Classification and Nomenclature of Plant

Pathogenic Bacteria A Review

David Stead
Central Science Laboratory
York, UK

Some Definitions
Taxonomy the principles and practice of classifying
bacteria

Classification arrangement of strains into natural

groups (taxa)-phenetic(phenotypic and genetic) and
phylogenetic
Nomenclature allocation of names to these groups
Identification- processes by which unknowns are
referred to known taxa

Some Terms
Identification of a bacterium
Diagnosis of a disease
Presumptive usually isolation plus identification
Confirmatory usually requires a host test

Detection demonstrating the presence of a

specific bacterium in a sample
Often based on a single test/method eg IF / PCR
Can give a presumptive diagnosis

Aims
To review the current state of classification of
plant pathogenic bacteria
To review the methods used for classification
To review current nomenclature

Useful literature
Names of Plant Pathogenic Bacteria(2006)
Young et al - ISPP website

Plant Pathogens within the Bacteria

There are 21 Phyla within the Bacteria
Plant pathogens are found in 3 of these:
The Firmicutes
The Actinobacteria
The Proteobacteria

The Gram positive taxa

Clavibacter
Curtobacterium
Rathayibacter
Leifsonia
Nocardia
Rhodococcus
Streptomyces

Bacillus
Clostridium
Spiroplasma
Candidatus Phytoplasma

Microbacteriaceae in
order Actinomycetales

Nocardiaceae in order
Actinomycetales
Streptomycetaceae in
order Actinomycetales
Firmicutes

Taxonomic information
Characteristic morphology
Chemical composition of cell walls
Fatty acids very useful for genus/family
differentiation - 15:1 anteiso A is a
chemotaxonomic marker for Clavibacter
Within families eg Microbacteriaceae, 16S
rRNA sequencing is the major basis for
differentiating genera

Differentiation of taxa within the Genera

Clavibacter (C.michiganensis)
monospecific genus with 5 subspecies
Some important damaging and statutory pathogens
well differentiated by classical methods and PCR based
methods/genetic fingerprints

Curtobacterium (C.flaccumfaciens)
1 pathogenic sp in a fairly large genus with 4 pvs
Only 1 important pathogen
Pathovars fairly well differentiated by traditional
morphological/physiological/nutritional tests, also by genetic
fingerprinting

Differentiation of taxa within the Genera

Rathayibacter ( R.rathayi, R.tritici, R.toxicus)
3 spp all nematode transmitted, in grasses
Produce toxins important in grazing
Not easy to differentiate other than by genetic
fingerprinting

Leifsonia (L.xyli)
Small genus with 1 pathogenic species with 2
subspecies
Important sugarcane pathogen
Traditional methods and genetic fingerprints useful

Differentiation of taxa within the Genera

Streptomyces
Huge genus (many hundreds of spp) with perhaps 20 pathogenic
spp, some of which are difficult for most labs to differentiate
All cause scabs of root crops-potato
Pathogenicity is associated with genes in pathogenicity islands
that may be readily transferred
Perhaps are non pathogenic strains within some species
The most recently described spp are differentiated on DNA
homology, 16SrDNA and 16S-23S ITS sequencing supported by
some morphological and nutritional differences
Genetic fingerprinting, 16SrDNA sequencing and fatty acid
analysis do not correspond well with current classification
Other gene sequencing, mlst, may solve the problem

Song et al,
2004
16SrDNA
phylogeny

Differentiation of taxa within the Genera

Rhodococcus (R.fascians)

Large genus with a single pathogenic sp

Can be damaging to some horticultural crops
Pathogenicity associated with a plasmid
Other Rhodococcus spp are often abundant on
plant surfaces and in soil
Can be difficult to differentiate R.fascians from
other spp

The Mollicutes

Firmicutes

Mollicutes

Mycoplasmatales

Entomoplasmatales

Spiroplasma

Anaeroplasmatales

Acholeplasmatales

Candidatus Phytoplasma

Candidatus Phytoplasma

20 species
No cell wall
Uncultivable
Detected by PCR based on 16SrDNA
Differentiated by sequencing or PCR - RFLP
of either the 16SrDNA or 16S-23S ITS

The Gram Negative Genera-all in the

Proteobacteria
25 years ago there were 4 main genera
(Agrobacterium, Erwinia, Pseudomonas and
Xanthomonas) in which host specificity dictated the
classification and nomenclature either at species or
pathovar level depending on your preference
Now there are more than 30 genera with plant
pathogenic spp
16SrDNA sequencing has become the gold standard
for genus differentiation and DNA homology at the
70% level that for species, often irrespective of the
fact that other methods for differentiating these
genera and species are not readily available

The sub classes of Proteobacteria

There are 5 sub classes recognised and referred to
as alpha, beta, gamma, delta and epsilon sub
classes
3 of these contain plant pathogenic taxa
Alphaproteobacteria
Agrobacterium, Sphingomonas, Candidatus Liberibacter

Betaproteobacteria
Acidovorax, Burkholderia, Ralstonia, Xylophilus

Gammaproteobacteria
Erwinias, Pseudomonas , Xanthomonas , Xylella

Alphaproteobacteria
5/6 genera contain plant pathogens
Acetobacter and Gluconobacter in
Acetobacteriaceae in Rhodospirillales
Sphingomonas in Sphingomonadales
Agrobacterium (Rhizobium)
Candidatus Liberibacter

Alphaproteobacteria- less well

known genera

Acetobacter and Gluconobacter spp associated with

fruit rots-opportunists?
Sphingomonas 2 plant pathogenic spp in a fairly
large genus
S.suberifaciens corky root
S. melonis fruit spot
These are differentiated form each other and from other
S.spp by nutritional and morphological tests

Candidatus Liberibacter 3 uncultivable species

causing citrus greening differentiated by 16SrDNA

Agrobacterium

Proposal to transfer to Rhizobium based largely on

lack of 16SrRNA sequences
Complex genus
Plasmid borne pathogenicity factors
2 main types of plasmid Ri,Ti
Ri plasmids increasingly often found in other genera
within Alphaproteobacteria (Rhizobium,Ochrobactrum
in Brucellaceae within Alphaproteobacteria)
Currently 5 spp recognised, some of which
correspond to Biovars defined by nutritional tests

Agrobacterium
A. radiobacter (A. tumefaciens, Biovar 1 )
Plasmid + and strains
Ri (Root mat )and Ti (crown gall)
Fatty acid and genetic fingerprinting show great
diversity
Many genomovars based on DNA homology at
70%

A. rhizogenes (biovar 2 )
Plasmid + and strains
Ri ( hairy root )and Ti (crown gall )
Fatty acid and genetic fingerprinting show some
homogeneity

Agrobacterium
A. vitis (Biovar 3)- Ti plasmid strains cause crown gall
of grape
A.rubi - Ti plasmid strains cause cane gall of some
Rubus spp
A.larrymoorei Ti plasmid strains cause
tumours/crown gall of Ficus benjamina
Sequencing of, or PCRRFLP of 16S and 16S + IGS
ribosomal regions and other house keeping genes
has value in classification and identification
Traditional morphological,nutritional and
physiological tests are useful for biovar determination
and for the species as currently defined but what we
call bv 1 is taxonomically diverse

Betaproteobacteria
6 genera contain pathogens and these
represent 4 of the 5 families in the
Burkholderiales
Acidovorax in Comamonadaceae
Burkholderia in Burkholderiaceae
Ralstonia in Ralstoniaceae
Herbaspirillum and Janthinobacterium in
Oxalobacteriaceae
Xylophilus (family not certain)

Differentiation of genera within

Betaproteobacteria
Morphological/physiological and nutritional methods
are useful in differentiating these genera
Fatty acid profiles are also very useful (hydroxy
acids)
10:0 12:0
16:0 16:0
3OH 2OH 2OH 3OH
Acidovorax

Burkholderia
Ralstonia

(+)
-

+
+

+
-

Herbaspirillum

Xylophilus

Acidovorax
At least 4 pathogenic spp in genus which
includes a few non pathogenic spp
Mostly from warm climates
A.avenae has several important pathogens eg
A.avenae subsp citrulli (watermelon blotch)
A.avenae subsp avenae almost certainly comprises
other species/subsp
Genus is differentiated from close relatives in
Comamonodaceae (Comamonas, Delftia) by simple
nutritional, physiological and morphological tests, by
fatty acid analysis and by 16SrRNA sequencing
Species and subsp can also be differentiated by
nutritional and physiological methods, fatty acids,
genetic fingerprints as well as host tests

Burkholderia
A few years ago there were less than 10 spp ,
mostly plant pathogens
Now there are c 50 spp, with no new plant
pathogenic spp
Highly diverse epidemiology
Abundant in soil /water/rhizospheres
Clinical strains from the B.cepacia complex
responsible for death in patients with
diseases eg cystic fibrosis

Burkholderia
B.cepacia complex strains important in nitrogen
fixation, bioremediation, biological control. Few used
commercially because of the link to clinical problems
Classification was difficult for the B.cepacia complex
and eventually solved by combination of phylogenetic
gene sequencing and DNA homology studies
Complex had some 8 genomovars, now all upgraded
to specific rank
Onion strains fall into 2/3 of these, B.cepacia
(genomovar 1) and B.cenocepacia (genomovar 3)

Classification of Burkholderias based on

16SrDNA

Burkholderia
B. gladioli 4 pvs , one of human health significance
(pv cocovenenans)
Burkholderia andropogonis
Burkholderia caryophylli
Burkholderia glumae
Burkholderia plantarii
Differentiation at genus level is by nutritional and
physiological tests, genus specific PCR primers
Differentiation at species level is by nutritional and
physiological tests, genetic fingerprints,
recA/rpoD/gyrB gene sequencing, host tests
Differentiation of pvs is perhaps best by genetic
fingerprinting

Ralstonia

Equally complex
2 pathogenic spp
R.solanacearum is a very complex species
R.syzygii
Bvs / races / phylotypes / sequevars
Identification of the species is not difficult,
nutritional/physiological methods, fatty acid analysis,
PCR are all good.
Race determination in tobacco is not always reliable
Biovar determination using sugar alcohols is not
always reliable and is slow
Phylotype determination uses 16S-23S ITS sequence
Sequevar determination uses endoglucanase gene
sequence

Phylotype II
American
(Division 2)
Bvs 1, 2, 2T)

R. pickettii

Prior & Fegan (2003)

ACH0732

0.01

Phylotype IV

Phylotype I

Phylogenetic tree and

multiplex-PCR based upon
the 16S-23S ITS region
sequences

African
Phylotype III

91 bp

ACH0732

African
Bvs 1 and 2T

Phylotype IV

Phylotype III
Phylotype I

Asian
(Division 1)
Bvs 3, 4, 5

213 bp
144 bp

Phylotype II

Indonesian
BDB, Bvs 2 and 2T

372 bp

759/760
Rs band
280 bp

Phylogenetic analysis of endoglucanase gene

Phylotype IV
sequence
Biovar 2 / BDB
Indonesian

Biovar 5 (race 5)

Phylotype I
(Division 1)
Asian

Biovar 3 (race 1)
Biovar 4 (race 4)
Biovar 2 (race 3)
Biovar 2 (race 3)

Sequevar 1
Sequevar 2

Biovar 2T
Biovar 1 (race 2)
Biovar 1 (race 2)

Sequevar 3
Sequevar 4

Biovar 1 (race 1)
Biovar 1 (race 2)
Biovar 1 (race 1)
Phylotype
III 2T
Biovars 1 and

Sequevar 5
Sequevar 6
Sequevar 7

0.01

Fegan & Prior (2003)

Phylotype II
(Division 2)
American

Phylotype III
African

Gammaproteobacteria
3 main families
Enterobacteriaceae 10 genera containing
pathogens
Pseudomonodaceae 1 genus
(Pseudomonas)
Xanthomonodaceae 2 genera
(Xanthomonas, Xylella)

Enterobacteriaceae
Brenneria 5 species
Dickeya - 6 species representing the original
E.chrysanthemi complex
Enterobacter 4 species
Erwinia 8 species
Ewingella 1 species on mushroom
Pantoea 3 species
Pectobacterium - 6 species
Candidatus Phlomobacter 1 species ("Candidatus
Phlomobacter fragariae" )
Samsonia 1 species
Serratia - 1 species

Determination of genera within

Enterobacteriaceae
The initial removal of the non pathogen, E. herbicola
into Pantoea based on 16SrDNA sequencing led to
the current division of Erwinia into at least 7 genera
based on 16S sequencing. Whereas some of these
may be justified at genus level , some perhaps are
not eg Pantoea and Samsonia and may be part of
other genera
There are no phenotypic methods for differentiating
some of these genera
Recent phylogenetic studies based
on several gene sequences show
that Erwinia amylovora and Pantoea
agglomerans are very similar

Identification of species within the genera

There is fairly good agreement between DNA
homology at the 70% level and 16SrDNA sequencing
for differentiation of the species in these genera
In most cases there are clear nutritional and
physiological differences between the species in all
the genera
This is supported by genetic fingerprinting, fatty acid
analysis and increasingly by sequencing of other
housekeeping genes ( tuf, atpD)

Pseudomonas
Complex taxonomy due to highly conserved
core genome but highly variable genetic
diversity of the virulence associated genes
through infraspecific recombination and
interspecific gene transfer

Taxonomic issues
Currently 21 validly named species pathogenic to
plants and fungi
Of these P.syringae (56) and P. savastanoi (6)and
P.marginalis (3)have multiple pathovars
P syringae comprises 9 genomospecies based on
70% DNA homology, some of which have been
elevated to species
There are no reliable phenotypic methods which
differentiate these genomospecies
Genetic fingerprinting shows some correlation with
genomospecies
RpoB, GyrB sequencing and ribotyping very useful
for species differentiation within Pseudomonas

Taxonomic issues
Recent phylogenetic studies on the P.syringae
complex based on 7/8 housekeeping gene
sequences show remarkable congruence within the
strains selected with 4 clades in one study and 3 in
another
One clade in both studies comprised excusively
monocot strains and is equivalent to genomospecies
IV
There was good correlation between the strains in
the 2 studies
With only 4 species in the P.syringae /P.viridiflava
complex it is clear that the genetic variation seen in
the core genome only weakly predicts host
association

What does this mean for a diagnostician?

Species
Pathovars
Genomospecies and
species
MLST clades / Phylotypes

Does it matter that we get

the species correct in the P
syringae complex?
LOPAT test with a host test
is still a very good way of
identification to pv level
Fatty acid profile plus
genetic fingerprint
comparison with reference
strains also very useful
For those able to do it or pay
to have it done, a gene
sequencing approach is a
cost effective method and
will increase confidence in
the identification

Xanthomonas
Slightly different story to that for
Pseudomonas
Currently 24 validly named species
Of these, 7 have multiple pathovars

X. arboricola ( 7), X. axonopodis (44)

X. campestris (7), X. hortorum (4)
X. oryzae (2), X. translucens (9)
X. vasicola (2)
C 50 other pathovars not yet classified

Taxonomic issues
Not much phenotypic evidence for differentiating the
24 species
Most are differentiated on basis of DNA homology at
70%
Most are differentiated on the basis of 16SrDNA,
16S-23S IS sequencer and other housekeeping gene
sequences, although not as much info as for
Pseudomonas
Thus unlike Pseudomonas where all the pathovars
fall into at least 4 species, in Xanthomonas they are
distributed between 24 even though only 7 of these
currently have pathovars
Gyr B sequencing indicates c 7/8 new species
The core genome of Xanthomonas species much
more closely predicts host association

What does this mean for a diagnostician?

A difference here is that the same symptoms can be
caused by several pathovars /species eg
tomato/pepper spot can be caused by
X.euvesicatoria, X.vesicatoria, X.gardneri, and X.
axonopodis pv vesicatoria
Thus for some , species identification is necessary,
but for most it is the pathovar which needs to be
identified
It is increasingly clear that for species identification, a
gene sequencing approach is potentially useful
For identification at pathovar level, it is clear that
there are few nutritional/physiological methods;
genetic fingerprinting and fatty acid analysis are both
useful. Fatty acid analysis to prove genus, followed
by a host test is a good compromise

Xylella fastidiosa
Close relatives of Xanthomonas in the
Xanthomonodaceae
3 subspecies with different host ranges
Difficult to culture
Differentiated mainly by PCR - RFLP

Conclusions
We are clearly in a taxonomic splitting phase
The classifications we are relying on as the
basis for identification are currently moving
towards phylogenetic classifications
This results in the increased use of
phylogenetic methods for identification
This favours fewer but larger centralised
diagnostic labs
Will classical diagnosticians disappear?

Conclusions
Need to consider the evolutionary stability of
the core genome in relation to the instability
of the horizontal transfer of virulence
associated genes (hrp, secretion systems)
both in the genome and in plasmids
Phylogenetic classification of species should
rely on core genome. Identification of
pathogens (pathovars) may be better
provided by analysis of virulence genes