Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
Edizioni ETS Pisa, 2005
249
INVITED REVIEW
SHORT TAXONOMIC GUIDE TO THE GENUS ‘CANDIDATUS PHYTOPLASMA’
G. Firrao1, K. Gibb2 and C. Streten2
1 Dipartimento
di Biologia Applicata alla Difesa delle Piante, Università degli Studi,
Via delle Scienze 208, 33100 Udine, Italy
2 Faculty of Education, Health and Science, Charles Darwin University, Darwin 0909, NT, Australia
SUMMARY
The category of Candidatus was introduced to allow
unambiguous reference to organisms that could not be
cultivated in vitro. In plant pathology, a major impact of
this novel taxonomic concept was to enable the classification of the diverse group of organisms, morphologically similar to the mycoplasmas, known by the trivial
name of phytoplasmas. These plant pathogens were
originally named according to the disease they caused.
Later, extensive sequence analysis of the ribosomal
RNA genes allowed the development of an evolutionbased classification and the grouping of the phytoplasmas into phylogenetically distinct clades. Unfortunately,
the phytopathological and the phylogenetic classification did not match and had striking contradictions.
With the adoption of the category Candidatus, the description of the genus ‘Ca. Phytoplasma’ and several
‘Ca. Phytoplasma’ species, the scientific community is
now attempting to provide a classification that takes into account both the phylogenetic and the biological/
ecological characteristics of the organisms. Here we
provide an outline of the characteristics and composition of the genus ‘Ca. Phytoplasma’.
Key words: Phytoplasma, Candidatus, taxonomy, yellows.
THE CATEGORY OF CANDIDATUS IN THE TAXONOMY OF NONCULTIVABLE BACTERIAL PLANT PATHOGENS
According to recent estimates, a large fraction of the
existing microorganisms cannot be cultivated in vitro.
The features of many bacteria, including economically
relevant plant pathogens, cannot be investigated using
the methodologies that are adopted conventionally for
the characterisation and differentiation of cultivable
Corresponding author: G. Firrao
Fax: +39.0432.558501
E-mail: [email protected]
prokaryotes. Nevertheless, molecular techniques are
now available for the molecular characterisation of organisms without their isolation in pure culture. Limited
but precise information on the taxonomy of several non
cultivable bacteria has been obtained in recent years,
particularly by the sequence analysis of DNA obtained
by PCR (Polymerase Chain Reaction)-based amplification of 16S rRNA (ribosomal RNA) genes. Phylogenetically significant information can be obtained by selective amplification of target DNA molecules in the presence of an excess of extraneous nucleic acids, thus circumventing the need for the in vitro cultivation of the
organism being studied. In plant pathology, phytoplasmas, a large group of plant pathogens, which, despite
several attempts during the last 30 years, still resist cultivation, have been the target of extensive nucleic acid
characterization. However, the information obtained is
not sufficient for a formal taxonomic description according to the minimum standards for descriptions of
new species of the class Mollicutes (International Committee on Systematic Bacteriology - Subcommittee on
the Taxonomy of Mollicutes, 1979). Therefore conventional Latin binomial nomenclature cannot be used for
those non-cultivable mollicutes, which are defined solely on the basis of their DNA sequences. The lack of a
conventional nomenclature has made difficult and confusing references to non-cultivable organisms defined
on a molecular basis. For instance, the practice of naming a phytoplasma on the basis of the associated plant
syndrome causes confusion because it has been shown
that phylogenetically distinct phytoplasmas can cause
similar symptoms on the same host plant species.
In a taxonomic note published in 1994 by the International Journal of Systematic Bacteriology, Murray and
Schleifer (1994) proposed the category Candidatus
(i) The Latin name Candidatus is not declined according to Latin
rules when it is included in the organism epithet (i.e.,‘Candidatus Phytoplasma’ is used instead of ‘Candidatum Phytoplasma’) to prevent indexing problems.
(ii) Candidata taxa cannot have a “type” as defined by rule 18a of the
International Code of Nomenclature of Bacteria (Lapage et al., 1992).
For practical purposes of comparison, it was agreed to define a “reference” strain for ‘Ca. Phytoplasma’ species. Some early description may
lack “reference” or define, improperly, a “type” strain.
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Taxonomy of Candidatus phytoplasma
“to provide a proper record of sequence-based potential
new taxa”. The introduction and ready acceptance
(Murray and Stackebrandt, 1995) of the concept of Candidatus defined a milestone in the nomenclature of bacteria, as unambiguous reference to organisms was no
longer hampered by the technical inability to provide
them with a suitable artificial environment for growth.
With reference to plant pathology, the introduction of
the Candidatus category had a major impact on the
nomenclature of the phytoplasmas, although its implementation was not obvious. A Candidata species should
be defined on the basis of habitat and nucleic acid sequence data. For the phytoplasmas, obligate pathogens,
the habitat translates as the host. Although the insect
host would have a greater significance, given that the
phytoplasma probably originated there, it is more convenient to give the plant host more emphasis in the description of ‘Ca. Phytoplasma’ species. With regard to
the nucleic acid sequences, the obvious reference is the
16S rRNA gene, the most studied among the prokaryotes. According to the original proposal (Murray and
Schleifer, 1994), a Candidata species description should
refer to a unique sequence deposited in public databases and also list unique and characteristic oligonucleotide
sequences. However, this approach did not fit particularly well for the phytoplasmas, whose extraordinary diversity was reflected in a large number of unique 16S
rDNA sequences, and therefore an impractical number
of species. From the several studies on phytoplasma diversity carried out in the last 15 years, it is apparent that
some strains are closely related even though their 16S
rRNA sequences are not identical and it would not be
useful to assign different names to each variant within a
16S rRNA group. On the other hand, the use of the
same name to refer to groups of related but different
strains would result in a loss of potentially significant biological information. The problems identified stimulated a large debate among the phytoplasmologists. Finally,
the ‘phytoplasmas, spiroplasmas, entomoplasmas and
mesoplasmas working team’ of the International Research Project for Comparative Mycoplasmology
(IRPCM; Anonymous, 2000), in agreement with the International Committee of Systematic Bacteriology
Subcommittee for the Taxonomy of Mollicutes (2001),
made some recommendations for the description of new
‘Candidatus Phytoplasma’ species. Accordingly, a strain
that shares more than 97.5% of its 16S rRNA gene sequence with an already described species should not be
described as a new Candidata species unless it is demonstrated that the organism belongs to an ecologically distinct population. Finally, the IRPCM Phytoplasma/
Spiroplasma Working Team Phytoplasma taxonomy
group (2004) coded the rules in a paper that describes
the properties and the composition of the genus ‘Candidatus Phytoplasma’.
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
THE GENUS ‘CANDIDATUS PHYTOPLASMA’
Non helical wall-less bacteria that colonize the plant
phloem were first reported almost 40 years ago when
Doi et al. (1967) detected by electron microscopy mycoplasma-like bodies in a mulberry plant showing yellows symptoms. About twenty-five years later, it was
proposed to rename these mycoplasma-like organisms
because they had been shown by molecular analyses not
to be phylogenetically related to the genus Mycoplasma.
The trivial name phytoplasmas was proposed by B.B.
Sears and B.C. Kirkpatrick to the International Committee on Systematic Bacteriology (ICSB) Subcommittee on the Taxonomy of Mollicutes (1993) and subsequently used (Sears and Kirkpatrick, 1994). Later, the
ICSB Subcommittee on the Taxonomy of Mollicutes
(1995) agreed that the phytoplasmas could be assigned
a provisional taxonomic status of Candidatus, since they
could only be defined on the basis of habitat, morphology and certain characteristics of the membrane and nucleic acids and not on the basis of biochemical, physiological and cultural characteristics because they could
not be cultivated in vitro. Phytoplasmas lack rigid cell
walls, are surrounded by a single unit membrane, and
are sensitive to the antibiotic tetracycline (Doi et al.,
1967). When observed by transmission electron microscopy, they appear as rounded to filamentous
pleiomorphic bodies with an average diameter ranging
from 200 to 800 nm (Kirkpatrick, 1991). They inhabit
the phloem sieve elements of infected plants and the
gut, haemolymph, salivary gland and other organs of
sap-sucking insect vectors (Kirkpatrick, 1991). The
genome of the phytoplasmas has a size of 530-1,350 kbp
(Marcone et al., 1999) and a G+C content of 23-29
mol% (Kollar and Seemüller, 1989). The phytoplasmas
have a tRNAILE in the spacer region between the 16S
and the 23S rRNA genes (Kuske and Kirkpatrick, 1992;
Smart et al., 1996). A major molecular characteristic for
their definition and identification is the sequence of
their 16S rRNA gene. Phylogenies based on this gene
showed that the phytoplasmas constitute a monophyletic group within the Mollicutes (Gundersen et al., 1994;
Seemüller et al., 1994), Acholeplasma palmae being their
closest cultivable relative. Due to the distinctive sequences in their 16S rRNA gene, several phytoplasmaspecific PCR assays have been developed (Ahrens and
Seemüller, 1992; Firrao et al., 1993; Lee et al., 1993;
Namba et al., 1993; Gibb et al., 1995; Lorenz et al.,
1995; Padovan et al., 1995; Smart et al., 1996). The phytoplasmas are a large and diverse group causing several
hundreds of plant diseases. More than 200 different,
nearly full length sequences of phytoplasma 16S rRNA
genes have been obtained to date (Seemüller et al.,
1998b; Lee et al., 2000; IRPCM Phytoplasma/Spiroplasma Working Team - Phytoplasma taxonomy group,
2004). Their analysis has delineated less than 20 clusters
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
of strains at 97.5% similarity. Selected representatives of
each cluster are described as ‘Ca. Phytoplasma’ species.
However, some groups defined at 97.5% or higher
similarity, include phytoplasmas with different biological, phytopathological, and molecular properties. In
some cases, phytoplasmas sharing high 16S rDNA sequence similarity may cause different plant diseases.
Hence, in addition to the minimum set of species belonging to ‘Ca. Phytoplasma’ defined at the 97.5% similarity of the 16S rDNA sequence, more Candidatae
species have been defined to distinguish between those
organisms that proved to be significantly different on
the basis of biological and genetic properties. In order
to prevent nomenclatural confusion arising from the description of poorly differentiated new taxa, the Phytoplasma/Spiroplasma Working Team of the International
Research Project for Comparative Mycoplasmology
(Anonymous, 2000; IRPCM Phytoplasma/Spiroplasma
Working Team – Phytoplasma taxonomy group, 2004)
suggested rules for the description as new Candidatae
species of phytoplasmas that share more than 97.5% of
their 16S rRNA gene. For such cases, the description of
Firrao et al.
251
two different species is recommended only when the
following three conditions apply: (i) the two phytoplasmas are transmitted by different vectors; (ii) the two
phytoplasmas have a different natural plant host (or at
least their behaviour is significantly different in the
same plant host); (iii) there is evidence of significant
molecular diversity from either hybridization tests with
cloned DNA probes, or serological reactions or PCRbased assays. At the time of writing, 21 Candidatae
species have been validly described and 6 additional
species have an informal description (Fig. 1). As a consequence of the taxonomic rules adopted for the classification of the phytoplasma, the plant host and the distinctive oligonucleotide signature given in the species
description should be verified, but not the identity of
the complete 16S rDNA sequence. Given the wide use
of the restriction analysis of the 16S rDNA, a presumptive identification of the isolate is most often done by
analyzing the 16S rDNA RFLP pattern. The next section summarizes the details needed for the identification
of each species presently recognized within the genus
‘Candidatus Phytoplasma’.
Fig. 1. Phylogenetic tree reporting the Candidatae species of the genus ‘Candidatus Phytoplasma’ and their relationships with the
groups identified by Lee et al. (1998, 2000) and Seemüller et al. (1994, 1998b). The asterisk (*) marks names which have been proposed at the X International Congress of the International Organization of Mycoplasmology (1994), but not yet formally described,
and are reported here as incidental citations which do not constitute prior citations, according to rule 28b of the bacteriological
code (Lapage et al., 1992). The tree was constructed using CLUSTALX (Thompson et al., 1997) from the alignment of the Candidatus species 16S rDNA sequences, publically available at TreeBase (http://www.treebase.org/treebase/console.html) as matrix
accession M1788.
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Taxonomy of Candidatus phytoplasma
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
‘CANDIDATUS PHYTOPLASMA’ SPECIES
reference strain. The description of this taxon is based
on the strain OAYR (=MIAY), associated with Oenothera
virescence in Michigan, USA. This strain belongs to the
Aster Yellows group (Seemüller et al., 1994, 1998b) or
16SrI group of Lee et al. (1998, 2000). Phylogenetic
studies based on sequences from different genes have
shown congruently that this group represents a distinct
clade within the phytoplasmas (Lee et al., 1998). ‘Ca. P.
asteris’ is the only species described within the 16SrI
group. However, this clade comprises a large number of
related phytoplasmas worldwide, representing the most
diverse and widespread phytoplasma group studied (Lee
et al., 2004a). The strains related to ‘Ca. P. asteris’, despite the relatively high similarity in the 16S rDNA sequence, occupy diverse ecological niches and show substantial genetic variations (Gundersen et al., 1994;
Seemüller et al., 1994, 1998b; Vibio et al., 1996; Lee et
al., 1998, 2000, 2002; Marcone et al., 2000; Jomantiene et
al., 2002). According to the rules for the description of
new Candidatae species this widely diverse group may
consist of more than one species. A specific primer pair,
designed by Lee et al. (1994) for the amplification of a ribosomal DNA fragment from strains belonging to the
16SrI group and named R16(I)F1/R1, is presently widely
used for the amplification from strains related to ‘Ca. P.
asteris’, although the 16S rDNA of strains belonging to
groups 16SrII and 16SrXII may also be a target. The 16S
rDNA of ‘Ca. P asteris’ has characteristic KpnI sites that
distinguish it from other species. Useful diagnostic enzymes for the assignment of isolates to subgroups within
the 16SrI group are MseI, HhaI and HpaII for the 16S
rDNA and MseI, Tsp509I and AluI for the ribosomal
protein gene operon (Lee et al., 1998, 2004a). Most ‘Ca.
P. asteris’ strains infect herbaceous dicotyledonous
plants. Nevertheless, some strains may infect monocotyledonous as well as woody plants. The host range of
this species is extremely large; a list of recognized host
species is given below but it may not be comprehensive:
Aconitum napellus, Allium cepa, Ambrosia artemisifolia,
Anemone coronaria, Anethum graveolens, Aphanostephus
skirrhobasis, Apium graveolens, Avena sativa, Brassica napus, Brassica oleracea capitata, Brassica oleracea italica,
Brassica oleracea palmifolia, Brassica rapa rapifera, Calendula officinalis, Callistephus chinensis, Carthamus tinctorius, Catharanthus roseus, Celtis australis, Chrysanthemum coronarium, Chrysanthemum frutescens, Cirsium arvense, Corinadrum sativum, Cornus racemosa, Cosmos
bippinatus, Cryptotaenia japonica, Cyclamen persicum,
Cynodon dactylon, Daucus carota, Delphinium sp.,
Diplotaxis eucoides, Echinacea purpurea, Epilobium sp.,
Erigeron canadensis, Eupatorium capillifolium, Fragaria x
ananassa, Gladiolus sp., Glycine max, Hyacinthus orientalis, Hydrangea macrophylla, Ipomoea obscura, Lactuca
sativa, Limonium sinuatum, Lycopersicon esculentum,
Medicago sativa, Morus bombycis, Nasturtium microphyllum, Oenothera hookeri, Olea europaea, Opuntia sp., Pa-
‘Ca. Phytoplasma allocasuarinae’ Marcone, Gibb,
Streten & Schneider (Marcone et al., 2003a). This phytoplasma was detected in naturally diseased Allocasuarina muelleriana (Slaty sheoak) plants growing in Australia and expressing a yellows symptom. It is associated
with the 16S rRNA gene sequence accession number
AY135523, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-TTTATTCGAGAGGGCG-3’. Sequence analysis of the 16S rRNA
gene shows that this phytoplasma is more closely related
to members of the apple proliferation (AP) group than
to other phytoplasma subclades (Gibb et al., 2003). Nucleotide sequence comparisons between the 16S rRNA
genes of ‘Ca. P. allocasuarinae’ and other phytoplasmas
assigned to the AP group revealed the buckthorn witches’-broom (BWB) agent is most closely related, sharing
96% similarity. The ‘Ca. P. allocasuarinae’ agent differs
from the BWB and spartium witches’-broom (SpaWB)
agents in 17 to 18% of the nucleotide positions on the
16S rRNA gene. The ‘Ca. P. allocasuarinae’ agent shared
94% similarity with apple proliferation (AP), pear decline (PD), European stone fruit yellows (ESFY) and
peach yellow leaf roll (PYLR) phytoplasmas, which are
also members of the AP-fruit tree group. ‘Ca. P. allocasuarinae’, ESFY, PD and PYLR phytoplasma have the
same banding pattern when digested with restriction enzyme ScfI and ‘Ca. P. allocasuarinae’ has a restriction site
for PvuII at position 518 that is unique to members of
the AP-group. The ‘Ca. P. allocasuarinae’ 16S rRNA
gene lacks restriction sites for SspI and AluI enzymes,
which are present in the SpaWB and BWB agents. ‘Ca.
P. allocasuarinae’ has an additional HpaII restriction site
that is not present in other AP-fruit tree agents. A specific PCR assay for ‘Ca. P. allocasuarinae’ is possible using the AP group-specific primer fO1 (Lorenz et al.,
1995) in combination with the reverse primer rAllo1 (5’GAGTCCCCAACTAAATGAT-3’) (Gibb et al., 2003).
‘Ca. P. asteris’ Lee, Gundersen, Rindal, Davis, Bottner, Marcone & Seemüller (Lee et al., 2004a). This
phytoplasma is associated with the 16S rRNA gene sequence accession M30790, and to the accession M74770,
corresponding to genes for ribosomal proteins rpl23 gene
(3’ end), rpl2, rps19, rpl22, and rps3 genes, and rpl16 (5’
end); oligonucleotide sequences of unique regions of 16S
rRNA gene are: 5’-GGGAGGA-3’ (positions 226-232),
5’-CTGACGGTACC-3’ (positions 476-485) and 5’CACAGTGGAGGTTATCAGTTG-3’ (positions 10081028); oligonucleotide sequences of unique regions of ribosomal protein genes are 5’-CCGCGAACAACTT-3’
and 5’-AGTAATAACTTCTAGCACAAACTTGC-3’ (positions 218-230 and 338-363) for rpl22 and 5’-AAAGAA
GATTTTTTAATTC-3’ and 5’-CTAGAAAATCGTATG
-3’ (positions 114-133 and 396-410) for rps3. OAY is the
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
paver rhoeas, Parthenium hysterophorus, Paulownia sp.,
Plantago coronopus, Plantago major, Poa pratensis, Populus nigra, Portulaca oleracea, Primula sp., Prunus aremeniaca, Prunus cerasus, Pyrus communis, Quercus robur, Ranunculus asiaticus, Ranunculus sp., Raphanus raphanistrum, Salix sp., Santalum album, Saponaria officinalis,
Schizanthus pinnatus, Sechium edule, Sechium tacaco,
Solanum melongena, Solanum tuberosum, Sonchus asper,
Symphytun sp., Tagetes erecta, Taraxacum officinale, Trifolium repens, Trifolium sativum, Trifolium sp., Vaccinium
sp., Valeriana officinalis, Vitis vinifera, Zea mays (refer to
Lee et al., 2004a, for details and references).
Due to the large host range, ‘Ca. P. asteris’ strains are
associated with several diseases worldwide, generally
named on the basis of symptoms and host. Typical symptoms caused by infection of ‘Ca. P. asteris’ include virescence (greening of flower petals) and phyllody (development of floral parts into leaf like structures), flower
streaking and malformation, yellowing and upright posture of leaves, elongation and etiolation of internodes,
excessive branching of axillary shoots, witches’-broom
and stunting. However, some infected plants may exhibit
only some of these symptoms. Plants infected with some
so-called “mild” strains may show no obvious symptoms.
‘Ca. Phytoplasma aurantifolia’ Zreik, Carle, Bové &
Garnier (Zreik et al., 1995). This phytoplasma was detected in Citrus aurantifolia affected by the witches’broom disease of small-fruited acid lime. The disease
appeared in Oman in the late 1970s, was later reported
in the United Arab Emirates and recently also in Iran
(Bové et al., 1998). ‘Ca. P. aurantifolia’ is associated with
the 16S rRNA gene sequence accession U15442, with
oligonucleotide sequence complementary to a unique
region of the 16S rRNA, 5’-GCAAGTGGTGAACCATTTGTTT-3’. The genome size is estimated to be 720
kbp. The phylogenetic analysis of the 16S rRNA gene
sequence assigns this phytoplasma to the faba bean
phyllody (FBP) group (Schneider et al., 1995; Seemüller
et al., 1998b) or to the 16SrIIB (according to Lee et al.,
2000). The reader is referred to ‘Ca. P. australasiae’ below for details about the identification and differentiation of strains of the FBP group using 16S rRNA gene
analysis. ‘Ca. P. aurantifolia’ could also be distinguished
from closely related strains by Southern blot – RFLP
analysis using cloned chromosomal probes and by monoclonal antibody reactions (Zreik et al., 1995).
‘Ca. Phytoplasma australasiae’ White, Blackall,
Scott, & Walsh (White et al., 1998) (revised nomenclature of ‘Ca. P. australasia’). This phytoplasma is associated with the 16S rRNA gene sequence accession
Y10097, with oligonucleotide sequence complementary
to unique region of the 16S rRNA 5’-TAAAAGGCATCTTTTATC-3’ and 5’-CAAGGAAGAAAAGCAAATGGCGAACCATTTGTTT-3’. Sequence analy-
Firrao et al.
253
sis of the 16S rRNA gene indicates that ‘Ca. P. australasiae’ and ‘Ca. P. aurantifolia’ are phylogenetically
closely related to each other and to other phytoplasmas,
collectively known as the Faba bean phyllody (FBP)
group (Schneider et al., 1995). The first reported phytoplasma of this group was implicated in naturally diseased tomato showing symptoms of tomato big bud
(TBB) disease (Bowyer et al., 1969). In a more recent
survey, phytoplasmas related to the TBB phytoplasma
were detected in 73 plant host species from 26 families
(Davis et al., 1997b; Schneider et al., 1999). Most of the
plant host species are members of the Fabaceae (Schneider et al., 1999). RFLP and sequence analysis of ribosomal genes indicate that a close relationship exists between the phytoplasmas associated with TBB, papaya
yellow crinkle (PYC), papaya mosaic (PM) and sweet
potato little leaf (SPLL) (Gibb et al., 1995, 1996; White
et al., 1998). Using these data the TBB type phytoplasmas from Australia were placed in the faba bean phyllody (FBP) strain cluster (Schneider et al., 1995), which
includes the type specimen FBP from Sudan, and the
lime witches’-broom phytoplasma WBDL), named ‘Ca.
Phytoplasma aurantifolia’ by Zreik et al. (1995). White
et al. (1998) proposed that the PYC, PM, and TBB phytoplasmas be assigned to a new species, ‘Ca. Phytoplasma australasia’ (sic), not in agreement with the recommended criteria (IRPCM Phytoplasma/Spiroplasma
Working Team - Phytoplasma taxonomy group, 2004).
Therefore the use of this species name is discouraged.
This phytoplasma was detected in Australia associated with diseases of Carica papaya L. named papaya yellow crinkle and mosaic. Since this Candidatus species
name was extended by the authors of the original paper
(White et al., 1998) to include other phytoplasmas in
addition to that associated with the sequence Y10097,
phytoplasmas isolated from other hosts might potentially be identified as ‘Ca. P. australasiae’. This is not recommended.
‘Ca. Phytoplasma australiense’ Davis, Dally, Gundersen, Lee & Habili (Davis et al., 1997a). This phytoplasma is associated with the 16S rRNA gene sequence
accession L76865, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’TTTATCTTTAAAAGACCTCGCAAGA-3’. According
to their phylogenetic studies, Lee et al. (2000) represented the ‘Ca. P. australiense’ as a member of the stolbur 16SrXII group, subgroup B. Diagnostic enzymes
for the identification of this phytoplasma by RFLP
analysis of amplified 16S rRNA gene are AluI, MseI and
TaqI. This phytoplasma was detected in naturally diseased ‘Chardonnay’ grapevines showing symptoms of
Australian grapevine yellows (AGY), and later in other
grapevine cultivars such as ‘Riesling’ and less frequently
‘Traminer’, ‘Montils’ and ‘Semillon’ (Padovan et al.,
1996). The disease looks very similar to European
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Taxonomy of Candidatus phytoplasma
grapevine yellows diseases caused by phylogenetically
different phytoplasmas. The diseases known as papaya
dieback (Carica papaya L.), Phormium yellow leaf
(Phormium tenax J.R. Forst. & G. Forst., P. cookianum
Le Jol.) and strawberry lethal yellows (Fragaria x
ananassa) were found to be associated with ‘Ca. P. australiense’ (Andersen et al., 1998; Liefting et al., 1998;
Padovan et al., 2000). ‘Ca. P. australiense’ is currently
restricted to Australia and New Zealand.
‘Ca. Phytoplasma brasiliense’ Montano, Davis, Dally, Hogenhout, Pimentel & Brioso (Montano et al.,
2001). This phytoplasma was found in Hibiscus rosasinensis L. showing symptoms of hibiscus witches’broom in Brazil (Montano et al., 2001). It is associated
with the 16S rRNA gene sequence accession AF147708,
with oligonucleotide sequence complementary to unique
region of the 16S rRNA 5’-GAAAAAGAAAG-3’, 5’TCTTTCTTT-3’, 5’-CAG-3’, 5’-ACTTTG-3’ and 5’GTCAAAAC-3’. Phylogenetic analysis of the hibiscus
witches’-broom phytoplasma 16S rDNA sequence showed that it was distinct from any other phytoplasma and
group 16SrXV was designated to accommodate it (Montano et al., 2001). Diagnostic enzymes for the identification of this phytoplasma by RFLP analysis of amplified
16S rRNA gene are MseI and HpaII.
‘Ca. Phytoplasma castaneae’ Jung, Sawayangi T,
Kakizawa, Nishigawa, Miyata, Oshima K, Ugaki, Lee,
Hibi & Namba (Jung et al., 2002). This phytoplasma
was found in Castanea sativa showing symptoms of
witches’-broom disease of chestnut in Japan. It is associated with the 16S rRNA gene sequence accession
AB054986, with oligonucleotide sequences complementary to unique regions of the 16S rRNA 5’-CTAGTTTAAAAACAATGATA-3’ and 5’-CTCATCTTCCTCCAATTC-3’. Sequence analysis revealed the Chestnut
witches’-broom (CnWB) phytoplasma shares from 86.8
to 94.9% similarity with other phytoplasmas. Phylogeny
and sequence analysis revealed the CnWB phytoplasma
is most closely related to the phytoplasmas that affect
coconut and it represents a distinct subgroup within the
group. The CnWB phytoplasma can be specifically amplified by using the primer pair CnWBF1/CnWBR1
(Jung et al., 2002). The phytoplasma signature sequence
ACTGGA (155-160) in the CnWB phytoplasma has a
‘G’ instead of a ‘A” at position 155 and a ‘T’ instead of
a ‘C’ at position 156. In addition, the phytoplasma signature sequences GTGT (276-279) and CCC (487-489)
have a ‘T’ at nucleotides 278 and 487 in the 16S rRNA
gene of the CnWB phytoplasma (Jung et al., 2002).
‘Ca. Phytoplasma cynodontis’ Marcone, Schneider
& Seemüller (Marcone et al., 2003b). This phytoplasma was detected in Cynodon dactylon exhibiting symptoms of chlorosis, proliferation of axillary shoots, bushy
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
growth habit, small leaves, shortened stolons and rhizomes, and stunting. The disease was named Cynodon
white leaf (CWL) or Bermuda grass white leaf (BGWL).
Its geographical distribution includes several countries
in Asia (Chen et al., 1972; Zahoor et al., 1995;
Viswanathan, 1997; Sdoodee et al., 1999), Italy (Marcone et al., 1997a), Sudan (Dafalla and Cousin, 1988),
and Australia (Tran-Nguyen et al., 2000; Blanche et al.,
2003). ‘Ca. P. cynodontis’ is associated with the 16S
rRNA gene sequence accession AJ550984, with oligonucleotide sequence complementary to unique region of
the 16S rRNA 5’-AATTAGAAGGCATCTTTTAAT-3’.
Phylogenetic studies assigned the Bermuda grass
white leaf (BGWL) phytoplasma to the 16SrXIVA
group, which includes blue grass (Poa annua) white leaf
and Brachiaria distachya white leaf (BraWL) phytoplasmas (Lee et al., 1998, 2000; Seemüller et al., 1998b;
Sdoodee et al., 1999). Although members of the 16SrXIV group share greater than 97.5% 16S rDNA similarity, data were provided showing that the group includes
ecologically and genetically distinct populations that
should correspond to distinct ‘Candidatus Phytoplasma’
species. Phylogeny studies and sequence analysis revealed that 16S rRNA genes of BGWL isolates from
Italy are identical while the Thai BGWL strain differed
from the Italian strain in four nucleotide positions. The
16S rRNA gene of the CWL phytoplasma differed from
the Thai isolate at one nucleotide and from the Italian
strains at three nucleotides. The BraWL agent is closely
related to the BGWL phytoplasma (99.4-99.7% similarity between 16S rRNA genes). The 16S rRNA gene of
SGS phytoplasma shares 98.3-98.5% similarity with the
BGWL strains while the Sugarcane White Leaf (SCWL)
phytoplasma shares 98.2-98.4% similarity with the BGWL isolates. Phylogeny derived from 16S-23S spacer region sequences confirmed the results of the 16S rRNA
gene analysis. Enzymatic RFLP analysis showed that
there is no variation between the phytoplasmas associated with BGWL, white leaf agent from annual blue
grass, BraWL, carpet grass white leaf (CGWL) and date
palm diseases. However, theoretical RFLP banding patterns showed that BGWL phytoplasma lacks a TaqI site
at position 1,424 that is present in the BraWL phytoplasma and that the BGWL phytoplasma also has a
MaeIII site not present in the BraWL agent. The BGWL
phytoplasma can be distinguished from other members
of the SCWL group including SCWL, SCGS, and SGS
phytoplasmas based on RFLP banding patterns.
The BGWL phytoplasma can be specifically amplified
using the fSCWL forward primer in combination with
the rCWL reverse primer (TranNguyen et al., 2000).
The estimated chromosome of the BGWL phytoplasma is 530 kbp (Marcone et al., 1999). Polyclonal antisera raised to the BGWL and SCWL phytoplasmas
showed no cross-reactivity (Sarindu and Clark, 1993;
Viswanathan, 1997, 2001). The BGWL antisera did not
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
react with the BraWL or Dactylocetenium aegyptum
white leaf (DacWL). The BGWL phytoplasma has not
been identified in other plant species and it is not transmitted by Matsumuratettis hiroglyphicus, which is the
natural vector for SCWL phytoplasma. Although sequence analysis indicated that the BraWL, CGWL and
agents associated with date palms are identical or nearly
identical to the BGWL phytoplasma, it has been suggested that these phytoplasmas should not be designated as the same ‘Candidatus Phytoplasma’ species name
as the BGWL phytoplasma because there is currently
insufficient evidence to confirm this relationship.
‘Ca. Phytoplasma fraxini’ Griffiths, Wayne, Sinclair,
Smart & Davis (Griffiths et al., 1999b). This phytoplasma was detected in Fraxinus americana L., Fraxinus velutina Torr., Fraxinus pennsylvanica Marsh., Fraxinus
bongeana A. DC., showing symptoms of ash yellows and
in Syringa patula (Palibin) Nakai, Syringa x prestoniae
McKelv., Syringa x josiflexa Preston ex Pringle with lilac
witches’-broom disease. It is restricted to the Americas
(Griffiths et al., 1999b, 2001). ‘Ca. P. fraxini’ is associated with the 16S rRNA gene sequence accession
AF092209, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-CGGAAACCCCTCAAAGGTTT-3’ and 5’-AGGAAAGTC-3’. In
phylogenetic analyses, strains associated with ash yellows and lilac witches’-broom constituted a clearly defined clade within the phytoplasmas (Griffiths et al.,
1999b), corresponding to 16SrVII group in Lee et al.
(1998, 2000). These strains were collectively named ‘Ca.
P. fraxini’, and strain AshY1 was selected as a type strain
(Griffiths et al., 1999b), the other strains differ from the
AshY1 type by 1 to 3 nucleotides in their 16S rRNA
gene sequence. However, the selection of a type strain is
not valid (see note 2) and strain AshY1R should more
properly referred as “reference strain”. Some recently
detected phytoplasmas were assigned to the 16SrVII
group (Meneguzzi et al., 2001; Barros et al., 2002; Bruni
et al., 2005), but those strains appear to be distinct from
‘Ca. P. fraxini’. The genome size of strain AshY3 is
around 645 kbp. Digestion of the PCR-amplified 16SrRNA gene with AluI is diagnostic for the identification
of this strain cluster (Lee et al., 1998; Griffiths et al.,
1999b). A specific PCR assay for AshY1 and closely related strains uses primers fB1/rAsYs (Smart et al., 1996).
‘Ca. Phytoplasma japonicum’ Sawayanagi, Horikoshi, Kanehira, Sinohara, Bertaccini, Cousin, Hiruki
& Namba (Sawayanagi et al., 1999). This phytoplasma
was detected in Hydrangea macrophylla (Thunb. ex J.
Murr.) Ser. f. normalis, Hydrangea serrata (Thunb.)
showing symptoms of Japanese Hydrangea phyllody, in
Japan. ‘Ca. P. japonicum’ is associated with the 16S
rRNA gene sequence accession AB010425, with oligonucleotide sequence complementary to unique region of
Firrao et al.
255
the 16S rRNA 5’-GTGTAGCCGGGCTGAGAGGTCA-3’ and 5’-TCCAACTCTAGCTAAACAGTTTCTG3’. In the phylogenetic analysis of the 16S rRNA gene sequence (Sawayanagi et al., 1999), this phytoplasma was
related to the strains in group 16SrI group but did not
cluster to any other strain and was therefore assigned to
a new sub-group. The most closely related phytoplasmas
were the stolbur phytoplasma and ‘Ca. P. australiense’
(Sawayanagi et al., 1999). Diagnostic enzymes for the
identification of this phytoplasma by RFLP analysis of
the amplified 16S rRNA gene are KpnI, EcoRI, HaeIII
and Tru9I (MseI). A specific amplification of this phytoplasma is achieved by using a nested PCR approach using the primer pair SN910601/SN910502 followed by
JHPF1/JHPR1 (Sawayanagi et al., 1999).
‘Ca. Phytoplasma mali’ Seemüller & Schneider (Seemüller and Schneider, 2004). This phytoplasma is associated with the economically important disease named apple proliferation. It occurs in a wide range of species of
the genus Malus (Kartte and Seemüller, 1991) and has
been detected occasionally in plants such as Pyrus communis, Pyrus pyrifolia, Prunus salicina, Corylus avellana,
Crataegus monogyna, Quercus robur, Quercus rubra,
Carpinus betulus, Convolvulus arvensis (Lee et al., 1995;
Marcone et al., 1996b; Schneider et al., 1997; Del Serrone et al., 1998; Seemüller, 2002) by serological and
DNA based techniques. ‘Ca. P. mali’ is restricted to Europe. It is associated with the 16S rRNA gene sequence
accession AJ542541, with oligonucleotide sequence
complementary to unique region of the 16S rRNA 5’AATACTCGAAACCAGTA-3’. The reference strain is
isolate AP15, which has a chromosome of 690 kb in
length. ‘Ca. P. mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’
are phylogenetically closely related, the interspecific differences in their 16S rDNA sequences range from 1.0 to
1.5%. The group was named ‘Apple proliferation strain
cluster’ (Seemüller et al., 1994, 1998b) or group 16SrX
(Lee et al., 1998, 2000). The AluI and RsaI restriction
patterns of the 16S rDNA are characteristic and diagnostic for this group (Schneider et al., 1993). The ‘Ca. P.
mali’ phytoplasma can be distinguished by 16S rDNA
digestion with SspI and BsaAI from ‘Ca. P. pyri’, and
from ‘Ca. P. prunorum’ with RsaI, BsaAI, SspI and SfcI.
Several diagnostic primers based on 16S rDNA sequences for the selective amplification of some or all
phytoplasmas of the Apple proliferation group have
been developed by Firrao et al. (1994), Jarausch et al.
(1994), Lee et al. (1995), Lorenz et al. (1995), Smart et
al. (1996) and Baric and Dalla Via (2004). AP-group
phytoplasma detection and differentiation can also be
carried out using randomly cloned chromosomal fragments, such as fragment IH196 of strain AT (Bonnet et
al., 1990). PCR amplification using primers AP9/AP10
followed by digestion with RsaI and HincII allows the
differentiation of ‘Ca. P. mali’ from ‘Ca. P. pyri’ and ‘Ca.
256
Taxonomy of Candidatus phytoplasma
P. prunorum’ (Jarausch et al., 1994, 2000a, b).
‘Ca. Phytoplasma oryzae’ Jung, Sawayanagi,
Wongkaew, Kakizawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Hibi & Namba (Jung et al., 2003b). This
phytoplasma was detected in Oryza sativa plants exhibiting symptoms of yellowing, stunted growth and
failure to produce rice, a disease named rice yellow
dwarf that occurs in Japan and Thailand. ‘Ca. P. oryzae’
is associated with the 16S rRNA gene sequence accession AB052873, with oligonucleotide sequences complementary to unique regions of the 16S rRNA 5’AACTGGATAGGAAATTAAAAGGT-3’ and 5’-ATGAGACTGCCAATA-3’. Phylogenetic analysis showed
that the closest relatives to rice yellow dwarf (RYD)
agent are the sugarcane white leaf (SCWL), sugarcane
grassy shoot (SCGS), annual blue grass white leaf
(ABGWL), Bermuda grass white leaf (BGWL) and
Brachiana grass white leaf (BraWL) phytoplasmas
(Nakashima et al., 1996; Lee et al., 1997; Wongkaew et
al., 1997; Tran-Nguyen et al., 2000). Sequence analysis
of the RYD phytoplasma 16S rRNA gene indicated the
presence of several signature sequences that are unique
to members of the RYD group, 5’-AACACTG-3’ and
5’-GCAA-3’, which are not found in phytoplasmas assigned to different groups within the subclade. The sequence 5’-TATCAGACTA-3’ at positions 626-635 is also conserved in the RYD group with the exception of
the SCWL and BVK phytoplasmas that have a ‘T’ instead of a ‘C’ at nucleotide 629. An additional sequence,
5’-AAATCTTCGGATTT-3’ (61-75) is also conserved
between members of the RYD group, except in RYD
isolates which have a ‘T’ instead of ‘C’ at nucleotide 65.
The ‘Ca. P. oryzae’ 16S rRNA gene contained additional nucleotide variations at positions 149 (G to A),
181 (C to T), 263 (A/C to T), 1,148, (G to A), 1,409 (A
to G) and 1,447 (G to A), which differentiated this isolate from other phytoplasmas including the other members of the RYD group. Sequence analysis of the 16S
rRNA genes from ‘Ca. P. oryzae’ and BGWL phytoplasma showed these phytoplasmas share 96.3-97.9% similarity.
As also discussed above (see ‘Ca. P. cynodontis’), the
data suggested that several ‘Ca. Phytoplasma’ species
should be described in this group despite their relatively
high 16S rDNA similarity. Southern blot analysis shows
chromosomal and extrachromosomal DNA extracted
from ‘Ca. P. oryzae’ only hybridises with DNA of RYD
isolates and not with that from the closely related SCWL
phytoplasma (Nakashima et al., 1993). ‘Ca. P. oryzae’ is
transmitted by three species of leafhoppers (Nephotettix
spp.) that are found only in Asia (Nakashima et al.,
1993). RYD disease has only been detected in Asia and it
is thought that rice is the only natural host of this phytoplasma (Nakashima et al., 1993).
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
‘Ca. Phytoplasma phoenicium’ Verdin, Salar, Danet,
Choueiri, Jreijiri, El Zammar, Gélie, Bové, Garnier
(Verdin et al., 2003). This phytoplasma was detected in
Prunus amygdalus showing symptoms of lethal disease
of almond trees in Lebanon and Iran (Abou-Jawdah et
al., 2002; Verdin et al., 2003). It is associated with the
16S rRNA gene sequence accession AF515636, with
oligonucleotide sequence complementary to the unique
region of the 16S rRNA 5’-CCTTTTTCGGAAGGTATG-3’. Phylogenetic analysis showed that this phytoplasma clusters with members of the pigeon pea
witches’-broom (PPWB) group (Schneider et al., 1995),
known as 16SrIX according to Lee et al. (2000). A
primer pair, AlmF1/AlmR1 was defined by Verdin et al.
(2003) for the specific amplification of a DNA fragment
of about 1.5 kb from the 16S rDNA of ‘Ca. P. phoenicium’. The phytoplasmas associated with Picris echioides
yellows (PEY) and Knautia arvensis phyllody (KAP)
share 99% identity in their 16S rDNA sequence, and
the PPWB phytoplasma shares 98.5% identity. They
should therefore be referred to as related to ‘Ca. P.
phoenicium’.
‘Ca. Phytoplasma pini’ Schneider, Torres, Martìn,
Schröder, Behnke & Seemüller (Schneider et al.,
2005). This phytoplasma was detected in Pinus halepensis, Pinus silvestris showing abnormal shoot branching
and dwarfed needles in Germany and Spain. ‘Ca. P. pini’ is associated with the 16S rRNA gene sequence accession AJ632155, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’GGAAATCTTTCGGGATTTTAGT-3’ (positions 6788) and 5’-TCTCAGTGCTTAACGCTGTTCT-3’ (positions 603-624). According to the phylogenetic analysis
carried out by Schneider et al. (2005), the pine phytoplasma strains form a distinct branch and are only distantly related to other phytoplasmas. The closest relatives, sharing 94.5% or less 16S rRNA gene sequence
identity, are the phytoplasmas associated with diseases
of rice (‘Ca. P. oryzae’), coconut and chestnut (‘Ca. P.
castaneae’), included in the 16SrIV or 16SrXI groups by
Lee et al. (1998, 2000). No specific PCR primers have
been developed for the selective amplification of ‘Ca. P.
pini’, neither has the use of diagnostic restriction enzymes been demonstrated. However, the restriction
map of the 16S rDNA gene deduced from the sequence
suggests that specific patterns should results from the
digestion with the enzymes commonly used for the
characterization of phytoplasmas (AluI, RsaI, MseI,
HhaI and HinfI) of an amplification product obtained
using general phytoplasma-specific primers. The reference strain is Pin127SR from Pinus halepensis.
‘Ca. Phytoplasma prunorum’ Seemüller & Schneider
(Seemüller and Schneider, 2004). This phytoplasma is
thought to cause all the yellows and decline diseases of
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
Prunus species in Europe known as apricot chlorotic leaf
roll, ‘Molières disease’ of sweet cherry, Japanese plum
leptonecrosis, peach yellows (in Europe), peach rosette
(in Europe), peach vein clearing, decline of almond and
decline of flowering cherry (Lorenz et al., 1994; Jarausch
et al., 2000a). Most, if not all Prunus species can support
the replication of ‘Ca. P. prunorum’ (Lorenz et al., 1994;
Jarausch et al., 2000a), although some species may not
show obvious symptoms. In addition, ‘Ca. P. prunorum’
was detected in Fraxinus excelsior, Rosa canina, Celtis
australis, Corylus avellana (Marcone et al., 1996b; Jarausch et al., 2001). It is restricted to Europe and Turkey
(Jarausch et al., 2000a). ‘Ca. P. prunorum’ is associated
with the 16S rRNA gene sequence accession AJ542544,
with oligonucleotide sequences complementary to unique
region of the 16S rRNA 5’-AATACCCGAAACCAGTA3’ and 5’-TGAAGTTTTGAGGCATCTCGAA-3’. The
reference strain is isolate ESFY-G1, which has a chromosome 630 kb in length. ‘Ca. P. prunorum’ is phylogenetically closely related to ‘Ca. P. mali’ and ‘Ca. P. pyri’. The
reader is referred to the above description of ‘Ca. P.
mali’ for details of the methods used to detect and differentiate this group of organisms. In particular, ‘Ca. P.
prunorum’ can be distinguished by 16S rDNA digestion
with RsaI, BsaAI, SspI and SfcI from ‘Ca. P. mali’, and
from ‘Ca. P. pyri’ by using RsaI and SfcI.
‘Ca. Phytoplasma pyri’ Seemüller & Schneider
(Seemüller and Schneider, 2004). This phytoplasma is associated with the pear decline, an economically important disease that affects all Pyrus species (Seemüller et al.,
1998a). In addition, ‘Ca. P. pyri’ was also detected in
Corylus avellana (Marcone et al., 1996b). ‘Ca. P. pyri’ is
spread in Europe, Asia and the Americas. In California, a
disease of peach, named Peach Yellow Leaf Roll, may be
caused by a phytoplasma closely related to ‘Ca. P. pyri’,
but, due to the lack of conclusive data, Seemüller and
Schneider (2004) did not include the Peach Yellow Leaf
Roll phytoplasma in this species. ‘Ca. P. pyri’ is associated
with the 16S rRNA gene sequence accession AJ542543,
with oligonucleotide sequences complementary to unique
region of the 16S rRNA 5’-AATACTCAAAACCAGTA3’ and 5’-ATACGGCCCAAACTCATACGGA-3’. The
reference strain is isolate PD1, which has a chromosome
660 kb in length. ‘Ca. P. pyri’ is phylogenetically closely
related to ‘Ca. P. mali’ and ‘Ca. P. prunorum’. The reader
is referred to the above description of ‘Ca. P. mali’ for details about the methods used to detect and differentiate
this group of organisms. In particular, the ‘Ca. P. pyri’
phytoplasma can be distinguished by 16S rDNA digestion with SspI and BsaAI from ‘Ca. P. mali’, and from ‘Ca.
P. prunorum’ by using RsaI and SfcI.
‘Ca. Phytoplasma rhamni’ Marcone, Gibb, Streten &
Schneider (Marcone et al., 2003a). This phytoplasma
was detected in Rhamnus catharticus affected by witch-
Firrao et al.
257
es’-broom disease of buckthorn in South-western Germany. It is associated with the 16S rRNA gene sequence
accession number X76431 and AJ583009, with oligonucleotide sequence complementary to unique region of
the 16S rRNA 5’-CGAAGTATTTCGATAC-3’. Sequence analysis of the Buckthorn witches’-broom (BWB)
agent shows that this phytoplasma is more closely related
to members of the AP group than to other phytoplasma
subclades. The 16S rRNA genes of ‘Ca. P. rhamni’ shares
96% similarity with the AP group phytoplasmas, AP, ESFY, PD and PYLR and 95% similarity with the Spartium
witches’-broom (SpaWB) phytoplasma (‘Ca. P. spartii’),
which is also a member of the AP group. The 16S-23S
spacer region ‘Ca. P. rhamni’ differs from the corresponding region of ‘Ca. P. spartii’ in 16% of the nucleotide
positions and in 14-17% of nucleotide positions of the
spacer regions from the AP, PD, ESFY and PYLR phytoplasmas. RFLP analysis of the 16S rRNA gene of the
BWB agent shows that this phytoplasma is indistinguishable from other members of the AP group when the gene
sequence is digested with AluI and RsaI (Maürer and
Seemüller, 1996). However, the BWB phytoplasma gene
lacks SspI and SfcI restriction sites that are present in the
16S rRNA genes of other AP-group phytoplasmas. Like
‘Ca. P. prunorum’, ‘Ca. P. rhamni’ DNA has an additional BsaAI site at position 428. ‘Ca. P. rhamni’ has additional PvuII and AluI restriction sites that do not occur in
the gene sequences of other AP-fruit tree agents. There
are currently no BWB phytoplasma-specific primers.
‘Ca. Phytoplasma spartii’ Marcone, Gibb, Streten &
Schneider (Marcone et al., 2003a). This phytoplasma
was detected in Spartium junceum and Sarothamnus scoparius showing witches’-broom in Italy and Spain. It is
associated with the 16S rRNA gene sequence accession
number X92869, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-TTATCCGCGTTAC-3’. Sequence analysis of the Spartium
witches’-broom (SpaWB) agent shows this phytoplasma
is more closely related to members of the AP group than
to other phytoplasma subclades. The SpaWB phytoplasma shares 97.1-97.2% similarity with the AP, PD, ESFY
and PYLR phytoplasmas, which are members of the AP
group (Marcone et al., 1996a; Seemüller et al., 1998b). In
the 16S-23S spacer region (approximately 210 bp), the
SpaWB agent differs from the AP and PD phytoplasmas
in 12% of the nucleotide positions. Whereas, the SpaWB
phytoplasma differs from ESFY and PYLR phytoplasmas in 13 and 18% of nucleotide positions of the 16S23S spacer regions, respectively (Marcone et al., 1996a;
Seemüller et al., 1998b). The SpaWB phytoplasma 16S
rRNA gene contains a PuvII site at position 518 that is
unique to members of the AP group. RFLP analysis
shows that the SpaWB phytoplasma 16S rRNA gene has
the same banding pattern as the AP phytoplasma when
digested with RsaI, Sau3AI, HhaI and BsaAI. However,
258
Taxonomy of Candidatus phytoplasma
the SpaWB and AP phytoplasmas give different banding
patterns when digested with AluI and SspI (Marcone et
al., 1996a, 1997b). The SpaWB phytoplasma can be distinguished from other phytoplasmas assigned to the AP
group based on differences in 16S rRNA gene SspI and
SfcI banding patterns. The 16S rRNA genes of SpaWB,
PD, ESFY and PYLR phytoplasmas lack an SspI restriction site at nucleotide 419 that is present in the AP phytoplasma. The PD, ESFY and PYLR phytoplasmas have
SfcI sites at positions 630 and 1,000, the SpaWB phytoplasma lacks the restriction site at position 1,000 and the
AP phytoplasma lacks the SfcI site at position 630. Specific detection of the SpaWB phytoplasma is possible by
using either the fU5 or P1 forward primers paired with
the rSP reverse primer (5’-GCTAATTAGAAATATCAACTA-3’) (Marcone et al., 1996a). The phytoplasma
associated with sarothamnus witches’-broom (SSWB)
has the same banding pattern as the SpaWB phytoplasma when digested with AluI, RsaI, Sau3AI, MseI, TaqI,
SspI and BsaI but differs from the SpaWB phytoplasma
when digested with HhaI (Marcone et al., 1997b). A
product can be amplified from the SSWB phytoplasma
by using the SpaWB specific primer combinations
(fU5/rSP or P1/rSP) (Marcone et al., 1997b).
‘Ca. Phytoplasma trifolii’ Hiruki & Wang (Hiruki
and Wang, 2004). This phytoplasma was detected in alsike clover (Trifolium hybridum) plants exhibiting symptoms of virescence and proliferation of shoots (Hiruki
and Chen, 1984). ‘Ca. P. trifolii’ is associated with the
16S rRNA gene sequence accession number AY390261,
with oligonucleotide sequences complementary to
unique regions of the 16S rRNA 5’-TTCTTACGA-3’
and 5’-TAGAGTAAAAGCC-3’. Phylogenetic analysis
shows the reference strain, CPR, and its close relatives
Brinjal little leaf (BLL), Fragaria multicipita phytoplasma
(FM) and Illinois elm yellows (ILEY) phytoplasmas
form a subcluster and were different from all other phytoplasmas, with sequence divergences of ≥2.5%. RFLP
analysis of 16S rRNA genes from CPR, the Alfalfa witches’-broom (AWB), Beet leafhopper-transmitted virescence (BLTV), Potato witches’-broom (PWB), Tomato
big bud from California (TBBc) and Potato yellows (PY)
phytoplasmas showed these phytoplasmas shared very
similar or identical banding patterns when digested with
EcoRII, HaeIII, HhaI, HinfI, HpaII, KpnI, RsaI, Sau3AI
and ThaI (Lee et al., 1993, 1998; Khadhair et al., 1997;
Wang et al., 1998; Wang and Hiruki, 2001). The AWB,
BLTV, CPR, PWB and TBBc phytoplasmas produced
unique RFLP profiles with restriction endonucleases
AluI and MseI (Lee et al., 1993, 1998; Khadhair et al.,
1997; Wang et al., 1998, Wang and Hiruki, 2001). The
banding patterns of the 16S rRNA genes of the Fragaria
multicipita phytoplasma (FM) and Illinois elm yellows
(ILEY) phytoplasmas were only differentiated from the
CPR phytoplasma by a single restriction site (AluI for
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
FM and HhaI for ILEY) (Jomantiene et al., 1998; Jacobs
et al., 2003). Based on RFLP banding profiles the phytoplasma strains named CPR, AWB, BLTV, PWB, PY, TBBc, FM, BLL, ILEY, ILEY1, ILEY2 are related to ‘Ca.
P. trifolii’. According to Hiruki and Wang (2004), they
were designated as different subgroups as follows: the
AWB, BLTV, CPR, PWB, PY and TBBc phytoplasmas
are assigned to CP subgroup A, the FM phytoplasma is
the only member of CP subgroup B, and BLL, ILEY,
ILEY1 and ILEY2 phytoplasmas are members of subgroup C. ‘Ca. P. trifolii’ and related strains are reported
to occur in Canada, United States of America and India.
‘Ca. Phytoplasma ulmi’ Lee, Martini, Marcone &
Zhu (Lee et al., 2004b). This phytoplasma is associated
with gene sequence accessions AY197655, AY197675,
AY197690, corresponding to 16S rRNA, rpl22-rps3 and
secY genes. Oligonucleotide sequences of unique regions
are 5’-GGAAA-3’ (positions 827-835) and 5’-CGTTAGTTGCC-3’ positions (1,098-1,108) in the 16S
rRNA gene, 5’-TTACGCTTGCC-3’ (positions 284-294),
5’-CATTTAATAAAATTGCTATT-3’ (positions 739758) and 5’-AAATTCTATTTCTATGGGAAT-3’ (positions 910-932) in the rpl22-rps3 gene fragment and 5’TTTGATCCAATGTTAA-3’ (positions 350-365), 5’GTCTTTCGGTCATGGATTGA-3’ (positions 595-614),
5’-ATTTAGTCTAAT-3’ (positions 616-627), 5’-CAAA
TAGAACAA-3’ (positions 1,053-1,064) in the secY
gene. By phylogenetic analysis of 16S rDNA sequences,
a distinct group of related phytoplasmas associated with
several destructive diseases of elm, grapevine and several fruit trees was identified and named Elm Yellows
strain cluster (Seemüller et al., 1994) or 16SrV (Lee et
al., 1998, 2000). The phytoplasmas assigned to 16SrV/
Elm Yellows group include those associated with flavescence dorée (FD) in grapevine (Daire et al., 1993),
Rubus stunt (RuS) in blackberries (Mäurer and
Seemüller, 1994), cherry lethal yellows (CLY) in cherry
(Zhu et al., 1997), jujube witches’-broom (JWB) in Ziziphus spp. (Zhu et al., 1997), alder yellows (ALY) in
alder (Mäurer et al., 1993), spartium witches’-broom in
Spartium sp. in Italy (SpaWB-EY, Marcone et al.,
1996a), eucalyptus little leaf phytoplasma in Eucalyptus
sp. in Italy (Marcone et al., 1996c), and the phytoplasmas detected in Apocynum cannabinum (Griffiths et al.,
1999a), in Virginia creeper (Parthenocissus quinquefolia)
(Harrison et al., 2001) and in tomato (Del Serrone et al.,
2001). Although the members of the 16SrV/Elm Yellows group share relatively high similarities in their 16S
rDNA sequence (98.6 to 99.9%), they constitute several
strain clusters with different ecological niches and geographical distribution.
Several ‘Ca. Phytoplasma’ species should therefore be
described within the group, and the task has only partially been accomplished at the time of writing. The phytoplasma associated with the elm yellows disease was
Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263
Firrao et al.
259
named ‘Ca. P. ulmi’. The other strains belonging to
16SrV could be differentiated from ‘Ca. P. ulmi’ by restriction analysis of 16S rDNA (diagnostic enzymes: RsaI
and BfaI), or the rpl22-rps3 ribosomal protein gene fragment (diagnostic enzymes: Tsp509I or MseI).
Elm Yellows/16SrV group-specific primer pair were
designed for both the 16S rRNA and the ribosomal protein gene fragment. Primers R16(V)R1/F1 (Lee et al.,
1994) were used to amplify an 1,100 bp fragment of the
16S rDNA gene. For the ribosomal protein gene operon,
Lee et al. (2004b) suggested a nested PCR using the ribosomal protein primer pair rp(V) F1/rpR1 specific to
the EY phytoplasma group followed by another EY
group-specific primer pair, rp(V)F1A/rp(V)R1A yielding
a DNA fragment of about 1.2 kb. ‘Ca. P. ulmi’ is widespread in Europe, Asia and North America.
Ahrens U., Seemüller E., 1992. Detection of DNA of plant
pathogenic mycoplasma-like organisms by a polymerase
chain reaction that amplifies a sequence of the 16S rRNA
gene. Phytopathology 82: 828-832.
‘Ca. Phytoplasma ziziphi’ Jung, Sawayanagi, Kakizawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Hibi &
Namba (Jung et al., 2003a). This phytoplasma was detected in Zizyphus jujuba affected by Jujube witches’broom (JWB) disease in China, Japan and Korea. ‘Ca. P.
ziziphi’ is associated with the 16S rRNA gene sequence
accession AB052879, with oligonucleotide sequence complementary to unique regions of the 16S rRNA 5’TAAAAAGGCATCTTTTTGTT-3’ and 5’-AATCCGGACTAAGACTGT-3’. Sequence comparisons showed
that the ‘Ca. P. ziziphi’ phytoplasma 16SrDNA sequences
were most similar to those of the strains belonging to the
Elm Yellows strain cluster or 16SrV group (see above ‘Ca.
P. ulmi’). Digestion with either HpaII or RsaI distinguished ‘Ca. P. ziziphi’ phytoplasmas from all other members of the Elm Yellows group. A primer set (JWBF1: 5’TAAAAAGGCATCTTTTTGTT-3’, JWBR1: 5’-AATCCGGACTAAGACTGT-3’) was designed by Jung et al.
(2003a) from the JWB phytoplasma 16S rDNA sequence to detect ‘Ca. P. ziziphi’ specifically. PCR using
primers JWBF1 and JWBR1 amplified an 1,119 bp
DNA fragment.
Barros T.S.L., Davis R.E., Resende R.O., Dally E.L., 2002.
Erigeron witches’-broom phytoplasma in Brazil represents
new subgroup VII-B in 16S rRNA gene group VII, the ash
yellows phytoplasma group. Plant Disease 86: 1142-1148.
ACKNOWLEDGEMENTS
Work on phytoplasmas in the Firrao lab is supported
by the “Ministero delle Politiche Agrarie e Forstali”,
Italy. Gibb and Streten thank the Cooperative Research
Centre Tropical Plant Protection and the Australian Research Council for financial support. The advice on restriction endonucleases by Dr. A. Bertaccini is gratefully
acknowledged.
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Received 20 September 2005
Accepted 3 November 2005
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