International Journal of Systematic and Evolutionary Microbiology (2012), 62, 2279–2285
DOI 10.1099/ijs.0.038273-0
Differentiation and classification of phytoplasmas in
the pigeon pea witches’-broom group (16SrIX): an
update based on multiple gene sequence analysis
I.-M. Lee,1 K. D. Bottner-Parker,1 Y. Zhao,1 A. Bertaccini2 and R. E. Davis1
Correspondence
I.-M. Lee
[email protected]
1
Molecular Plant Pathology Laboratory, USDA, ARS, Beltsville, MD 20705, USA
2
DiSTA, Patologia Vegetale, Alma Mater Studiorum, University of Bologna, 40126 Bologna, Italy
The pigeon pea witches’-broom phytoplasma group (16SrIX) comprises diverse strains that
cause numerous diseases in leguminous trees and herbaceous crops, vegetables, a fruit, a nut
tree and a forest tree. At least 14 strains have been reported worldwide. Comparative
phylogenetic analyses of the highly conserved 16S rRNA gene and the moderately conserved rplV
(rpl22)–rpsC (rps3) and secY genes indicated that the 16SrIX group consists of at least six
distinct genetic lineages. Some of these lineages cannot be readily differentiated based on
analysis of 16S rRNA gene sequences alone. The relative genetic distances among these closely
related lineages were better assessed by including more variable genes [e.g. ribosomal protein
(rp) and secY genes]. The present study demonstrated that virtual RFLP analyses using rp and
secY gene sequences allowed unambiguous identification of such lineages. A coding system is
proposed to designate each distinct rp and secY subgroup in the 16SrIX group.
INTRODUCTION
Phytoplasmas are unculturable, wall-less, plant-pathogenic
bacteria that have been shown to cause diseases in more
than 1000 plant species (Lee et al., 2000; Bertaccini, 2007;
Hogenhout et al., 2008). Molecular methods using highly
conserved biomarkers, notably the 16S rRNA gene, have
been employed for the detection, differentiation and classification of phytoplasmas (Lee et al., 1993, 1998; Schneider
et al., 1995; Seemüller et al., 1994, 1998). So far, 31 major
phytoplasma groups (termed 16Sr groups) have been identified and designated based on RFLP analysis of 16S rRNA
gene sequences (Lee et al., 1998, 2000; Wei et al., 2007; Zhao
et al., 2009). Thirty-one ‘Candidatus Phytoplasma’ species
have been proposed in accordance with the guidelines established by the IRPCM Phytoplasma/Spiroplasma Working
Team – Phytoplasma Taxonomy Group (2004). However,
the resolving power of the highly conserved 16S rRNA gene
sequence is relatively limited for classification of closely
related strains. Therefore, many biologically and ecologically
distinct strains that are closely related cannot be readily
differentiated on the basis of the 16S rRNA gene sequence
alone. Multilocus analyses using the 16S rRNA gene and less
conserved genetic markers, such as ribosomal protein (rp),
secA and secY genes, have been employed recently for finer
Abbreviation: rp, ribosomal protein.
The GenBank/EMBL/DDBJ accession numbers for the sequences
determined in this study are given in Fig. 2.
Two supplementary figures are available with the online version of this
paper.
038273 G
Printed in Great Britain
differentiation of distinct strain lineages in many important
phytoplasma groups (Lee et al., 2006, 2010; Hodgetts et al.,
2008; Martini et al., 2007). Extended classification schemes
based on multilocus analyses have greatly enhanced the
capacity for differentiation and identification of closely
related, distinct phytoplasma strains and may be highly
significant for disease epidemiological studies or for
quarantine regulations.
Strain differentiation and classification in the pigeon pea
witches’-broom group (16SrIX) have been based on
sequence analyses of two conserved biomarkers, the 16S
rRNA and rp genes (Martini et al., 2007). However, analysis
based on secY gene sequences is still lacking for this group.
Recently, many additional novel strains in group 16SrIX
have been identified, including strains associated with
juniper witches’-broom (Davis et al., 2010) and blueberry
stunt (P. Bagadia and others, unpublished) diseases. In this
communication, we report an updated classification scheme
for the pigeon pea witches’-broom group phytoplasmas
based on 16S rRNA, rp and secY gene sequences.
METHODS
Phytoplasma strains and nucleic acid preparation. All 16SrIX
phytoplasma strains used in this study are listed in Table 1. Many of
the phytoplasma strains are available in our inventory (as extracted
total nucleic acid preparations) or from the International Phytoplasmologist Working Group (IPWG) collection at the University of
Bologna. Total nucleic acid was extracted according to the methods
described by Lee et al. (1993) and Green et al. (1999) using leaf
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 10:33:44
2279
I.-M. Lee and others
Table 1. Strain designations, associated diseases, origins, phytoplasma 16Sr group/subgroup affiliations and rp and secY subgroup
classifications of phytoplasma strains used in this study
After strain names, c1, c2, c3, etc. denote different clones.
Strain
PPWB
PPWB c1
PPWB c2
PPWBfl
PPWBja
PPWBpr
RLL-FL
PW1
BrazHLB
AlmWB1
AlmWB2
AlmWB3
AlmWB-A112
AlmWB112 c3
AlmWB112 c4
AlmWB112 c5
‘Ca. P. phoenicium’
strain A4
‘Ca. P. phoenicium’
strain 21
KAP
KAP539 c1
KAP585 c1
KAP585 c2
PEY
PEY1 c3
PEY2 c3
PEY2 c4
PEY2 c5
PEYAB c2
NaxY
NaxY c1
NaxY c2
NaxY c3
NaxY c4
BraR
EchinWB
LactPh
JunWB-2A
JunWB-2A c1
JunWB-2A c2
JunWB-2C
JunWB-2C c1
JunWB-2C c2
JunWB-2C c3
BBS3NJ
BBS3NJ c1
BBS40NJ c1
BBS3NJ c5
BBS3NJ c6
2280
Associated disease
Geographical
origin
16S rRNA gene RFLP
classification
Subgroup classification
rp
secY
Pigeon pea witches’-broom
Pigeon pea witches’-broom
Pigeon pea witches’-broom
Pigeon pea witches’-broom
Pigeon pea witches’-broom
Pigeon pea witches’-broom
Rhynchosia little leaf
Periwinkle yellows
Brazilian HLB disease-associated
phytoplasma
Almond witches’-broom
Almond witches’-broom
Almond witches’-broom
Almond witches’-broom
Almond witches’-broom
Almond witches’-broom
Almond witches’-broom
Almond witches’-broom
Florida, USA
Florida, USA
Florida, USA
Florida, USA
Jamaica
Puerto Rico
Florida, USA
Colombia
Brazil
16SrIX-A
16SrIX-A
16SrIX-A
16SrIX-A
16SrIX-A
16SrIX-A
16SrIX-A
16SrIX-A*
16SrIX-A
rp(IX)-A1
–
–
rp(IX)-A1
rp(IX)-A1
rp(IX)-A1
rp(IX)-A1
–
–
–
secY(IX)-A1
secY(IX)-A1
–
–
–
–
–
–
Lebanon
Lebanon
Lebanon
Lebanon
Lebanon
Lebanon
Lebanon
Lebanon
16SrIX-B
16SrIX-B
16SrIX-B
16SrIX-B
16SrIX-B
16SrIX-B
16SrIX-B
16SrIX-B
–
–
–
rp(IX)-B1
–
–
–
–
–
–
–
–
secY(IX)-B1
secY(IX)-B1
secY(IX)-B1
–
Almond witches’-broom
Iran
16SrIX-B
–
–
Knautia arvensis phyllody
Knautia arvensis phyllody
Knautia arvensis phyllody
Knautia arvensis phyllody
Picris echioides yellows
Picris echioides yellows
Picris echioides yellows
Picris echioides yellows
Picris echioides yellows
Picris echioides yellows
Periwinkle virescence
Periwinkle virescence
Periwinkle virescence
Periwinkle virescence
Periwinkle virescence
Brassica rapa phyllody
Echinops witches’-broom
Lactuca sativa phyllody
Juniper witches’-broom
Juniper witches’-broom
Juniper witches’-broom
Juniper witches’-broom
Juniper witches’-broom
Juniper witches’-broom
Juniper witches’-broom
Blueberry stunt
Blueberry stunt
Blueberry stunt
Blueberry stunt
Blueberry stunt
Italy
Italy
Italy
Italy
Italy
Italy
Italy
Italy
Italy
Italy
Italy
Italy
Italy
Italy
Italy
India
Oman
Iran
Oregon, USA
Oregon, USA
Oregon, USA
Oregon, USA
Oregon, USA
Oregon, USA
Oregon, USA
New Jersey, USA
New Jersey, USA
New Jersey, USA
New Jersey, USA
New Jersey, USA
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-C
16SrIX-CD
16SrIX-C
16SrIX-D
16SrIX-D
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-E
rp(IX)-C2
–
–
–
rp(IX)-C1
–
–
–
–
–
–
rp(IX)-C1
rp(IX)-C1
–
–
–
–
–
–
–
–
–
rp(IX)-E1
rp(IX)-E1
rp(IX)-E1
–
rp(IX)-E2
rp(IX)-E2
–
–
–
secY(IX)-C2
secY(IX)-C2
secY(IX)-C2
–
secY(IX)-C1
secY(IX)-C1
secY(IX)-C1
secY(IX)-C1
secY(IX)-C1
–
secY(IX)-C1
secY(IX)-C1
–
–
–
–
–
–
secY(IX)-E1
secY(IX)-E1
secY(IX)-E1
secY(IX)-E1
–
–
–
–
secY(IX)-E2
secY(IX)-E2
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 62
IP: 88.99.165.207
On: Sun, 18 Jun 2017 10:33:44
Multiple gene-based 16SrIX phytoplasma phylogeny
Table 1. cont.
Strain
BBS41NJ
BBS41NJ c3
BBS41NJ c6
GLL-Hon
Associated disease
Blueberry stunt
Blueberry stunt
Blueberry stunt
Honduran Gliricidia little leaf
Geographical
origin
New Jersey, USA
New Jersey, USA
New Jersey, USA
Honduras
16S rRNA gene RFLP
classification
16SrIX-E
16SrIX-E
16SrIX-E
16SrIX-F
Subgroup classification
rp
secY
–
–
–
rp(IX)-F1
–
secY(IX)-E2
secY(IX)-E2
–
*Variant of subgroup 16SrIX-A.
DVariant of subgroup 16SrIX-C.
midribs or other tissues from the original hosts that were infected by
these phytoplasma strains. The strains were previously characterized
and identified by RFLP or sequence analysis of the 16S rRNA gene
(Abou-Jawdah et al., 2002; Davis et al., 2010; Lee et al., 1998, 2000;
Martini et al., 2007; Seemüller et al., 1998). Blueberry stunt phytoplasma strains were prepared in a separate study (P. Bagadia and
others, unpublished).
PCR amplification, cloning and sequencing
16S rRNA and rp genes. A nested PCR using primer pair P1/16S-SR
(Deng & Hiruki, 1991; Lee et al., 2004) followed by P1A/16S-SR (Lee
et al., 2004) was used to amplify nearly full-length phytoplasma 16S
rRNA genes (about 1.5 kb). A nested PCR using primer pair rpF1/
rpR1 (Lim & Sears, 1992) followed by rpF1/rp(I)R1A (Lee et al.,
2003) was used to amplify a phytoplasma DNA segment (about
1.2 kb) of the rp operon that encompassed genes rplV (rpl22) and
rpsC (rps3). For PCR amplification, 38 cycles were conducted in an
automated thermal cycler (MJ Research DNA Thermal Cycler PTC200) with GeneAmp High Fidelity polymerase (Life Technologies).
PCR mixtures contained 1 ml undiluted DNA preparation (approx.
10–30 ng), 200 mM each dNTP, 0.4 mM each primer and 1.25 U
polymerase. The following conditions were used: denaturation at 94 uC
for 1 min, annealing for 2 min at 55 uC (50 uC for rp sequence
amplification) and primer extension for 3 min (7 min in the final
cycle) at 72 uC. A negative control devoid of DNA template in the
reaction mixture was included in all PCR assays. One microlitre of
diluted (1 : 30) PCR product from the first amplification was used as
the template in the nested PCR. PCR products (3 ml) were electrophoresed through a 1 % agarose gel, stained in ethidium bromide and
visualized with a UV transilluminator. For strains whose sequences
were unavailable in GenBank, the amplicons were purified using PCR
Kleen Spin Columns (Bio-Rad) and cloned into Escherichia coli TOP10
by using the TOPO TA Cloning kit (Invitrogen) according to the
manufacturer’s instructions. The clones were sequenced with an
automated DNA sequencer (ABI Prism model 3730) at the Center for
Biosystems Research, University of Maryland, College Park, MD, USA.
secY gene. A nested PCR using primer pair L15F1/MapR1 (Lee et al.,
2010) followed by primer pair L15F2(IX)/MapR2(IX) (Table 2) was
used to amplify a phytoplasma DNA segment (about 1.9 kb) of the
partial spc operon that includes the complete secY gene from some of
the phytoplasma strains listed in Table 1. Subsequently, additional
primers were designed within the L15F1/MapR1 amplicon to facilitate
sequencing of the spc operon (Table 2). All primer positions are
indicated on Fig. 1. For PCR amplification, 35 cycles were conducted
in an automated thermal cycler (MJ Research DNA Thermal Cycler
PTC-200) with TaKaRa LA Taq polymerase (Takara Mirus Bio). The
PCR was performed in mixtures containing 1 ml DNA extract
(approx. 10–30 ng), 400 mM each dNTP, 0.8 mM each primer and
2.5 U polymerase. The following conditions were used: denaturation
at 94 uC for 30 s (1 min for the first cycle), annealing for 1 min at
50 uC and primer extension for 5 min at 68 uC (10 min in the final
cycle at 72 uC). A negative control devoid of DNA template in the
reaction mixture was included in all the PCR assays. PCR products
(3 ml) were electrophoresed through a 1 % agarose gel, stained in
ethidium bromide and visualized with a UV transilluminator. The
amplicons were purified and cloned using the methods described in
the previous section.
Phylogenetic analyses. Sequences of 16S rRNA (1348 bp), rp
(1156 bp containing rplV and rpsC genes) and secY (complete secY
gene, ranging in length from 1260 to 1311 bp) genes from
representative phytoplasma strains in the 16SrIX group were trimmed
from the respective amplicons obtained in the present study or from
the GenBank database (accession numbers listed in Fig. 2) and used
for phylogenetic analyses. Sequences from 16SrIX group phytoplasma
strains and from clover proliferation phytoplasma CP (16SrVI-A)
Table 2. Primers designed in this study for amplification of the partial spc operon of the 16SrIX phytoplasma group
Primer(s)
L15F2(IX)/MapR2(IX)
L15F4(IX)/MapR7(IX)
MapR4(IX)
secYF2(IX)
secYF4(IX)
secYR1(IX)
secYR2(IX)
http://ijs.sgmjournals.org
Sequence(s) (5§–3§)
Annealing
temperature (6C)
TTCAAAGAATTCCTAAAAGAGG/GTACAACTGCTTCGTTTACAGA
GGAAATGTTGAAATAATAAGGTA/TGTAACCAAAACAAAATAGAACC
CAATTCAGCTGCTATCATATCTA
CTTAGCTTTTGGATATGGTTT
ATCCCTATTCTTTATTATACTA
CCCACTATAATTAAAAGACT
TAGTATAATAAAGAATAGGGAT
50
50
49
48
40
41
40
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 10:33:44
2281
I.-M. Lee and others
Fig. 1. Schematic gene arrangement and
positions of primers designed in this study for
amplification of the partial spc operon for
group 16SrIX phytoplasma strains. Positions
to which each amplicon was trimmed for in
silico RFLP analysis are indicated by arrowheads.
were aligned using CLUSTAL V from the LaserGene software MEGALIGN
program (DNASTAR). Cladistic analyses were performed with PAUP
version 4.0 (Swofford, 1998) on a Power Mac G4. Uninformative
characters were excluded from analyses. Phylogenetic trees were
constructed by a heuristic search (or neighbour-joining algorithm)
via random stepwise addition, implementing the tree bisection and
reconnection branch-swapping algorithm to find the optimal tree(s)
(Gundersen et al., 1994). CP phytoplasma was used as the outgroup to root the trees. The analysis was replicated 1000 times.
Bootstrapping was performed to estimate the stability and support for
the inferred clades.
Strain differentiation by computer-simulated RFLP analysis of
16S rRNA, rp and secY gene sequences. Virtual RFLP analyses of
16S rRNA gene sequences were performed by use of the iPhyClassifier
(Zhao et al., 2009). Collective RFLP patterns were based on analysis
with 17 restriction enzymes. For virtual RFLP analyses, rp DNA
fragments [rpF1/rp(I)R1A amplicons ranging in length from 1233 to
1257 bp], containing the rplV and rpsC genes, and the complete secY
gene (ranging in length from 1260 to 1311 bp) were used. In silico
restriction digestion and pairwise virtual RFLP pattern comparison were
performed using a modified Perl program developed previously (Wei
et al., 2008), with the inclusion of an additional restriction enzyme,
Tsp509I. Virtual gel images were generated using the program VGelME
and key enzymes that distinguish different (group/subgroup) pattern
types were identified by using the program VGelMS. Both VGelME and
VGelMS programs were described previously (Zhao et al., 2009).
RESULTS
rp and secY gene sequences of 16SrIX group
phytoplasmas
Cloned rp DNA fragments (1233–1257 bp) contained both
rplV and rpsC open reading frames. The rplV gene from all
16SrIX group strains contained 381 bp, and the rpsC gene
contained 747 bp. The length of the secY gene nucleotide
sequence was variable among strains in the 16SrIX group:
strains BBS3NJ c5, BBS3NJ c6, BBS41NJ c3, BBS41NJ c6,
JunWB-2A c1, JunWB-2A c2, JunWB-2C c1 and JunWB2C c2 contained 1275 bp; strains NaxY c1, NaxY c2, PEY1
c3, PEY2 c3, PEY2 c4, PEY2 c5 and PEYAB c2 contained
1290 bp; strains PPWB c1 and PPWB c2 contained
1278 bp; strains KAP539 c1, KAP585 c1 and KAP585 c2
contained 1260 bp; and strains AlmWB112 c3, AlmWB112
c4 and AlmWB112 c5 contained 1311 bp.
Comparative phylogenetic analyses based on 16S
rRNA, rp and secY genes
16S rRNA gene-based phylogenetic analysis using available
sequences from GenBank and new sequences obtained in
2282
the present study resolved one representative tree (Fig. 2a).
It showed that the 22 strains belonging to the pigeon pea
phytoplasma witches’-broom group (16SrIX) were delineated into two major phylogenetic subclades and at least
six distinct lineages, which correspond to subgroups 16SrIXA (strains PPWB, BrazHLB, RLL-FL and subgroup variant
PW1), 16SrIX-B (strains ‘Ca. Phytoplasma phoenicium’ A4,
‘Ca. P. phoenicium’ 21, AlmWB1, AlmWB2 and AlmWB3),
16SrIX-C (strains KAP, BraR, PEY, NaxY, NaxY c3 and
subgroup variant NaxY c4) and 16SrIX-E (strains JunWB2A, JunWB-2C, BBS3NJ and BBS41NJ). Strains EchinWB
and LuctPh-Iran represent a novel 16SrIX subgroup,
16SrIX-D, based on virtual RFLP analysis. Strain GLL-Hon
represents a tentative novel 16SrIX subgroup, 16SrIX-F. rp
and secY gene-based trees (Fig. 2b, c) have overall topologies
similar to that of the 16S rRNA gene-based tree, containing
two major subclades that separated the ‘Ca. P. phoenicium’
strain cluster from the remaining members of the 16SrIX
group.
Because of the greater variability of secY and rp genes, the
16Sr subgroup delineation based on these two genes was
highly discriminating, as reflected by the identification of
two additional distinct lineages (with high bootstrap values)
within subgroups 16SrIX-C and 16SrIX-E. The mean genetic
distances among 16SrIX subgroups as assessed by sequence
similarities of 16S rRNA (based on a 1384 bp DNA fragment), rp (based on a 1156 bp fragment containing rplV and
rpsC) and secY (based on entire secY gene sequences of 1260–
1311 bp) genes were 98.3–99.8 % (16S rRNA), 89.9298.2 %
(rp) and 82.0–97.3 % (secY). The sequence variation of the
secY and rp genes among 16SrIX subgroups was much
greater than that of the 16S rRNA gene, allowing identification of distinct genetic lineages that could not be readily
resolved by analysis of the 16S rRNA gene sequence. For
example, within subgroup 16SrIX-C, the sequence similarities between strains of Picris echioides yellows and Naxos
yellows and strains of Knautia arvensis phyllody are 99.6 %
(based on 16S rRNA gene), 94.5 % (rp) and 91.1 % (secY)
and, within 16SrIX-E, the sequence similarities between
strains of juniper witches’-broom phytoplasma and strains
of blueberry stunt phytoplasma are 99.5 % (16S rRNA gene),
98.2 % (rp) and 97.2 % (secY). Based on analysis of rp and
secY gene sequences, strains of Picris echioides yellows and
Naxos yellows phytoplasmas clearly represent one distinct
lineage and strains of Knautia arvensis phyllody another
distinct lineage. In addition, strains of juniper witches’broom phytoplasma and strains of blueberry stunt phytoplasma represent two separate lineages.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 62
IP: 88.99.165.207
On: Sun, 18 Jun 2017 10:33:44
74
CP (AY500130)
(a)
1 change
90
99
(b)
BrazHLB (HQ423159)
JunWB-2C c1 (JN712785)
100
GLL-Hon (AF361017)
JunWB-2C c2 (JN712784)
10 changes
PW1 (EU816776)
99
JunWB-2C
c3 (JN712783)
PPWB (AF248957)
RLL-FL (AF361019
100 BBS3NJ c1 (JN712787)
BBS3NJ (JN791268)
93
BB40NJ c1 (JN712786)
BBS41NJ (JN791267)
99
KAP
(EF186801)
JunWB-2A (GQ925919)
98
NaxY c1 (JN712782)
JunWB-2C (GQ925918)
100
71 BraR (GU111554)
NaxY c2 (JN712781)
KAP (EF186823)
PEY (EF186802)
NaxY (HQ589191)
PPWB (EF193383)
PEY (Y16389)
NaxY c3 (JN791266)
PPWBfl (EF183495)
NaxY c4 (JN791265)
PPWBja (EF183496)
94
EchinWB (GU902973)
RLL-FL (EF186799)
LactPh (DQ889748)
100
AlmWB1 (AF390136)
PPWBpr (EF183497)
AlmWB3 (AF455038)
GLL-Hon (EF186800)
AlmWB2 (AF390137)
AlmWB-A112
(EF186803)
‘Ca. P. phoenicium’ A4 (AF515636)
CP (EF183486)
‘Ca. P. phoenicium’ 21 (AF515637)
(c)
100
BB3NJ c5 (JN791260)
BB3NJ c6 (JN791261)
BBS41NJ c3 (JN791259)
BBS41NJ c6 (JN791258)
93
JunWB-2A c1 (JN791257)
10 changes
100 JunWB-2A c2 (JN791256)
JunWB-2C c1 (JN791255)
JunWB-2C c2 (JN791254)
NaxY c2 (JN791249)
NaxY c1 (JN791250)
PEY1 c3 (JN791248)
100
85
PEY2 c3 (JN791247)
PEY2 c4 (JN791246)
PEY2 c5 (JN791245)
PEYAB c2 (JN791244)
PPWB c1 (JN791243)
100
PPWB c2 (JN791242)
KAP539 c1 (JN791253)
100
KAP585 c1 (JN791252)
KAP585 c2 (JN791251)
86 AlmWB112 c3 (JN791264)
100
AlmWB112 c5 (JN791262)
AlmWB112 c4 (JN791263)
CP (GU004315)
Multiple gene-based 16SrIX phytoplasma phylogeny
Strain differentiation by virtual RFLP analyses of
16S rRNA, rp and secY genes and subgroup
designations
To compare the efficacy of 16S rRNA, rp and secY genes for
differentiation of 16Sr IX group phytoplasma strains,
http://ijs.sgmjournals.org
Fig. 2. Phylogenetic trees reconstructed by parsimony analyses of
partial 16S rRNA (a), rp (1156 bp containing rplV and rpsC genes)
(b) and full secY (c) gene sequences from group 16SrIX
phytoplasma strains. Clover proliferation (CP) phytoplasma was
used as the outgroup to root the trees. Branch lengths are
proportional to the number of inferred character state transformations. Bootstrap values are shown on the main branches. Bars, 1
(a) and 10 (b, c) inferred character state changes. GenBank
accession numbers are indicated in parentheses. Sequences
submitted to GenBank in this study are highlighted in bold.
Phytoplasma strain abbreviations are defined in Table 1.
computer-simulated virtual RFLP patterns were generated
using the iPhyClassifier (Zhao et al., 2009). Based on the
data available, cut-off similarity coefficients that separate
subgroups are ¡97 % (for 16S rRNA gene sequence),
¡91 % (rp genes) and ¡90 % (secY) (Fig. S1, available in
IJSEM Online). In the present study, based on virtual RFLP
analysis of 16S rRNA gene sequences (about 1.2 kb), we
identified that strains LactPh and EchinWB, whose sequences
are available in GenBank, represented a novel 16SrIX subgroup. Because of the presence of insufficient characters on
the 1.2 kb fragment of the 16S rRNA gene sequence, RFLP
analysis was unable to differentiate among closely related
lineages, as shown on the phylogenetic tree (Fig. 2a). In contrast, rp or secY gene-based RFLP analysis readily identified
such genetically distinct lineages, as shown in Fig. 2(b, c).
Based on cut-off similarity coefficients that separate subgroups, two distinct lineages were identified within two
16SrIX subgroups, 16SrIX-C and 16SrIX-E. Examples of key
enzymes used for virtual RFLP analyses that separated these
subgroups are AluI, TaqI and HaeIII (16SrIX subgroups),
AluI, HhaI and RsaI [rp(IX) subgroups] and AluI, HhaI and
MseI [secY(IX) subgroups] (Fig. S2). rp- and secY-based
distinct lineages identified within each 16SrIX subgroup
were designated rp and secY subgroups, respectively.
The coding systems for rp and secY subgroups were designed
to reflect the 16Sr group and subgroup of the phytoplasma
strain described. The codes for the proposed rp and secY
subgroups in the 16SrIX group were as follows: rp(IX)-A1
(strains PPWB, PPWBfl, PPWBja, RLL-FL and PPWBpr),
rp(IX)-B1 (strain AlmWB-A112), rp(IX)-C1 (strains PEY,
NaxY c1 and NaxY c2), rp(IX)-C2 (strain KAP), rp(IX)-E1
(strains JunWB-2C c1, JunWB-2C c2 and JunWB-2C c3),
rp(IX)-E2 (strains BBS3NJ c1 and BBS3NJ40 c1), rp(IX)-F1
(strain GLL-Hon), secY(IX)-A1 (strains PPWBc1 and
PPWBc2), secY(IX)-B1 (strains AlmWB112 c3, AlmWB112
c4 and AlmWB112 c5), secY(IX)-C1 (strains PEY1 c3, PEY2
c3, PEY2 c4, PEY2c5, PEYAB c2, NaxY c1 and NaxY c2),
secY(IX)-C2 (strains KAP539 c1, KAP585 c1 and KAP585
c2), secY(IX)-E1 (strains JunWB-2A c1, JunWB-2A c2,
JunWB-2C c1 and JunWB-2C c2) and secY(IX)-E2 (strains
BBS3NJ c5, BBS3NJc6, BBS41NJ c3 and BBS41NJ c6) (Table
1). The Roman numeral (IX) and capital letter (e.g. A, B, C,
etc.) assigned in the rp and secY coding systems refer to the
strain’s 16Sr group and subgroup affiliations. The numbers
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 10:33:44
2283
I.-M. Lee and others
(e.g. 1, 2, 3, etc.) following the capital letter in the rp and secY
coding respectively represent the rp and secY subgroup affiliation determined by consensus RFLP pattern type generated by virtual and/or actual gel RFLP analysis. An example
of the coding system using all three genetic markers for the
PPWB phytoplasma strain is as follows: 16SrIX-A; rp(IX)A1; secY(IX)-A1. This code depicts the taxonomic affiliation
status of strain PPWB based on the three selected housekeeping genes with increasing sequence variability levels.
DISCUSSION
The pigeon pea witches’-broom phytoplasma group (16SrIX)
consists of diverse phytoplasma strains that cause numerous
diseases in leguminous trees and herbaceous plants (family
Fabaceae), vegetable crops (Brassicaceae and Dipsacaceae), a
nut crop (almond), herbs and weeds (Asteraceae) and,
recently, a forest tree (juniper) and a fruit crop (blueberry)
in various geographical regions including North and South
America, Europe, Asia and the Middle East. Some diseases
may be specific to particular geographical regions or hosts.
Some members of the 16SrIX group are genetically closely
related but cause different diseases. Often, they cannot be
readily differentiated with confidence by analysis of conserved 16S rRNA gene sequences. For quarantine purposes,
it is necessary to develop a better classification system that
allows the unambiguous identification of these strains of
interest. An approach using multiple genetic markers for
analysis has been applied successfully to the differentiation
of closely related strains in numerous phytoplasma groups.
In the present study, we demonstrated that including lessconserved genes such as rp or secY for phylogenetic analyses
is essential in order to achieve more detailed and accurate
assessments of relative genetic distances among members of
the 16SrIX group.
Currently, phytoplasma speciation is based solely on the
highly conserved 16S rRNA gene as a phylogenetic parameter to assess genetic distance. The threshold value of 16S
rRNA gene sequence similarity set to designate a novel
taxon is too rigid to draw a definite line that can be used to
determine accurately the taxonomic rank of a given strain.
According to the consensus guidelines proposed by the
International Phytoplasma Working Group (IPWG), phytoplasma speciation (‘Candidatus Phytoplasma’ species designation) has been based solely on the highly conserved
16S rRNA gene sequence, with an arbitrary cut-off similarity of ¡97.5 % to separate two species. Strains that share
more than 97.5 % 16S rRNA gene sequence similarity may
be designated a novel candidate species only when specific
ecological and biological properties (i.e. specific insect
vector or plant host) are demonstrated. These guidelines
potentially exclude many distinct strains that warrant a
novel taxon designation because their vectors are unknown
and comprehensive plant host ranges are difficult, if not
impossible, to determine. Previous studies and the present
study have indicated that distinct lineages can be recognized unambiguously by also including the less-conserved
2284
rp and/or secY genes as genetic markers. Therefore, including an additional gene, such as rp or secY, in assessing the
genetic distance of a potential candidate for a novel taxon
should alleviate the ambiguity resulting from the use of the
16S rRNA gene alone. In the existing taxonomy of prokaryotes (including cell-wall-less mollicutes), many culturable species have been designated that share 16S rRNA gene
sequence similarities ranging from 98.8 to 99.4 % and secY
sequence similarities ranging from 85.5 to 89.5 %, based on
sequences available in GenBank. These values can serve as
reference points for defining new guidelines for designating
a novel phytoplasma taxon, so that the requirement for
specific biological properties can be omitted in cases in
which the genetic distance of the candidate strain from
existing ‘Candidatus Phytoplasma’ species, as assessed by
multiple genes, is obvious.
ACKNOWLEDGEMENTS
We thank all the individuals who provided phytoplasma strains used
in this study. We thank Prachi Bagadia for her technical assistance in
sequence assembly and phylogenetic analysis and for permission to
use unpublished blueberry stunt phytoplasma data.
REFERENCES
Abou-Jawdah, Y., Karakashian, A., Sobh, H., Martini, M. & Lee, I.-M.
(2002). An epidemic of almond witches’-broom in Lebanon: classi-
fication and phylogenetic relationships of the associated phytoplasma.
Plant Dis 86, 477–484.
Bertaccini, A. (2007). Phytoplasmas: diversity, taxonomy, and
epidemiology. Front Biosci 12, 673–689.
Davis, R. E., Dally, E. L., Zhao, Y., Lee, I. M., Jomantiene, R.,
Detweiler, A. J. & Putnam, M. L. (2010). First report of a new
subgroup 16SrIX-E (‘Candidatus Phytoplasma phoenicium’-related)
phytoplasma associated with juniper witches’-broom disease in
Oregon, USA. Plant Pathol 59, 1161.
Deng, S. & Hiruki, C. (1991). Genetic relatedness between two non-
culturable mycoplasmalike organisms revealed by nucleic acid hybridization and polymerase chain reaction. Phytopathology 81, 1475–
1479.
Green, M. J., Thompson, D. A. & MacKenzie, D. J. (1999). Easy and
efficient DNA extraction from woody plants for the detection of
phytoplasmas by polymerase chain reaction. Plant Dis 83, 482–485.
Gundersen, D. E., Lee, I.-M., Rehner, S. A., Davis, R. E. & Kingsbury,
D. T. (1994). Phylogeny of mycoplasmalike organisms (phytoplas-
mas): a basis for their classification. J Bacteriol 176, 5244–5254.
Hodgetts, J., Boonham, N., Mumford, R., Harrison, N. & Dickinson, M.
(2008). Phytoplasma phylogenetics based on analysis of secA and
23S rRNA gene sequences for improved resolution of candidate
species of ‘Candidatus Phytoplasma’. Int J Syst Evol Microbiol 58,
1826–1837.
Hogenhout, S. A., Oshima, K., Ammar, D., Kakizawa, S., Kingdom,
H. N. & Namba, S. (2008). Phytoplasmas: bacteria that manipulate
plants and insects. Mol Plant Pathol 9, 403–423.
IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma
Taxonomy Group (2004). ‘Candidatus Phytoplasma’, a taxon for the
wall-less, non-helical prokaryotes that colonize plant phloem and
insects. Int J Syst Evol Microbiol 54, 1243–1255.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 62
IP: 88.99.165.207
On: Sun, 18 Jun 2017 10:33:44
Multiple gene-based 16SrIX phytoplasma phylogeny
Lee, I.-M., Davis, R. E., Sinclair, W. A., DeWitt, N. D. & Conti, M. (1993).
Genetic relatedness of mycoplasmalike organisms detected in Ulmus
spp. in the United States and Italy by means of DNA probes and
polymerase chain reactions. Phytopathology 83, 829–833.
Lee, I.-M., Gundersen-Rindal, D. E., Davis, R. E. & Bartoszyk, I. M.
(1998). Revised classification scheme of phytoplasmas based on RFLP
analyses of 16S rRNA and ribosomal protein gene sequences. Int J Syst
Bacteriol 48, 1153–1169.
Lee, I.-M., Davis, R. E. & Gundersen-Rindal, D. E. (2000). Phyto-
plasma: phytopathogenic mollicutes. Annu Rev Microbiol 54, 221–255.
Lee, I.-M., Martini, M., Bottner, K. D., Dane, R. A., Black, M. C. &
Troxclair, N. (2003). Ecological implications from a molecular
analysis of phytoplasmas involved in an aster yellows epidemic in
various crops in Texas. Phytopathology 93, 1368–1377.
Lee, I.-M., Martini, M., Marcone, C. & Zhu, S. F. (2004). Classification
Martini, M., Lee, I.-M., Bottner, K. D., Zhao, Y., Botti, S., Bertaccini, A.,
Harrison, N. A., Carraro, L., Marcone, C. & other authors (2007).
Ribosomal protein gene-based phylogeny for finer differentiation and
classification of phytoplasmas. Int J Syst Evol Microbiol 57, 2037–2051.
Schneider, B., Seemüller, E., Smart, C. D. & Kirkpatrick, B. C. (1995).
Phylogenetic classification of plant pathogenic mycoplasma-like
organisms or phytoplasmas. In Molecular and Diagnostic Procedures
in Mycoplasmology, vol. 2, pp. 369–380. Edited by S. Razin & J. G. Tully.
New York: Academic Press.
Seemüller, E., Schneider, B., Mäurer, R., Ahrens, U., Daire, X., Kison, H.,
Lorenz, K. H., Firrao, G., Avinent, L. & other authors (1994). Phylo-
genetic classification of phytopathogenic mollicutes by sequence
analysis of 16S ribosomal DNA. Int J Syst Bacteriol 44, 440–446.
Seemüller, E., Marcone, C., Lauer, U., Ragozzino, A. & Göschl, M.
(1998). Current status of molecular classification of the phytoplasmas.
J Plant Pathol 80, 3–26.
of phytoplasma strains in the elm yellows group (16SrV) and proposal
of ‘Candidatus Phytoplasma ulmi’ for the phytoplasma associated
with elm yellows. Int J Syst Evol Microbiol 54, 337–347.
Swofford, D. L. (1998). Phylogenetic analysis using parsimony (PAUP),
Lee, I.-M., Zhao, Y. & Bottner, K. D. (2006). SecY gene sequence
Wei, W., Davis, R. E., Lee, I.-M. & Zhao, Y. (2007). Computer-
analysis for finer differentiation of diverse strains in the aster yellows
phytoplasma group. Mol Cell Probes 20, 87–91.
simulated RFLP analysis of 16S rRNA genes: identification of ten new
phytoplasma groups. Int J Syst Evol Microbiol 57, 1855–1867.
Lee, I.-M., Bottner-Parker, K. D., Zhao, Y., Davis, R. E. & Harrison,
N. A. (2010). Phylogenetic analysis and delineation of phyto-
Wei, W., Lee, I.-M., Davis, R. E., Suo, X. & Zhao, Y. (2008). Automated
version 4. Sunderland, MA: Sinauer Associates.
plasmas based on secY gene sequences. Int J Syst Evol Microbiol 60,
2887–2897.
RFLP pattern comparison and similarity coefficient calculation for
rapid delineation of new and distinct phytoplasma 16Sr subgroup
lineages. Int J Syst Evol Microbiol 58, 2368–2377.
Lim, P. O. & Sears, B. B. (1992). Evolutionary relationships of a plant-
Zhao, Y., Wei, W., Lee, I.-M., Shao, J., Suo, X. & Davis, R. E. (2009).
pathogenic mycoplasmalike organism and Acholeplasma laidlawii
deduced from two ribosomal protein gene sequences. J Bacteriol 174,
2606–2611.
Construction of an interactive online phytoplasma classification tool,
iPhyClassifier, and its application in analysis of the peach X-disease
phytoplasma group (16SrIII). Int J Syst Evol Microbiol 59, 2582–2593.
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 10:33:44
2285