International Journal of Systematic and Evolutionary Microbiology (2016), 66, 492–513 DOI 10.1099/ijsem.0.

000726

The underestimated diversity of phytoplasmas

in Latin America
Edel Pérez-López,1 Mauricio Luna-Rodrı́guez,2 Chrystel Y. Olivier3 and
Tim J. Dumonceaux3,4
1
Correspondence Instituto de Biotecnologı́a y Ecologı́a Aplicada (INBIOTECA), Universidad Veracruzana, Avenida de
Edel Pérez-López Las Culturas Veracruzanas, Xalapa, Veracruz, Mexico
edellopez1987@gmail.com 2
DGI-LATEX, Universidad Veracruzana, Avenida de Las Culturas Veracruzanas, Xalapa, Veracruz,
Mexico
3
Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, Saskatchewan,
Canada
4
Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada

Phytoplasmas (‘Candidatus Phytoplasma’) are insect-transmitted, cell-wall-less, plant-

pathogenic bacteria that cause economically important crop diseases. Because phytoplasmas
are difficult or impossible to culture in vitro, they are classified taxonomically according to the
convention used for unculturable micro-organisms. The first coherent scheme of classification of
phytoplasmas, based on the RFLP pattern of the 16S rRNA-encoding gene generated with 17
restriction endonucleases, was updated several times until the development of the
i PhyClassifier. i PhyClassifier is an interactive online tool capable of determining the species,
group and subgroup of ‘Candidatus Phytoplasma’ of unknown samples using the 16S F2nR2
sequence. Latin America, an important geographical area in relation to food production, has a
high incidence of plant diseases caused by phytoplasmas. However, many phytoplasmas
associated with these diseases have not been properly classified. An extensive literature review
and the use of i PhyClassifier allowed us to identify two new tentative groups (16SrXXXIII-A and
16SrXXXIV-A) and the following tentative new subgroups among Latin American strains that
were either previously unclassified or misclassified: six in 16SrI, six in 16SrII, one in 16SrIII, one
in 16SrVII, one in 16SrIX, one in 16SrXII and two in 16SrXIII.

Phytoplasmas comprise a broad group of small, wall-less,
insect-vectored bacteria that cause disease in a diverse
array of economically important crops around the world
(Lee et al., 2000; Harrison et al., 2014). In plants, phyto-
plasmas are located in the sieve elements of phloem
tissue while, in insect vectors, mostly leafhoppers, they
are detected at higher concentrations in the salivary
glands (Hogenhout et al., 2008). Phytoplasmas are mem-
bers of the class Mollicutes with small, A+T-rich genomes
derived from a Gram-positive-walled bacterium, possibly
an Acholeplasma-like ancestor (Zhao et al., 2005, 2014;
Wei et al., 2008a). With a distinctive genome architecture
characterized by the presence of sequence-variable mosaics
(Jomantiene & Davis, 2006) thought to be vestiges of
ancient phages (Wei et al., 2008a) and a trans-kingdom
parasitism, phytoplasmas differ from all other members
of the order Acholeplasmatales (Zhao et al., 2014, 2015).
Details of i PhyClassifier analyses are available with the online
Supplementary Material.
Because of the difficulty in establishing axenic cultures of
phytoplasmas, measurable phenotypic characters are largely
inaccessible for their taxonomic classification. The Inter-
national Committee of Systematic Bacteriology Subcommittee
for the Taxonomy of Mollicutes and the International
Research Program for Comparative Mycoplasmology
(IRPCM) adopted the taxonomic convention established for
uncultured micro-organisms (Murray & Stackebrandt,
1995), and proposed the erection of a provisional genus ‘Can-
didatus Phytoplasma’ (IRPCM, 2004). To date, 37 candidate
species of ‘Ca. Phytoplasma’ have been formally described,
and more than ten potentially novel species have been
suggested based on the similarity of 16S rRNA-encoding
gene sequences (IRPCM, 2004; Davis et al., 2013; Nejat
et al., 2013; Harrison et al., 2014). Phylogenetic analysis of
16S rRNA-encoding gene sequences from reference strains of
candidate species of ‘Ca. Phytoplasma’ divides the phyto-
plasma clade into three subclades (Hogenhout et al., 2008;
Zhao et al., 2014). It has been suggested that each subclade
may represent a genus-level taxon and that the ‘Ca.

492 000726 G 2015 IUMS Printed in Great Britain


Phytoplasma’ clade may represent a family-level taxon (Zhao
et al., 2014, 2015; Wei et al., 2015).
Besides the taxonomic (Candidatus species) assignment,
phytoplasmas are classified into groups and subgroups
based on RFLP analysis of their 16S rRNA-encoding loci.
A comprehensive phytoplasma classification scheme was
established on the basis of RFLP analysis of the PCR-
amplified F2nR2 fragment of 16S rRNA-encoding genes
with a set of 17 restriction endonucleases: Alu I, Bam HI,
Bfa I, Bst UI (Tha I), Dra I, Eco RI, Hae III, Hha I, HinfI,
Hpa I, Hpa II, Kpn I, Sau3AI (Mbo I), Mse I, Rsa I, Ssp I
and Taq I (Lee et al., 1993, 1998, 2000). In 1993, the first
16S rRNA RFLP (16Sr) group was delineated (Lee et al.,
1993) and, in 2009, Zhao and colleagues reported the exist-
ence of 29 16Sr groups designated 16SrI–16SrXXIX (Zhao
et al., 2009). Recently, this has been expanded to include
groups up to 16SrXXXII (Nejat et al., 2013), highlighting
the vast diversity among phytoplasmas strains worldwide.
The development and validation of computer-simulated
RFLP analysis, along with an automated RFLP pattern-
comparison program that facilitates the calculation of simi-
larity coefficients for differentiating phytoplasma groups/
subgroups, was an important advancement in the field of
phytoplasma characterization (Wei et al., 2007, 2008b).
The i PhyClassifier is an interactive online tool for quick
identification and classification of diverse phytoplasmas.
By comparing RFLP profiles and the 16S rRNA gene
sequences archived in a database of known phytoplasma
strains, i PhyClassifier can determine the 16Sr group/sub-
group classification status of a query strain and assign the
strain to a candidate species of ‘Ca. Phytoplasma’ (Zhao
et al., 2009). Several novel 16Sr groups and dozens of
novel subgroups have been delineated using the i PhyClas-
sifier (Zhao et al., 2009, 2014; Fernández et al., 2015).
Latin America is characterized by high agricultural pro-
ductivity supported by a primarily tropical climate.
Mexico is one of the main producers of corn, tomatoes,
coffee and avocados, and Brazil is one of the main produ-
cers of a wide variety of citrus crops, soybean and many
other economically important crops (FAO, 2012). Latin
America, like the rest of the world, has been affected by
plant diseases caused by phytoplasmas, with a significant
impact on the production of corn, coconut, papaya and
strawberries (Nault, 1983; Acosta et al., 2011; Harrison
et al., 2014; Fernández et al., 2015; Luis-Pantoja et al.,
2015). Therefore, Latin America is an important area for
the development and application of effective strategies for
the detection, characterization and management of phyto-
plasmal diseases. To manage phytoplasmal diseases effec-
tively, it is vital to understand the distinctions between
biologically different strains, emphasizing the importance
of accurate phytoplasma identification and classification.
In the 1990s, the phytoplasmology community made
major achievements in the study of phytoplasmas, includ-
ing the creation of the scheme to classify phytoplasmas,
and the design and validation of several sets of ‘universal
primers’ to detect diverse phytoplasmas through PCR
http://ijs.microbiologyresearch.org
New phytoplasma groups and subgroups in Latin America
(Lee et al., 1993, 1998; Gundersen & Lee, 1996; Smart
et al., 1996). However, in Latin America, it was not until
the 2000s that the molecular identification and characteriz-
ation of phytoplasmas became routine in phytopathology
studies. The rapid emergence of diseases associated with
phytoplasma infections and their economic impact has
led many researchers to publish short notes and new dis-
ease reports describing the symptoms, the methodology
used to detect the pathogens associated with the symptoms
and, in certain cases, the identification of the phytoplasmas
detected. Through a broad literature review of studies pub-
lished in peer-reviewed and indexed journals about phyto-
plasma characterization, we noted a large number of strains
that were unclassified in Latin America. Here, with the help
of the interactive online phytoplasma classification tool
i PhyClassifier, we have classified 18 strains and reclassified
11 previously reported strains and delineated two tentative
new groups and the following tentative new subgroups: six
in 16SrI, six in 16SrII, two in 16SrIII, one in 16SrVII, one
in 16SrIX, one in 16SrXII and two in 16SrXIII. The find-
ings we obtained using i PhyClassifier were supported by
computer-simulated RFLP and phylogenetic analysis.
Phytoplasmas in Latin America
Despite its comparatively small geographical area, Cuba is
the country in Latin America with the largest number of
reports of phytoplasmal diseases by far (Fig. 1). Brazil,
Argentina, Mexico and Bolivia, in descending order,
make up the rest of the top five in terms of the number
of phytoplasmal diseases reported in the literature. One
remarkable phenomenon that we observed during this
study was the preferential distribution of certain phyto-
plasma groups in the southern or northern hemisphere
(Table 1). For example, members of the 16SrII groups
have been detected in Argentina or Chile, the most south-
erly countries in Latin America, while members of the
16SrI group were not detected until after 2000 in those
countries located in the southern hemisphere (Torres
et al., 2004; Gajardo et al., 2009). On the other hand, the
detection of group 16SrVII phytoplasmas has hitherto
been restricted to the southern hemisphere countries
Brazil, Chile and Argentina (Table 1). Such differential dis-
tribution of phytoplasmas is probably due to the diversity
and preferential distribution of their insect vectors. While
some leafhopper vectors of phytoplasma, such as species
of Dalbulus, have been detected in all Latin American
countries (Nault, 1980, 1983), others, such as Amplicephalus
curtulus, have been detected only in Chile and Colombia
(Alvarez et al., 2009; Arismendi et al., 2014).
Six of the 37 formally described candidate phytoplasma
species have been detected in Latin America. The associ-
ation of ‘Ca. Phytoplasma caricae’ with papaya (Caricae
papaya) and the association of ‘Ca. Phytoplasma graminis’
with sugarcane (Saccharum officinarum), Cynodon dactylon,
Conyza canadensis, Sorghum halepense, Macroptilium
lathyroides, Saccharosydne saccharivora and species of
493
E. Pérez-López and others

Guatemala
Uruguay
Argentina
Mexico
Cuba Costa Rica
Cuba
Guatemala Colombia
Costa Rica
Chile
Colombia

Peru Brazil

Bolivia Mexico
Brazil

Chile
Uruguay
Bolivia
Argentina Argentina

Fig. 1. Numbers of reports of plant diseases associated with phytoplasmas by country based on a literature review between
2000 and 2015.

Cedusa were detected in Cuba (Arocha et al., 2005a); the
association of ‘Ca. Phytoplasma brasiliense’ with plants of
the genus Hibiscus and the association of ‘Ca. Phytoplasma
sudamericanum’ with Passiflora edulis f. flavicarpa were
detected in Brazil (Montano et al., 2001; Davis et al.,
2012); the association of ‘Ca. Phytoplasma lycopersici’
with alfalfa (Medicago sativa) and the weeds Morrenia
variegata and Serjania perulacea was detected in Bolivia
(Arocha et al., 2007a); and the association of ‘Ca. Phyto-
plasma costaricanum’ with soybean (Glycine max), sweet
pepper (Capsicum annuum) and Passiflora edulis was
detected in Costa Rica, as the epithet suggests (Lee et al.,
2011). It is important to note that, contrary to many
other phytoplasma species, the species newly described in

Table 1. Phytoplasma 16Sr RFLP groups detected in Latin American countries

Results were taken from a review of the literature.

Country I II III IV V VI VII IX XII XIII XIV XV XVI XXXI

Argentina + + + + +
Bolivia + + + + +
Brazil + + + + + + + +
Chile + + +
Colombia + +
Costa Rica + + +
Cuba + + + + + + + +
Guatemala +
Mexico + + + + +
Peru + + +
Uruguay + +

494 International Journal of Systematic and Evolutionary Microbiology 66


Latin America have not been detected in any other conti-
nent or geographical location.
Another intriguing finding was that phytoplasmas in the
Mexican periwinkle virescence group (16SrXIII) have
been found almost exclusively in Latin America. Reports
of plants affected by phytoplasmas affiliated with group
16SrXIII have been observed in Florida (USA) and from
Mexico to Argentina, associated mainly with strawberry
plants (Jomantiene et al., 1998; Lee et al., 1998; Fernández
et al., 2015).
Exploring the genetic diversity of previously
unclassified, misclassified and incompletely
classified phytoplasmas
As mentioned above, previously published studies on phyto-
plasmas and phytoplasmal diseases in Latin America are
mainly new disease reports or short communications. After
an extensive review of this literature, we found that most
of the reports had no or incomplete information regarding
classification of the respective phytoplasmas. These studies
were published in peer-reviewed and indexed journals in
English, Spanish or Portuguese with relevant nucleotide
sequences deposited in public databases including GenBank,
EMBL and/or DDBJ. All of the accession numbers reported
in the literature we analysed are shown in Table 2, together
with the original classification and references. All of the
sequences were retrieved from the databases and were
screened for adequate length required for analysis using
i PhyClassifier (encompassing the full 16S F2nR2 region of
approximately 1240 bp). As i PhyClassifier is a DNA
sequence-based research tool, the quality of the input
sequence is critical for proper operation and interpretation
of results (Zhao et al., 2009). We also corroborated in
GenBank the origin of each sequence analysed as strains
detected in Latin America. Taking that into account, we
only considered sequences referred to as consensus sequences
resulting from the alignment of at least three sequences.
New subgroups in the aster yellows phytoplasma
group (16SrI)
The aster yellows group (16SrI) is the largest and most
diverse phytoplasma group distributed worldwide
(Gundersen et al., 1996; Lee et al., 2004; Wei et al., 2011).
Phytoplasmas in this group have been detected in both
herbaceous and woody plants (Moreira et al., 2010; Pérez-
López et al., 2013a). Recently, the identification of two new
subgroups of the 16SrI group has been reported, identified
as 16SrI-W and 16SrI-Y, using up almost the entire alphabet
for designating subgroups (Acosta et al., 2015). We propose
in this study the use of an extended, two-letter system to des-
ignate new subgroups in this phytoplasma group, for
example, 16SrI-AB, 16SrI-AC, 16SrI-AD, (...), 16ASrI-AZ,
and then continue with 16SrI-BC, and so on. To avoid con-
fusion, we will not use the same two letters to designate new
subgroups, for example 16SrI-AA. Based on this system, the
http://ijs.microbiologyresearch.org
New phytoplasma groups and subgroups in Latin America
six new 16SrI subgroups delineated in this study from the
classification and reclassification of Latin America strains
were named 16SrI-Z 16SrI-AB, 16SrI-AC, 16SrI-AD, 16SrI-
AE and 16SrI-AF. Using i PhyClassifier, in silico 16S F2nR2
RFLP patterns were generated for eight of the at least 25 pre-
viously described 16SrI subgroups, along with the RFLP pat-
terns of the six new subgroups detected in this study (Fig. 2).
The new subgroup 16SrI-Z is represented by Bolivia alfalfa
phytoplasma (GenBank accession no. AY725211), which
was not previously classified (Jones et al., 2005a). The new
subgroup 16SrI-Z is also represented by ‘brotes grandes’
(big bud) of potato phytoplasma (GenBank accession no.
AY725209), and was also detected in mora-mora vine (Ser-
jania perulacea) (GenBank accession no. AY725210) in the
same study (Jones et al., 2005a). With 0.92 as the highest
similarity with a previously described 16SrI subgroup
(16SrI-B, GenBank accession no. NC_005303), and a dis-
tinctly different RFLP pattern, those strains could potentially
be placed in the proposed new subgroup 16SrI-Z (Fig. 2).
Subgroup 16SrI-AB is represented by Bougainvillea shoot
proliferation phytoplasma strain BSP-Br1 (GenBank acces-
sion no. EU423898), which was previously classified as a
member of 16SrI-B (Silva et al., 2009). With a similarity coef-
ficient of 0.94 with the reference strain member of the 16SrI-
B subgroup, strain BSP-Br1 is not a member of subgroup
16SrI-B or related strains. Strain BSP-Br1 had a similarity
coefficient less than or equal to 0.97 with any other described
16SrI subgroup, and a unique RFLP pattern, allowing its
differentiation into a new subgroup (Fig. 2). Subgroup
16SrI-AC is represented by Fraxinus uhdei and Populus
nigra phytoplasmas (GenBank accession nos JQ730859 and
JQ730860) (Perilla-Henao et al., 2012), with a similarity
coefficient of 0.95 with the 16SrI-B reference strain (Gen-
Bank accession no.NC_005303), and a unique RFLP pattern
(Fig. 2). New subgroups 16SrI-AD and 16SrI-AE were
detected in Cuba, where 16SrI-AD is represented by basil
little leaf phytoplasma (GenBank accession no. DQ286577)
and sweet pepper phytoplasma (GenBank accession no.
DQ286947), while 16SrI-AE is represented by broad bean
phytoplasma (GenBank accession no. DQ286953). None of
these strains was previously classified, and the geographical
relationship between sweet pepper phytoplasma (16SrI-
AD) and broad bean phytoplasma (16SrI-AE) suggests the
possibility of one strain with a heterogeneous rrn operon,
as reported previously in other studies (Zhao et al., 2009).
However, the original authors of the report of the members
of subgroups 16SrI-AD and 16SrI-AE made reference to the
analysis of more than 50 symptomatic samples of each of
basil, sweet pepper and broad bean and, in each case, they
found the same 16S rRNA gene sequence, depositing only
the consensus 16S rRNA gene sequence in GenBank
(Arocha et al., 2006a, 2007b). For this reason, we dismiss
the probability of the presence of a heterogeneous rrn
operon. Subgroup 16SrI-AF detected in this study is rep-
resented by marigold phyllody phytoplasma (GenBank acces-
sion no. AY249247), with a unique RFLP pattern and the
highest similarity coefficient of 0.94 with the 16SrI-B reference
strain (Fig. 2). With the exception of subgroups 16SrI-AD and
495
 Table 2. Phytoplasmas detected in Latin America

496
Both the original and the new classifications, if applicable, are shown. Our new classification is based on the use of i PhyClassifier software; i PhyClassifier output can be found in the online
Supplementary Material. NC , Not classified; NV , not verified.

GenBank accession no(s). Host(s) 16Sr classification Reference(s)

Previous This study

E. Pérez-López and others

Argentina
AY081817 Chinaberry (Melia azedarach) 16SrIII-B 2 Galdeano et al. (2013)
KC412024 Daisy (Bellis perennis) 16SrIII-J 2 Galdeano et al. (2013)
AY081816, KC412032 Garlic (Allium sativum) 16SrIII-J 2 Galdeano et al. (2004, 2013)
KC202812 ‘Lagaña de perro’ (Caesalpinia gilliesii) 16SrIII-B 2 Galdeano et al. (2013)
KC412025 Madagascar periwinkle (Catharanthus roseus) 16SrIII-B 2 Galdeano et al. (2013)
KC412026, KC412027 ‘Rama negra’ (Conyza bonariensis) 16SrIII-X 2 Galdeano et al. (2013)
KC412029 ‘Romerillo’ (Heterothalamus alienus) 16SrIII-W 2 Galdeano et al. (2013)
KC412028 Summer squash (Cucurbita maxima) 16SrIII-J 2 Galdeano et al. (2013)
KC412031 Tomato (Solanum lycopersicum) 16SrIII-J 2 Galdeano et al. (2013)
KF640663, KF640662 Sunflower (Helianthus annuus) 16SrIII-J 2 Guzmán et al. (2014)
JQ359014 Peach (Prunus persica) 16SrIII-B 2 Fernández et al. (2013a)
AF105315, AF105316, AF092209 Strawberry (Fragaria 6 ananassa) 16SrVII-A 2 Fernández et al. (2013b)
DQ989178 ‘Sweet annie’ (Artemisia annua) 16SrVII-B 2 Meneguzzi et al. (2008)
DQ989179, DQ989180 Conyza bonariensis 16SrVII-B 2 Meneguzzi et al. (2008)
DQ989178, DQ989179, DQ989180 Strawberry (Fragaria 6 ananassa) 16SrVII-B 2 Fernández et al. (2013b)
AY147038, JN368423 Strawberry (Fragaria 6 ananassa) 16SrVII-C 2 Fernández et al. (2013b)
AY147038 Alfalfa (Medicago sativa) 16SrVII-C 2 Conci et al. (2005)
DQ444264, DQ444265 Chinaberry (Melia azedarach) 16SrXIII-C 16SrXIII-G* Arneodo et al. (2007); this
study
KJ921641, KJ921642, KJ921643, KJ921644 Strawberry (Fragaria 6 ananassa) 16SrXIII-F 2 Fernández et al. (2015)
Bolivia
AY725211 Alfalfa (Medicago sativa) 16SrI-NC 16SrI-Z* Jones et al. (2005a); this study
AY725209, AY725210 Potato (Solanum tuberosum), mora-mora vine (Serjania 16SrI-B 16SrI-Z* Jones et al. (2005b); this study
perulacea)
FJ207457, FJ207453, FJ207452 Podocarpus macrophyllus, rose (Rosa sp.), tomatillo 16SrII-NC NV Arocha et al. (2010a)
(Physalis ixocarpa)
FJ207456, FJ207455, FJ207454, FJ207451 Bell pepper (Capsicum annuum), strawberry (Fragaria 16SrIII-NC NV Arocha et al. (2010b)
chiloensis),
Schinus molle, Arracacia xanthorrhiza
AF495657 Chinaberry (Melia azedarach) 16SrIII-J 16SrIII-B Harrison et al. (2002); this
study
AY725212 Peach (Prunus persicae) 16SrXII-NC 16SrXII-J* Jones et al. (2005b); this study
AF495882 Chinaberry (Melia azedarach) 16SrXIII-C 2 Harrison et al. (2002)
Brazil
EU423898 Bougainvillea (Bougainvillea spectabilis) 16SrI-B 16SrI-AB* Silva et al. (2015); this study
JX626330 Broccoli (Brassica oleracea) 16SrI-NC 16SrI-B Eckstein et al. (2013), this study
EU423900 Sugarcane (Saccharum sp.) 16SrI-B 2 Silva et al. (2009)
EF647744 Tabebuia pentaphylla 16SrII-NC 16SrII-O* Mafia et al. (2007); this study
AF147706, AF147707 Chayote (Sechium edule) 16SrIII-J 2 Montano et al. (2001)

International Journal of Systematic and Evolutionary Microbiology 66

 Table 2. cont.

GenBank accession no(s). Host(s) 16Sr classification Reference(s)

Previous This study

EU423899 Bougainvillea (Bougainvillea spectabilis) 16SrIII-B 2 Silva et al. (2015)
JX626327 Broccoli (Brassica oleracea) 16SrIII-NC 16SrIII-Z* Eckstein et al. (2013); this study
JX626331, JX626332 Broccoli (Brassica oleracea) 16SrIII-NC NV Eckstein et al. (2013)
GU193977, GU193976 Cassava (Manihot esculenta) 16SrIII-B 2 Flôres et al. (2013)
JQ742084, JQ742080, JQ742079, Empoasca sp., Planicephalus flavicosta, Agallia albidula, 16SrIII-NC NV Eckstein et al. (2014)

http://ijs.microbiologyresearch.org
JQ742081, JQ742083 Agalliana sticticollis, Atanus nitidus
JX028239 Honeyweed (Leonurus sibiricus) 16SrIII-B 2 Flôres & Bedendo (2013)
AY034608, AF411592 Erigeron sp. 16SrVII-B 2 Barros et al. (2002)
KJ831066 Erigeron sp. 16SrVII-D 2 Flôres et al. (2015)
EU266074 Sweet orange (Citrus sp.) 16SrIX-NC 16SrIX-H* Teixeira et al. (2008); this study
JN792515, JN792516 Madagascar periwinkle (Catharanthus roseus) 16SrIX-A 2 Barbosa et al. (2012)
JN574433 Celosia argentea, Celosia spicata 16SrXIII-J 2 Eckstein et al. (2012)
JX626329, JX626326 Broccoli (Brassica oleracea) 16SrXIII-NC 16SrXIII-H* Eckstein et al. (2013); this study
EU719111, JQ792171 Papaya (Carica papaya) 16SrXIII-E 2 Melo et al. (2013)
Chile
AY741532, DQ177149 Grapevine (Vitis vinifera) 16SrI-B NV Gajardo et al. (2009)
FM200179 Murta (Ugni molinae) 16SrVII-A NV Arismendi et al. (2010)
AM980866 Chaura (Gaultheria phillyreifolia) 16SrVII-A NV Arismendi et al. (2010)
DQ232752 Grapevine (Vitis vinifera) 16SrVII-A 16SrXXXIII-A* Gajardo et al. (2009); this study
AY741531, DQ194355 Grapevine (Vitis vinifera) 16SrVII-A 16SrVII-E* Gajardo et al. (2009); this study
DQ177148 Grapevine (Vitis vinifera) 16SrXII-A NV Gajardo et al. (2009)
Colombia
JQ730861 Pittosporum undulatum 16SrI-B 2 Perilla-Henao et al. (2012)
JQ730859, JQ730860 Fraxinus uhdei, Populus nigra 16SrI-B 16SrI-AC* Perilla-Henao et al. (2012); this
study
HQ530152 Corn (Zea mays) 16SrI-B 2 Alvarez et al. (2014)
AY737646, EU346761, AY737647 Cassava (Manihot esculenta) 16SrIII-L 16SrIII-B Alvarez et al. (2009)
JX681021 Oil palm (Elaeis guineensis) 16SrI-B 2 Alvarez et al. (2014)
Costa Rica
FJ603645 Common bean (Phaseolus vulgaris) 16SrI-B 2 Moreira et al. (2010)
FJ226068 toFJ226073 Soybean (Glycine max), passion fruit (Passiflora edulis), 16SrXXXI-A 2 Villalobos et al. (2009)
sweet pepper (Capsicum annuum)
HQ258882, HQ258883 Guacimo (Guazuma ulmifolia) 16SrXV-B 2 Villalobos et al. (2011)
Cuba
DQ286577 Basil (Ocimum basilicum) 16SrI-NC 16SrI-AD* Arocha et al. (2006a); this study
DQ286953 Broad bean (Vicia faba) 16SrI-NC 16SrI-AE* Arocha et al. (2007b); this study
DQ286947 Sweet pepper (Capsicum annuum) 16SrI-NC 16SrI-AD* Arocha et al. (2007b); this study
EU328254, EU328252, EU328251, EU328256 Carrot (Daucus carota), cabbage (Brassica oleracea), beetroot 16SrI-NC NV Arocha et al. (2009a)
(Beta vulgaris), cassava (Manihot esculenta)
JN383914 Common bean (Phaseolus vulgaris) 16SrI-NC NV Zamora et al. (2012)
KC825053 Strawberry (Fragaria 6 ananassa) 16SrI-NC NV Ferriol-Marchena et al.(2013)
KC513772 Macadamia nut (Macadamia integrifolia) 16SrI-NC NV Pérez-López et al. (2013a)
New phytoplasma groups and subgroups in Latin America

497
 Table 2. cont.

498
GenBank accession no(s). Host(s) 16Sr classification Reference(s)

Previous This study

JF781311 Papaya (Carica papaya) 16SrI-Z 2 Acosta et al. (2013); this study
JF781307 Papaya (Carica papaya) 16SrI-B 16SrI-L Acosta et al. (2013)
E. Pérez-López and others

JF781308 Papaya (Carica papaya) 16SrI-X 2 Acosta et al. (2013)

EU328253 Radish (Raphanus sativus) 16SrII-NC NV Arocha et al. (2009a)
EU869220 Sapodilla (Manilkara zapota) 16SrII-NC NV Acosta et al. (2008)
EU350565, EU350564, EU350566 Papaya (Carica papaya) 16SrII-NC NV Arocha et al. (2009b)
DQ286948, DQ286949, DQ868531, DQ868532, Papaya (Carica papaya), Empoasca sp., Anoda acerifolia, 16SrII-NC 16SrII-P* Arocha et al. (2006b, 2007c);
DQ286950, DQ286952, DQ286951, Euphorbia heterophylla, Malvastrum coromandelianum, this study
DQ868533 Rhynchosia minima
JF781310 Papaya (Carica papaya) 16SrII-A 16SrII-Q* Acosta et al. (2013); this study
JF781309 Papaya (Carica papaya) 16SrII-N 2 Acosta et al. (2013)
KF500549 Sapodilla (Manilkara zapota) 16SrV-NC NV Pérez-López et al. (2013b)
AY725238 Bermuda-grass (Cynodon dactylon) 16SrXIV-NC NV Arocha et al. (2005b)
AY257547 Papaya (Carica papaya) 16SrX-NC NV Arocha et al. (2003)
AY725237, AY257548 Planthopper (Saccharosydne saccharivora), sugarcane 16SrXVI-NC NV Arocha et al. (2005c)
(Saccharum sp.)
Mexico
EU310514 Amaranth (Amaranthus hypochondriacus) 16SrI-B NV Rojas-Martı́nez et al. (2009)
AY249248 Marigold (Tagetes erecta) 16SrI-B 2 Rojas-Martı́nez et al. (2003)
AY249249 Marigold (Tagetes erecta) 16SrI-B NV Rojas-Martı́nez et al. (2003)
AY249247 Marigold (Tagetes erecta) 16SrI-B 16SrI-AF* Rojas-Martı́nez et al. (2003);
this study
AY265208 Corn (Zea mays) 16SrI-B 2 Lee et al. (2004)
FJ914638, FJ914654, FJ914649, FJ914648, Potato (Solanum tuberosum) 16SrI-S 2 Santos-Cervantes et al. (2010)
FJ914653, FJ914640, FJ914652, FJ914641
FJ914639 Potato (Solanum tuberosum) 16SrI-T 2 Santos-Cervantes et al. (2010)
FJ914642 Potato (Solanum tuberosum) 16SrI-V 2 Santos-Cervantes et al. (2010)
FJ914650 Potato (Solanum tuberosum) 16SrI-U 2 Santos-Cervantes et al. (2010)
AF217247, AF329315 Potato (Solanum tuberosum) 16SrI-NC NV Leyva-López et al. (2002)
AY144608 Vinca minor 16SrII-NC 16SrII-C Leyva-López et al. (2002); this
study
DQ535899 Opuntia sp. 16SrII-NC NV Aviña-Padilla et al. (2009)
DQ535900 Echinopsis sp. 16SrII-NC 16SrII-R* Aviña-Padilla et al. (2009); this
study
FJ357164, FJ357167, FJ390055 Amaranth (Amaranthus hypochondriacus) 16SrII-NC 16SrII-S* Ochoa-Sánchez et al. (2009);
this study
FJ914643, FJ914644 Potato (Solanum tuberosum) 16SrII-M 2 Santos-Cervantes et al. (2010)
EU125184 Pepper (Capsicum annuum) 16SrII-NC 16SrXXXIV-A* Tapia-Tussell et al. (2012); this
study
EU125185 Tomatillo (Physalis ixocarpa) 16SrII-NC 16SrII-T* Tapia-Tussell et al. (2012); this
study
EU165357, EU165358 Euphorbia pulcherrima 16SrIII-NC NV Aviña-Padilla et al. (2009)

International Journal of Systematic and Evolutionary Microbiology 66

 New phytoplasma groups and subgroups in Latin America

16SrI-AE, all the new 16SrI subgroup lineages delineated in this

González-Pacheco et al. (2014)

study were detected in geographically unrelated locations

Santos-Cervantes et al. (2010)

throughout Latin America and were associated with different

Aviña-Padilla et al. (2009)

Tapia-Tussell et al. (2010)
plant hosts. Further details of the assignment of these strains
Reference(s)

Hodgetts et al. (2009)

Hodgetts et al. (2009)

Hodgetts et al. (2009)
Harrison et al. (2002)
is available in the online Supplementary Material.

New subgroups in the peanut witches’-broom

phytoplasma group (16SrII)
The peanut witches’-broom phytoplasma group has been
detected in many economically important crops worldwide
(Gibb et al., 1999; Cai et al., 2008; Mitrović et al., 2012),
This study

and two of the subgroups have been proposed as ‘Ca. Phy-

16Sr classification

16SrIII-L

toplasma sp.’, ‘Ca. Phytoplasma aurantifolia’ (16SrII-B)

and ‘Ca. Phytoplasma australasia’ (16SrII-D) (Zreik et al.,
NV

NV

NV

NV
NV

1995; White et al., 1998). With at least 14 subgroups ident-

ified (16SrII-A to 16SrII-N), this RFLP-based 16Sr group is
16SrXIII-D
Previous
16SrIII-NC
16SrIII-NC

16SrIV-NC

16SrIII-NC

one of the most highly variable groups detected so far.

16SrII-NC
16SrIV-B

Potato (Solanum tuberosum), papaya (Carica papaya), carrot 16SrI-NC

In this study, through the reclassification or classification

of previously reported strains in Latin America, we have
identified six new 16SrII subgroups. These new subgroups
(Daucus carota), oat (Avena sativa), Madagascar periwinkle

are 16SrII-O, represented by Tabebuia pentaphylla phyto-

plasma (GenBank accession no. EF647744) with no previous
classification; subgroup 16SrII-P, represented by Cuban
Dandelion (Taraxacum officinale), tomato (Solanum

papaya phytoplasma (GenBank accession no. DQ286948);

subgroup 16SrII-Q, represented by papaya bunchy top phy-
toplasma strain BTSpHav02-IIA (GenBank accession no.
JF781310), which was erroneously classified as 16SrII-A;
and subgroups 16SrII-R, 16SrII-S and 16SrII-T, which
were identified from reports made in Mexico, identifying
Host(s)

this country as the main source of diversity of the peanut

Tomato (Solanum lycopersicum)

witches’-broom group in Latin America. Subgroup 16SrII-

Potato (Solanum tuberosum)

Potato (Solanum tuberosum)

R is represented by Echinopsis sp. yellow patch phytoplasma

Coconut (Cocos nucifera)

(Catharanthus roseus)

(GenBank accession no. DQ535900); subgroup 16SrII-S is

Euphorbia pulcherrima

represented by Amaranthus hypochondriacus phytoplasma

strain 52A (GenBank accession no. FJ357164) and subgroup
Agave tequilana

16SrII-T is represented by tomatillo witches’-broom phyto-

lycopersicum)

plasma (GenBank accession no. EU125185). Echinopsis

sp. yellow patch phytoplasma, Amaranthus hypochondriacus
phytoplasma and tomatillo witches’-broom phytoplasma
were only classified to the group level in the original reports
*New groups and/or subgroups detected in this study.

(Aviña-Padilla et al., 2009; Ochoa-Sánchez et al., 2009; Tapia-

Tussell et al., 2012). Many reports of the 16SrII group have
AF500329, AF500330, AF500331, AF500332,

been made in Latin America, but the report of an exception-

FJ951626, FJ951627, FJ951628, FJ951629

ally short F2nR2 sequence made classification or reclassifica-

tion impossible in certain cases (Table 2). All the new 16SrII
EU882814, EU882812, EU350561,

subgroups reported here have a unique RFLP pattern com-

pared with other previously described members of the
GenBank accession no(s).

16SrII group and a similarity coefficient of less than or

EU350560, EU882815
AF500333, AF500334

equal to 0.97 to other 16SrII members (Fig. 3). Further details

EU165359, EU165360

EU350563, EU882813
KJ156364, KJ156365

of the assignment of these strains is available in the online

Supplementary Material.
Table 2. cont.

One finding consistent with the literature was the detection

EU350562

of two new 16SrII subgroups associated with papaya plants

FJ914647

(16SrII-P and 16SrII-Q). In Cuba, papaya bushy stunt

Peru

disease has been associated with several unculturable

pathogens such as Rickettsia-related proteobacterium and

http://ijs.microbiologyresearch.org 499
E. Pérez-López and others

Fig. 2. Distinctive RFLP patterns obtained with i PhyClassifier from in silico digestion of 16S rRNA gene F2nR2 fragments
from eight representative 16SrI strains of previously characterized subgroups and six new 16SrI subgroups delineated in this
study. §, New subgroup. In the computer-simulated digestions, we used the set of 17 enzymes previously defined in the
scheme of phytoplasma classification. Lanes labelled MW represent Hae III-digested phage w X174 DNA.

phytoplasmas grouped in the 16SrI-I, 16SrII and 16SrXVII
(sub)groups (Acosta et al., 2011; Luis-Pantoja et al., 2015).
The detection of several subgroups affecting the same plant
host has been observed before (Cai et al., 2008).
New subgroup in the peach X-disease
phytoplasma group (16SrIII)
Along with the aster yellows phytoplasma group and the
peanut witches’-broom phytoplasma group, peach X-dis-
ease phytoplasma is among the more diverse phytoplasma
groups in Latin America that is associated with a large
number of plant diseases (Lee et al., 1998; Jomantiene
et al., 2002; Lee et al., 2014). To the 14 subgroups defined
before the creation of i PhyClassifier, Zhao and colleagues
added four new subgroups in 2009, and the 16SrIII-Y sub-
group was identified in 2014, the last subgroup proposed in
the 16SrIII group (Lee et al., 2014). In this study, we have
identified one new 16SrIII subgroup, 16SrIII-Z, rep-
resented by broccoli stunt phytoplasma strain BSP-21
(GenBank accession no. JX626327), which has previously
been classified only at the group level (Eckstein et al.,

500 International Journal of Systematic and Evolutionary Microbiology 66


Fig. 3. Distinctive RFLP patterns obtained with i PhyClassifier from in silico digestion of 16S rRNA gene F2nR2 fragments
from eight representative 16SrII strains of previously characterized subgroups and six new 16SrII subgroups delineated in
this study. See Fig. 2 for further details.
2013). Group 16SrIII is broadly represented in Latin
America, with an enormous diversity in Brazil and
Argentina (Table 2). The new 16SrIII-Z subgroup showed
a distinctive RFLP pattern compared with previously
described 16SrIII subgroups (Fig. 4).
New subgroup in the ash yellows phytoplasma
group (16SrVII)
With only four subgroups described, 16SrVII-A (‘Ca.
Phytoplasma fraxinis’), 16SrVII-B (Erigeron witches’-broom
phytoplasma), 16SrVII-C (Argentinian alfalfa witches’-
broom phytoplasma) and the recently identified 16SrVII-D
http://ijs.microbiologyresearch.org
New phytoplasma groups and subgroups in Latin America
(EboWB-Br01), group 16SrVII is one of the least diverse
groups in terms of distribution and plant hosts affected
(Griffiths et al., 1999; Conci et al., 2005; Meneguzzi et al.,
2008; Flôres et al., 2015). The new subgroup detected in this
study, subgroup 16SrVII-E, is represented by Chilean grapevine
yellows phytoplasma (GenBank accession no. AY741531).
This strain, previously classified as 16SrVII-A (Gajardo et al.,
2009), showed a maximum similarity coefficient of only 0.94
with the reference pattern of subgroup 16SrVII-A (GenBank
accession no. AF092209) (see the supplementary information).
These results were supported by the distinctive RFLP pattern
of the new subgroup 16SrVII-E compared with the RFLP
pattern of the four known 16SrVII subgroups (Fig. 5).
501
E. Pérez-López and others

Fig. 4. Distinctive RFLP patterns obtained with i PhyClassifier from in silico digestion of 16S rRNA gene F2nR2 fragments
from six representative 16SrIII strains of previously characterized subgroups and one new 16SrIII subgroup delineated in this
study. See Fig. 2 for further details.

New subgroup in the pigeon pea witches’-broom
phytoplasma group (16SrIX)
The pigeon pea witches’-broom phytoplasma group,
similar to the ash yellows phytoplasma group, is one of
the less widely distributed strains worldwide. Only ten
16SrIX subgroups (A–G) have been described, and ‘Ca.
Phytoplasma phoenicium’ (16SrIX-A) is the candidate
species associated with this group (Verdin et al., 2003;
Davis et al., 2010; Lova et al., 2011). In this study, Brazilian
huanglongbing disease-associated phytoplasma (GenBank
accession no. EU266074) (Teixeira et al., 2008), previously
identified at the group-taxon level, was classified as a new
subgroup 16SrIX-H, with a unique RFLP pattern and a
similarity coefficient of 0.92 compared with the reference
pattern of the subgroup 16SrIX-A (GenBank accession
no. AF248957) (Supplementary information, Fig. 6).
New subgroup in the ’stolbur’ phytoplasma group
(16SrXII)
Members of group 16SrXII have been detected in a large
number of plants worldwide, with a high incidence in
grapevines (Davis et al., 1997; Cheng et al., 2012; Quaglino
et al., 2013). With five candidate species of ‘Ca.

502 International Journal of Systematic and Evolutionary Microbiology 66

 New phytoplasma groups and subgroups in Latin America

Fig. 5. Distinctive RFLP patterns obtained with i PhyClassifier from in silico digestion of 16S rRNA gene F2nR2 fragments
from four representative 16SrVII strains of previously characterized subgroups and one new 16SrVII subgroup delineated in
this study. See Fig. 2 for further details.

Fig. 6. Distinctive RFLP patterns obtained with i PhyClassifier from in silico digestion of 16S rRNA gene F2nR2 fragments
from three representative 16SrIX strains of previously characterized subgroups and one new 16SrIX subgroup delineated in
this study. See Fig. 2 for further details.

http://ijs.microbiologyresearch.org 503
E. Pérez-López and others
Phytoplasma’ formally assigned to the 16SrXII group, ‘Ca.
Phytoplasma australiense’ (16SrXII-B) (Davis et al., 1997),
‘Ca. Phytoplasma japonicum’ (16SrXII-D) (Sawayanagi
et al., 1999), ‘Ca. Phytoplasma fragariae’ (16SrXII-E)
(Valiunas et al., 2006), ‘Ca. Phytoplasma convolvuli’
(16SrXII-H) (Martini et al., 2012) and ‘Ca. Phytoplasma
solani’ (16SrXII-A) (Quaglino et al., 2013), and another
group that may represent a separate candidate species,
‘Ca. Phytoplasma luffae’ (16SrXII-A) (IRPCM, 2004),
group 16SrXII is one of the most diverse based on 16S
rRNA gene nucleotide identity. Recently, the diversity of
this group has increased with the detection of new sub-
groups 16SrXII-F and 16SrXII-G through the analysis of
single nucleotide polymorphisms in the 16S rRNA gene
(Quaglino et al., 2009), and subgroup 16SrXII-I, associated
with potato plants in China (Cheng et al., 2012).
In Latin America, members of group 16SrXII have been
detected in only two countries (Chile and Bolivia), associated
with grapevine and peach, respectively (Jones et al., 2005b;
Gajardo et al., 2009). In this study, we identified the Isidro
peach phytoplasma (GenBank accession no. AY725212) as
Fig. 7. Distinctive RFLP patterns obtained with i PhyClassifier from in silico digestion of 16S rRNA gene F2nR2 fragments
from four representative 16SrXII strains of previously characterized subgroups and one new 16SrXII subgroup delineated in
this study. See Fig. 2 for further details.
504
a new subgroup (16SrXII-J). This strain showed a distinct
RFLP pattern (Fig. 7) compared with the previously
described subgroups and had a similarity coefficient of 0.95
with the reference pattern of subgroup 16SrXII-C (GenBank
accession no. AJ243045) (Supplementary information).
New subgroup in the Mexican periwinkle
phytoplasma group (16SrXIII)
The Mexican periwinkle phytoplasma group has been
found to be associated with disease in Latin America.
As the name implies, the first plants affected by this
group were collected in Mexico, and further reports were
made mainly in countries from the USA (Florida) to
Argentina (Jomantiene et al., 1998; Lee et al., 1998;
Harrison et al., 2002). Six subgroups have been identified
within group 16SrXIII, 16SrXIII-A to 16SrXIII-F, and
subgroups 16SrXIII-B and 16SrXIII-F have been shown to
affect strawberry plants (Jomantiene et al., 1998; Fernández
et al., 2015). Subgroup 16SrXIII-A was detected in periwinkle
plants (Lee et al., 1998), subgroup 16SrXIII-C in chinaberry
plants (Harrison et al., 2002) and subgroup 16SrXIII-D in
International Journal of Systematic and Evolutionary Microbiology 66
 New phytoplasma groups and subgroups in Latin America

samples collected from potato plants (Santos-Cervantes et al.,
2010). Using i PhyClassifier, we identified two new 16SrXIII
subgroups in Latin American samples in this study. The new
subgroup 16SrXIII-G is represented by chinaberry yellows
phytoplasma strain ChTYXIII-4 (GenBank accession no.
DQ444264). Strain ChTYXIII-4 was previously classified as
a member of subgroup 16SrXIII-C, but our results showed
a similarity coefficient of 0.95 with the reference pattern of
subgroup 16SrXIII-C (GenBank accession no. AF495882)
(Supplementary information), and a distinctive RFLP pattern
compared with the previously described 16SrXIII subgroups
(Fig. 8). The second new 16SrXIII subgroup identified in
this study was 16SrXIII-H, represented by the broccoli
stunt phytoplasma strain BSP-33 (GenBank accession no.
JX626329). The previously unclassified strain BSP-33
(Eckstein et al., 2013) showed a unique RFLP profile and a
similarity coefficient of 0.95 with the reference pattern of
subgroup 16SrXIII-A (GenBank accession no. AF248960)
(Fig. 8; Supplementary information).
New groups 16SrXXXIII and 16SrXXXIV
Based on the threshold similarity coefficient for a new group
stablished at 0.85 (Wei et al., 2008b), i PhyClassifier can

Fig. 8. Distinctive RFLP patterns obtained with i PhyClassifier from in silico digestion of 16S rRNA gene F2nR2 fragments
from six representative 16SrXIII strains of previously characterized subgroups and two new 16SrXIII subgroups delineated in
this study. See Fig. 2 for further details.

http://ijs.microbiologyresearch.org 505
E. Pérez-López and others
suggest new 16Sr phytoplasma groups. So far, 32 16Sr
groups have been formally described, which are generally
associated with the description of novel candidate species
of ‘Ca. Phytoplasma’ (Zhao et al., 2009; Lee et al., 2011;
Nejat et al., 2013). In this study, two novel 16Sr groups
were identified from previous reports. The novel group
16SrXXXIII is represented by the Chilean grapevine phyto-
plasma (GenBank accession no. DQ232752). This strain
was previously classified as a member of the subgroup
16SrVII-A (Table 2), but our analysis showed that, though
this strain is most similar to the reference pattern of sub-
group 16SrVII-A (GenBank accession no. AF092209), the
similarity coefficient is only 0.79. Moreover, this strain had
a unique RFLP pattern compared with other groups pre-
viously identified (Fig. 9). The other novel group described
in this study is the group 16SrXXXIV, represented by
pepper witches’-broom phytoplasma (GenBank accession
no. EU125184), which was previously classified as a
member of group 16SrII. Our results confirmed that this
strain is most similar to the reference pattern of the sub-
group 16SrII-F (GenBank accession no. EU099556), with a
similarity coefficient of 0.85. Pepper witches’-broom
Fig. 9. Distinctive RFLP patterns obtained with i PhyClassifier from in silico digestion of 16S rRNA gene F2nR2 fragments
from ten representative strains of previously characterized 16Sr groups and two groups delineated in this study. See Fig. 2
for further details.
506
phytoplasma had a distinctive RFLP pattern compared
with other phytoplasma groups (Fig. 9).
Reclassifications to previously reported
subgroups
In this study, besides detecting novel phytoplasma subgroups
and groups, we reclassified certain strains to already known
groups. The chinaberry yellows phytoplasma strain CbY17
(GenBank accession no. AF495657) was reclassified from
16SrIII-J to 16SrIII-B; broccoli stunt phytoplasma strain
BSP-3 (GenBank accession no. JX626330), which had no
previous classification, was classified as a member of sub-
group 16SrI-B; cassava frogskin disease phytoplasma strains
FSDY17, FSDY15 and FSDY29 (GenBank accession nos
AY737646, EU346761 and AY737647, respectively) were
reclassified from 16SrIII-L to 16SrIII-B; papaya bunchy top
phytoplasma strain BTSpHav35-IB (GenBank accession
no. JF781307), previously classified as 16SrI-B, was reclassified
as a member of subgroup 16SrI-L; Vinca minor phytoplasma
(GenBank accession no. AY144608), which was previously
classified to the group-taxon level, was classified in this
International Journal of Systematic and Evolutionary Microbiology 66
 New phytoplasma groups and subgroups in Latin America

Table 3. Analysis of the gain and loss of restriction sites in the reference sequences of new subgroups detected in this study

Restriction enzyme Recognition sequence Restriction site Subgroup (sequence at the restriction site)

Gained Lost New subgroup (this study) Reference subgroup

16SrI
16SrI-Z (AY725211) 16SrI-B (NC_005303)
Hae III GGQCC + CGCC GGCC
Rsa I GTQAC + GTAC GTAG
16SrI-AB (EU423898) 16SrI-B (NC_005303)
HinfI GQAnTC + CAATC GAATC
Mse I TQTAA + TCAA TTAA
16SrI-AC (JQ730859) 16SrI-B (NC_005303)
Hpa I GTTQAAC + GTTGAC GTTAAC
16SrI-AD (DQ286577) 16SrI-B (NC_005303)
Bfa I CQTAG + CTTG CTAG
Eco RI GQAATTC + AAATTC GAATTC
16SrI-AE (DQ286953) 16SrI-B (NC_005303)
Hpa II CQCGG + CTGG CCGG
16SrI-AF (JF781311) 16SrI-B (NC_005303)
Bst UI CGQCG + CGCG CGTG
16SrI-AG (AY249247) 16SrI-B (NC_005303)
Kpn I GGTACQC + GGCACC GGTACC
Rsa I GTQAC + GCAC GTAC
16SrII
16SrII-O (EF647744) 16SrII-C (AJ293216)
Hae III GGQCC + GGCC GCCC
16SrII-P (DQ286948) 16SrII-A (L33765)
Sau3AI QGATC + GATC CTTC
Mse I TQTAA + GTAA TTAA
16SrII-Q (JF781310) 16SrII-A (L33765)
Hha I GCGQC + GCGC GGGC
16SrII-R (DQ535900) 16SrII-C (AJ293216)
Alu I AGQCT + AGCT AGTT
Hae III GGQCC + GGCC AGCC
16SrII-S (FJ357164) 16SrII-C (AJ293216)
Rsa I GTQAC + GTAC GGAC
16SrII-T (EU125185) 16SrII-F (EU099556)
Bst UI CGQCG + CGCG CGCA
16SrIII
16SrIII-Z (JX626327) 16SrIII-J (AF147706)
Bst UI CGQCG + CGCG CGCA
16SrVII
16SrVII-E (AY741531) 16SrVII-A (AF092209)
HinfI GQAnTC + GAATC GAATT
Taq I TQCGA + TCGG TCGA
16SrIX
16SrIX-F (EU266074) 16SrIX-A (AF248957)
Hpa II CQCGG + CCGG TCGG
Rsa I GTQAC + GTGC GTAC
16SrXII
16SrXII-J (AY725212) XII-C (AJ243045)
Eco RI GQAATTC + GTTTTC GAATTC
16SrXIII
16SrXIII-G (DQ444264) 16SrXIII-C (AF495882)
Hae III GGQCC + GTCC GGCC
Mse I TQTAA + TTAA TTAG
16SrXIII-H (JX626329) 16SrXIII-A (AF248960)
Alu I AGQCT + AGTT AGCT
Mse I TQTAA + TTAG TTAA

http://ijs.microbiologyresearch.org 507
E. Pérez-López and others

68 ‘Ca. Phytoplasma graminis’ (AY725228) R

99 ‘Ca. Phytoplasma caricae’ (AY725234) R
57 ‘Ca. Phytoplasma solani’ (AJ964960) R
0.01
68 ‘Ca. Phytoplasma americanum’ (DQ174122) R
16SrXII
89 ‘Ca. Phytoplasma australiense’ (L76865) R
100 Isidro peach phytoplasma (16SrXII-JS, AY725212) RS
36 ‘Ca. Phytoplasma convolvuli’ (JN833705) R
Mexican periwinkle virescense (AF248960) RS
53 100 Broccoli stunt phytoplasma strain BSP-33 (16SrXIII-HS, JX626329) RS 16SrXIII
63 Chinaberry yellows phytoplasma strain Ch TYXIII-4 (16SrXIII-G§, DQ444264) RS
‘Ca. Phytoplasma japonicum’ (AB010425) R
64
43 ‘Ca. Phytoplasma fragariae’ (DQ086423) R
‘Ca. Phytoplasma lycopersici’ (EF199549) R
‘Ca. Phytoplasma asteris’ (M30790) R
100
Bougainvillea shoot proliferation phytoplasma strain BSP-Br1 (16SrI-ACS, EU423898) RS
56
Broad bean phytoplasma (16Srl-AF§, DQ288953) RS
99
46 Fraxinus uhdei phytoplasma (16Srl-AD§, JQ730859) RS
16SrI
Basil little leaf phytoplasma (16Srl-AE§, DQ286577) RS
49
Marigold phyllody phytoplasma (16Srl-AG§, AY249247) RS
‘Brotes grandes’ of potato phytoplasma (16Srl-AB§, AY725209) RS
100 Bolivia alfalfa phytoplasma (16Srl-AB§, AY725211) RS
‘Ca. Phytoplasma costaricanum’ (HQ225630) R
76 ‘Ca. Phytoplasma pyri’ (AJ542543) R
96 ‘Ca. Phytoplasma mali’ (AJ542541) R
90 ‘Ca. Phytoplasma prunarum’ (AJ542544) R
39 ‘Ca. Phytoplasma s partii’ (X92869) R

94 ‘Ca. Phytoplasma allocasuarinae’ (AY135523) R

96 ‘Ca. Phytoplasma rahmini’ (X76431) R

‘Ca. Phytoplasma tamaricis’ (FJ432664) R

98 Tomatillo witches’ broom phytoplasma (16Srl-AB§, AY725209) RS

96 Pepper witches’ broom phytoplasma (16SrXXXIV-A§, EU125184) RS

Amaranthus hypochondriacus phytoplasma strain 52A (16Srlll-S§, FJ357164) RS

98
62 Echinops is sp. yellow patch phytoplasma (16Srll-R§, DQ535900) RS

84 ‘Ca. Phytoplasma aurantifolia’ (U15442) R 16SrII

97 65
Tabebuia pentaphylla phytoplasma (16Srll-O§, EF647744) RS

97 Cuban papaya phytoplasma (16Srll-P§, DQ286948) RS

100
Papaya bunchy top phytoplasma strain BTSpHav02-IIA (16Srll-Q§, JF781310) RS
100
‘Ca. Phytoplasma australasia’ (Y100097) R

‘Ca. Phytoplasma brasiliense’ (AF147708) R

100 Broccoli stunt phytoplasma strain BSP-21 (16SrIII-Z§, JX826327) RS

99 16SrIII
‘Ca. Phytoplasma pruni’ (JQ044393) R

‘Ca. Phytoplasma palmicola’ (KF751387) R

97 100 Brazilian Huanglongbing disease-associated phytoplasma (16SrlX-H§, EU266074) RS

99
16SrIX
‘Ca. Phytoplasma phoenicium’ (AF515636) R
34 ‘Ca. Phytoplasma omanense’ (EF666051) R
38
‘Ca. Phytoplasma pini’ (AJ632155) R
91 ‘Ca. Phytoplasma castaneae’ (AB054986) R
99 ‘Ca. Phytoplasma oryzae’ (D12581) R
27
‘Ca. Phytoplasma cynodontis’ (AJ550984) R
‘Ca. Phytoplasma malaysianum’ (EU371934) R
51 ‘Ca. Phytoplasma rubi’ (AY197648) R
85
81 ‘Ca. Phytoplasma ulmi’ (AF122910) R
96 98
‘Ca. Phytoplasma ziziphi’ (AY072722) R
‘Ca. Phytoplasma balanitae’ (AB689678) R
70 ‘Ca. Phytoplasma sudamericanum’ (GU292081) R
‘Ca. Phytoplasma trifolii’ (AY390261) R
64
53 ‘Ca. Phytoplasma fraxini’ (AF092209) R 16SrVII
99 Chilean grapevine yellows phytoplasma (16SrVll-E§, AY741531) RS
75 Chilean grapevine phytoplasma (16SrXXXIll-A§, DQ232752) RS

Acholeplasma laidlawii PG8 (U14905)

508 International Journal of Systematic and Evolutionary Microbiology 66


Fig. 10. Phylogenetic tree reconstructed through the neighbour-joining method of the 16S rRNA gene sequences of the 37
candidate species of ‘Ca. Phytoplasma’ so far formally described (labelled R), the 16S rRNA gene sequences of the refer-
ence strains of the new 18 subgroups delineated in this study and the 16S rRNA gene sequences of the reference of the
new two groups also delineated in this study (labelled RS, and the new groups are also labelled by filled triangles). Accession
numbers are indicated in parentheses, and the strain acronym is given if applicable. Acholeplasma laidlawii PG8 was used as
an outgroup. The phylogenetic tree was bootstrapped 1000 times to achieve reliability. Bar, 1 substitution in 100 positions.
study as a member of subgroup 16SrII-C (Supplementary
information). Finally, poinsettia branch-inducing phyto-
plasma strains MJMC-A and MJMC-B (GenBank accession
nos EU165359 and EU165360) were classified as members
of subgroup 16SrIII-L (Supplementary information). All of
the sources of the original reports and details of the plant
host affected by each strain are shown in Table 2.
Confirmatory analysis
To confirm that our results using i PhyClassifier were
correct and consistent with previously described strains,
we repeated the same analysis with F2nR2 sequences
taken at random belonging to groups 16SrI, II, III, VII,
IX, XII and XIII described by different authors such as
Wei et al. (2007), Zhao et al. (2009) and Quaglino et al.
(2009). Our results agreed with the results described by
those authors and the classification made by them.
To provide evidence that the sites responsible for any novel
RFLP profiles were not due to errors in the reported
sequences, we identified endonucleases capable of differen-
tiating all the novel subgroups identified in this study.
We aligned the reference sequences of the novel subgroups
detected in this study to the reference sequences of the
subgroup with the highest similarity coefficient to that
sequence. After trimming both sequences to the F2nR2 frag-
ment, the restriction sites were localized using pDRAW32
software (AcaClone Software; http://www.acaclone.com).
The results obtained showed that, with the exception of
new subgroups 16SrI-AD and 16SrXII-J, analysis of the
restriction sites in the novel subgroups was not related to
any sequencing errors (Table 3). The presence of CTTG,
which removes the restriction site for Bfa I in 16SrI-AD
(GenBank accession no. DQ286577), may suggest the pre-
sence of a poly(T) run, but this change of A to T is also
detected in the other strain classified in subgroup 16SrI-
AD (DQ286947). The presence of GTTTTC in 16SrXII-J
(AY725212) may suggest the presence of a poly(T) run,
but this change of AA to TT and the loss of the Eco RI
restriction site can also be detected in the reference strain
for subgroup 16SrXII-D (AB010425). Our results supported
the conclusion that all the novel subgroups detected in this
study are not generated by i PhyClassifier because of mistakes
during sequencing.
We also analysed all F2nR2 sequences through the DECIPHER
Find Chimeras web tool (http://decipher.cee.wisc.edu/
FindChimeras.html). We did not find any chimeras in any
of the novel subgroups that we identified for groups 16SrI,
http://ijs.microbiologyresearch.org
New phytoplasma groups and subgroups in Latin America
II, III, VII, IX, XII and XIII, or the novel groups detected
in this study.
Phylogenetic analysis of 16S rRNA gene sequences of all the
strains identified as members of novel groups and subgroups
in this study was reconstructed by the neighbour-joining
method, using the tree-bisection-and-regrafting (TBR) algor-
ithm available in MEGA 6 (Tamura et al., 2013), bootstrapped
1000 times. The phylogenetic analysis was congruent with the
new classifications (Fig. 10). The novel strains branched with
the corresponding candidate species of ‘Ca. Phytoplasma’;
for example, the new 16SrI subgroups branched with ‘Ca.
Phytoplasma asteris’, the new 16SrII subgroups branched
with ‘Ca. Phytoplasma aurantifolia’ and ‘Ca. Phytoplasma
australasia’, and so on (Fig. 10).
Conclusions
Accurate identification of biologically distinctive phyto-
plasma strains is very important for the development of
management strategies, vector studies and further epidemio-
logical research. With more than 10 years of molecular
studies of phytoplasmas in Latin America, a mean of seven
emerging diseases related to phytoplasmas has been reported
per year (Table 2). Issues such as easy internet access or the
relatively high price of sequencing could previously have
affected the use of online tools in countries such as Cuba,
but recent improvements in internet access and continuing
decreases in sequencing prices will ensure that phytoplasmas
can be identified using online tools. Another problem in
Latin America is the lack of funds and research projects
dedicated to the study of phytoplasmas and diseases related
to these pathogens, leading to the underestimation of the
real problems associated with phytoplasmas. The groups
and subgroups that we have identified in the current study
reveal how the real diversity of phytoplasmas can be easily
underestimated. With the use of i PhyClassifier, a free,
user-friendly and interactive tool shown to be capable of
accurately classifying phytoplasma strains, we have expanded
the known diversity of groups 16SrI, 16SrII, 16SrIII, 16SrVII,
16SrIX, 16SrXII and 16SrXIII, and have described two novel
phytoplasma groups. This study does not yet allow us to
distinguish a relationship between ecosystem distribution
and phytoplasma diversity, such as a correlation between
the host range and insect vectors of diverse phytoplasma
strains. More data are needed.
These results suggest that the diversity of phytoplasma strains
in Latin America is much higher than was previously thought,
and underscore the importance of exploiting the tools that
509
E. Pérez-López and others
are available in order to classify accurately strains of phyto-
plasma as they are detected in plant and insect hosts.
Acknowledgements
This work was supported by the Genomic Research and Development
Initiative for the shared priority project on quarantine and invasive
species. We sincerely thank the critical review and suggestions made
by Dr Yan Zhao. E.P.-L. thanks CONACYT for a PhD scholarship
(CVU: 517835).
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