.
Molecular species:
Molecular species is the term applied to a group of strains with a high degree of
nucleic acid (DNA) or (RNA) homology. The sequence of nucleic acids
establishes and preserves the identity of a species. The species in
phylogenetic systematics corresponds to this

27.
Identification and classification of plant pathogenic bacteria
The concept of species in Bacteria…continued
 Bacteria taxonomy and names are less clear and stable than in other
organisms
 It is difficult to verify all characteristics of a bacterium, and
differentiation between species is not always apparent.
 Intermediate strains occur
 Bergey’s Manual of Systematic Bacteriology is the basic manual
for the classification of bacteria.
 New names and taxonomic changes in bacterial classification are
published in the International Journal of Systematic Bacteriology.
 .

28.
Identification and classification of plant pathogenic bacteria
The concept of species in Bacteria…continued
 Some groups of phytopathogenic bacteria can only be differentiated by their
pathogenecity on plants; an intraspecific division called pathovar. Some
phytopathogenic bacteria attack only specific plant species; some have a
narrow host range while others have a wide host range
 In plants and animal, species is defined as a population of individuals in
which the fundamental characteristics are stable, and intraspecific crosses
do not cause sterility nor reduce reproduction
 In bacteria, the concept of species is less clear compared with that of higher
organisms because of their simple cell morphology and the primitive form of
sexual reproduction

29.
Identification and classification of plant pathogenic bacteria
The concept of species in Bacteria…continued
 Taxospecies:
 Taxospecies refers to a group of strains that have a number of phenotypic
characteristics in common and can be grouped into distinct phenotypic groups
 The bacterial species which are operationally and routinely used in
bacteriology for example Erwinia amylovora or Ralstonia solanacerum or
Xanthomonas campestris
 The named group in a formal taxonomy is referred to as a taxon (plural taxa).

30.
Identification and classification of plant pathogenic bacteria
The concept of species in Bacteria…continued
 Genospecies: A genospecies is a group of strains that can
accomplish genetic exchange in some way
 Because genetic exchange occurs not only at species level but also
at genus level or even at the family level, genospecies has not been
considered of high practical value.

31.
Methods of classification
 Conventional taxonomy:
 Numerical taxonomy:
 Similarity index: S = [Ns / Ns + Nd ] x 100
 Matching index: Ssm = [Ns / No / Ns + Nd + No ] x 100
 Molecular taxonomy:: compositions expressed as guanaine plus
cytocine content (G + C%),
 DNA-DNA or DNA-rRNA homology,
 DNA bas composition:
 DNA-DNA homology:
 Restriction fragment-length polymorphism (RFLP):
 Protein profiles:
 Isozymes: