Folia Microbiologica

https://doi.org/10.1007/s12223-020-00772-x

ORIGINAL ARTICLE

Detection of 16SrVI and 16SrIX phytoplasma groups in pot marigold

and tickseed plants in northeastern Iran
Sara Gharouni-Kardani 1 & Mahnaz Ashnayi 2 & Assunta Bertaccini 3

Received: 5 June 2019 / Accepted: 16 January 2020

# Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2020

Abstract
Pot marigold and tickseed are ornamental plants with many medicinal and cosmetic uses and for landscape, respectively. During a
survey in 2018, phyllody symptoms were observed in high percentages in these plants in some regions of the Razavi Khorasan
province (northeastern Iran). Total DNA was extracted from symptomatic and asymptomatic plants and polymerase chain
reaction was carried on using universal phytoplasma primer pairs P1/P7 and nested primer pairs R16F2n/R16R2. The nested
amplification of 1200-bp fragments confirmed the presence of phytoplasmas only in the symptomatic plants. BLAST search,
phylogenetic analysis, and virtual RFLP patterns of cloned amplicons allowed to classify the pot marigold phyllody phytoplasma
in the 16SrVI-A subgroup while tickseed phyllody was enclosed in the 16SrIX-I subgroup. This is the first report of the
association of a 16SrVI phytoplasma with pot marigold phyllody in Iran and of the phytoplasma presence in tickseed.

Keywords Pot marigold . Tickseed . Calendula officinalis . Coreopsis grandiflora . 16SrIX . In silico RFLP

Introduction assays greatly improved the phytoplasma detection

Phytoplasmas are plant pathogens insect-transmitted associat-
ed with diseases in hundreds of plant species worldwide
(Bertaccini et al. 2014; Maejima et al. 2014; Namba 2019)
that are also efficiently spread via vegetative propagation such
as cutting, grafting, and micropropagation practices
(Bertaccini 2007). Their detection was initially based on elec-
tron microscopy images of diseased phloem tissue and on
some biological properties, such as unique symptomatology,
specific insect vector, and plant host range. In recent years, the
development of serological and molecular tools such as mono-
clonal antibodies and cloned DNA probes together with PCR
* Sara Gharouni-Kardani
s.gharooni@areeo.ac.ir
1 tive to southern Europe and cultivated in temperate regions
Plant Protection Research Department, Khorasan Razavi
Agricultural and Natural Resources Research and Education Center,
AREEO, Mashhad, Iran
2
Department of Plant Protection, College of Agriculture, Ferdowsi
University of Mashhad, Mashhad, Iran
3
Department of Agricultural and Food Sciences, Alma Mater
Studiorum University of Bologna, Bologna, Italy
(Bertaccini and Lee 2018).
The economic importance of ornamental plants has been
progressing significantly in many countries with the interna-
tional trade expanding continuously. Among a number of
pathogens such as viruses, fungi, bacteria, the phytoplasmas
drastically damage growth, and marketing parameters of or-
namental plants affecting their commercial value. The epi-
demics of phytoplasma-associated diseases have been com-
pelled the withdrawal of many ornamental varieties from cul-
tivation and market (Priya and Rao 2017). General yellowing
and stunted growth of plants, proliferation of shoots, phyllody,
virescence, reduced flower size, and reddening of leaves are
the most common symptoms observed in phytoplasma-
infected ornamental plants (Khan et al. 2016). The number
of phytoplasmas identified in ornamental species has greatly
increased over the last decades as a consequence of the in-
creased worldwide production (Bellardi et al. 2018).
Pot marigold (Calendula officinalis L., Asteraceae) is na-
since it is among the easiest and most versatile flowers to be
grown. The flowers were used in ancient cultures as a medic-
inal herb as well as a dye for fabrics, foods, and cosmetics.
Compounds of marigold flowers exhibit anti-inflammatory,
anti-tumor-promoting, and cytotoxic activities (Ukiya et al.
2006). Coreopsis grandiflora Hogg ex Sweet, also called

tickseed, is a flowering plant native to the USA used as orna-
mental plant for the landscape for its attractive ornamental
foliage.
During a survey on ornamental plants for landscape in the
Khorasan Razavi province (northeastern Iran) in 2018, pot
marigold phyllody (PMP) and Coreopsis phyllody (CorP)
were observed. Hence, this study was planned to identify the
phytoplasmas associated with those two ornamental plants.
Materials and methods
Plant material and DNA extraction
During a survey in gardens producing flowers, phytoplasma-
suspected symptoms with high incidence (nearly 70%) were
observed in pot marigold and tickseed plants. Leaf petioles
and midribs from 12 symptomatic plants (seven from PMP
and five from CorP) were used for DNA extraction.
Approximately 0.4 g of fresh tissue was used for each extrac-
tion, according to the method described by Zhang et al.
(1998). Samples collected from 2 asymptomatic plants, one
per species, collected from the same fields were prepared in
the same manner and used as negative controls. DNA extract-
ed from cowpea phyllody phytoplasma strain (16SrVI-A
group) maintained in laboratory served as positive control
(Gharouni-Kardani and Jamshidi 2018a).
Phytoplasma detection by PCR assays
The universal primer pair P1 (Deng and Hiruki 1991) and P7
(Schneider et al. 1995) was employed in PCR to amplify an
1800-bp fragment that extends from the 5′ end of the 16S
rRNA-encoding DNA to the 5′ region of the 23S rRNA-
encoding DNA. A 30-fold dilution of the product was then
used as template for nested PCR using primer pair R16F2n/
R16R2 which amplify an internal fragment of 1250 bp from
the 16S rRNA-encoding DNA (Gundersen and Lee 1996).
Each 25 μL PCR mix contained 1 × PCR Master Mix red
containing; 2 mmol/L MgCl2, Tris-HCl pH 8.5, (NH4)2S04,
4 mmol/L MgCl2, 0.2% tween 20, 0.4 mmol/L dNTPs
(Ampliqon, Korea), 0.4 μmol/L of each primer, and 25 ng of
DNA template. PCR products were separated on 1.2% aga-
rose gels in TBE buffer, stained with 1 μL/ml DNA Staining
Dye (Green Viewer™, Genet Bio, Korea) and visualized with
a UV transilluminator. Distilled water and DNA of the two
asymptomatic plants were included as templates in the PCRs
as negative controls.
Cloning and sequencing
P1/P7 amplicons were selected (one fom PMP and one from
CorP), extracted by GenCatch™ Gel Extraction Kit (Epoch
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Life Science, USA) ligated into pTZ57R/T plasmid using
T/Aclone™ PCR Product Cloning Kit following the manufac-
turer protocol (Thermo Fisher, USA), transformed into
Escherichia coli DH5-ɑ cells (Invitrogen Life Technologies,
USA). The plasmid DNA from cultures of recombinant colo-
nies was purified using High Pure Plasmid Isolation kit (Epoch
Life Science, USA). The presence of the insert was checked by
digesting the plasmid DNA with PstI and EcoRI (Fermentas,
Lithuania). Sequencing was performed in both directions by a
commercial service (Macrogen Inc., Korea) using M13 forward
and reverse primers and internal primers designed by the se-
quencing company. The sequences were assembled using DNA
baser V.4 program. Aligned and edited sequences were depos-
ited in NCBI. GenBank Nucleotide Basic Local Alignment
Search Tool (BLAST) was employed to compare phytoplasma
sequences with those available in the National Center for
Biotechnology Information (NCBI).
Phylogenetic and in silico RFLP analysis
The phylogenetic relationship of the phytoplasmas under
study and of some selected from the GenBank was studied
by a phylogenetic tree generated using the neighbor-joining
method with the program MEGA (Molecular Evolutionary
Genetics Analysis), version 7 (Kumar et al. 2016).
Acholeplasma laidlawii was employed as outgroup to root
the trees. Bootstrapping (1000 replicates) was performed to
estimate the stability and support for the inferred clades.
Moreover, the partial sequences of the 16S rRNA gene of
nested PCR product were subjected to virtual restriction frag-
ment length polymorphism (RFLP) analysis with 17 restric-
tion enzymes (AluI, BfaI, TaqI, DraI, EcoRI, HaeIII, HhaI,
HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, BamHI, RsaI,
BstUI, and SspI), using the iPhyClassifier to assess ribosomal
group affiliation (Zhao et al. 2009).
Results
The symptoms of PMP and CorP diseases were similar and in
both cases were observed in the flowers as phyllody, vires-
cence, proliferation, and sterility; leaf size reduction and axil-
lary bud production along the stem with witches’ broom for-
mation were also present (Fig. 1).
Direct PCR using P1/P7 universal primers resulted in the
expected size bands of approximately 1.8 kb from some of the
symptomatic plants in both species. The nested PCR using uni-
versal primer pairs of R16F2n/R16R2 resulted in bands of ap-
proximately 1.25 kb respectively in all symptomatic plants. No
product was obtained from any of the symptomless plants or
distilled water (data not shown). The P1/P7 PCR products of
two strains (one from PMP and one from CorP) were cloned;
one of the clones for each strain was sequenced and designed as
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Fig. 1 Symptoms associated with

phytoplasma presence in pot
marigold represented by phyllody
and witches’ broom (a, b)
compared with the healthy plant
(c). Leaf size reduction, phyllody,
virescence, and witches’ broom in
Coreopsis grandiflora plants (d,
e) compared with the healthy
plant (f) collected from Khorasan
Razavi province (northeastern
Iran)

PMP-Khoshab and CorP-Mashhad, and deposited in GenBank
under the accession numbers MK433199 and MK454714, re-
spectively. Results of BLAST searches using PMP-Khoshab
phytoplasma strain sequence showed its close relationship to
tomato big bud phytoplasma (JF508511), vinca virescence phy-
toplasma (AY500817), and other members of the clover prolif-
eration group (16SrVI) with 99% identity. The CorP-Mashhad
phytoplasma strain (MK433199) has the maximum identity to
Bushehr (Iran) eggplant big bud phytoplasma (JX483702), per-
iwinkle virescence phytoplasma (HQ589191), and other mem-
bers of the pigeon pea witches’ broom group (16SrIX) with 98–
99% identity.
The phylogenetic trees showed that the two phytoplasma de-
tected cluster with members of 16SrVI and 16SrXI groups
(Fig. 2). The results of phylogenetic analysis indicated that
PMP from Iran clusters with the “Candidatus Phytoplasma
trifolii”-related strains enclosed in the subgroup 16SrVI-A
(JF508507 and KC633092) associated with diseases in tomato
in the Kermanshah province and cowpea in East Azerbaijan
province, Iran, respectively, 16SrVI-E (AY270156) from yellow
starthistle, Italy, and 16SrVI-H (EF651786) from Portulaca
grandiflora, India (Fig. 2). The phytoplasma strain CorP-
Khoshab clusters with strains enclosed in group 16SrIX in the
“Candidatus Phytoplasma phoenicium”-related strains clade
(JF508515, KX011516, DQ195209, KU892213, AF515637,
HQ407532, HQ407514, and AF515636). Results of the virtual
RFLP utilizing the online iPhyClassifier program indicated that
PMP-Khoshab strain is identical (similarity coefficient 1.00) to
the “Candidatus Phytoplasma trifolii” (AY390261) that is
assigned to the subgroup 16SrVI-A (Fig. 3a, b). The same tool
encloses the phytoplasma strain CorP-Khoshab in subgroup
16SrIX-J (similarity coefficient 1) to the reference pattern of
16Sr group IX, subgroup J that is a clone of a phytoplasma from
chichory (KY986922) (Fig. 3c, d). However, considering the
lack in the tool of the newly designed subgroup 16SrIX-I a
further alignment allow to allocate it in subgroup 16SrIX-I from
Onobrychis viciifolia (KX461906) from Iran and show that this
and other strains from Iran cluster together in a separate branch
from strain ChicBS from Saudi Arabia (KY986922), and their
alignment show that they have in common four SNPs to the
strain classified as 16SrIX-J (Fig. 4).
Discussion
In this study, the presence of a phytoplasmas in phyllody af-
fected pot marigold and tickseed plants was confirmed by
PCR followed by in silico RFLP of the cloned amplicons that
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Fig. 2 Phylogenetic tree 75 16SrIX-C Sesame phyllody phytoplasma Iran (JF508515)

constructed by neighbor-joining 90 16SrIX-C Grapevine yellows phytoplasma Shiraz (KX011516)

analysis of partial 16S rRNA- 88
16SrIX-C Khafr (Iran) almond witches’ broom phytoplasma (DQ195209)
encoding DNA sequences of
63 16SrIX-H Sarson phyllody Pakistan (KU892213)
phytoplasma strains obtained
from PMP and CorP 16SrIX-I Iranian Coreopsis grandiflora phyllody phytoplasma strain CoP-Mashhad (MK454714)
phytoplasmas (shown by ●), other 100
76 16SrIX-J ‘Candidatus Phytoplasma phoenicium’ clone ChicBS192-C4 (KY986922)
sequences retrieved from
GenBank (shown by 16Sr 16SrIX-D Almond witches’ broom Lebanon (AF515637)

subgroup affiliations and 16SrIX-F Almond and stone fruit witches’ broom Lebanon (HQ407532)
accession number), and 81
99 16SrIX-G Almond and stone fruit witches’ broom Lebanon (HQ407514)
Acheleplasma laidlawii as the 87
outgroup 72 16SrIX-B ‘Candidatus Phytoplasma phoenicium’ (AF515636)

16SrV ‘Candidatus Phytoplasma ulmi’ (EU184021)

100 16SrVI-H Portulaca little leaf phytoplasma India (EF651786)

99 100 16SrVI-E Centaurea solstitialis virescence phytoplasma Italy (AY270156)

16SrVI-A Tomato big bud phytoplasma Iran (JF508507)

99

16SrVI-A Calendula officinalis phyllody phytoplasma strain PMP-Khoshab (MK433199)

67
16SrVI-A cowpea phyllody phytoplasma strain Nazarlou (KC633092)

16SrII-D Calendula officinalis phyllody phytoplasma strain Yazd (KU297202)

100 16SrII-D Calendula officinalis phyllody phytoplasma strain Ashkezar (MH065715)

16SrI-B Aster yellows phytoplasma watercress Hawaii - USA (AY665676)

16SrXII-A ‘Candidatus Phytoplasma solani’ (AF248959)

100
100 16SrXII-A Eggplant big bud phytoplasma Bushehr (Iran) (JX483703)

Acholeplasma laidlawii (M23932)

0.02

Fig. 3 Comparison of computer-

simulated virtual RFLP patterns
derived from in silico RFLP of
phytoplasma 16S rRNA gene
(1.2-kb fragment) between
reference strain of 16SrVI
subgroup A (a) “Candidatus
Phytoplasma trifolii”
(AY390261) and a PMP-Khoshab
strain (b) and between strain
chicory (KY986922) of 16SrIX-J
(c) and CorP-Mashhad strain (d)
using the iPhyClassifier. MW,
molecular weight marker
phiX174 HindIII digested.
Fragment sizes in base pair from
top to bottom: 1353; 1078; 872;
603; 310; 281; 271; 234; 194;
118; and 72
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70 16SrIX-C Grapevine yellows phytoplasma Shiraz (KX011516)

16SrIX-C Sesame phyllody phytoplasma (JF508515)
82 16SrIX-C Tomato big bud phytoplasma strain STBB (JF508510)

50 16SrIX-C Sesame phyllody phytoplasma strain Sarvestan(JX464670)

16SrIX-C Almond witches’ broom phytoplasma Khafr (DQ195209)
16SrIX-H Sarsoon phyllody Pakistan (KU892213)
16SrIX-A Pigeon pea witches’ broom phytoplasma (AF248957)

16SrIX-E Juniperus occidentalis witches’ broom phytoplasma (GQ925918)

16SrIX-J ‘Candidatus Phytoplasma phoenicium’ clone ChicBs192-C4 (KY986922)

16SrIX-I Iranian Lactuca sativa phyllody phytoplasma (DQ889748)

83 16SrIX-I Iranian Lactuca serriola phyllody phytoplasma (DQ889749)
16SrIX-I Eggplant big bud phytoplasma Bushehr (JX483702)
56
16SrIX-I Onobrychis viciifolia phytoplasma (KX461906)
16SrIX-I Iranian Coreopsis grandiflora phyllody phytoplasma strain CoP-Mashhad (MK454714)
16SrIX-C Periwinkle virescence phytoplasma strain Naxos Italy (HQ589191)

16SrIX-D ‘Candidatus Phytoplasma phoenicium’ (AF515637)

16SrIX-B ‘Candidatus Phytoplasma phoenicium’ (AF515636)
95
16SrIX-F ‘Candidatus Phytoplasma phoenicium ‘(HQ407532)
92
16SrIX-G ‘Candidatus Phytoplasma phoenicium’ (HQ407514)
Acholeplasma laidlawii (M23932)

0.02
a

Fig. 4 a Phylogenetic tree constructed by neighbor-joining analysis of
partial 16S rRNA-encoding DNA sequences of phytoplasma strains
obtained from phytoplasma strains enclosed in the 16SrIX group
retrieved from GenBank (shown by 16Sr subgroup affiliations and
accession number) and A. laidlawii as the outgroup; b alignment of
b
selected sequences at the 3′ of the 16S rDNA of some phytoplasma
strains classified in the subgroups 16SrIX-I and IX-J and the strain
CorP-Khoshab: the latter show 4 SNPs in the initial 8 nucleotides as in
other strains reported from different crops in Iran

allow to identify in the two plant species 16SrVI and 16SrIX
group of phytoplasmas. Phytoplasmas enclosed in three ribo-
somal groups were identified in pot marigold 16SrII-D from
Yazd and Khuzestan provinces in Iran and from Saudi Arabia
(Esmailzadeh Hosseini et al. 2011, 2016; Azimi et al. 2018;
Omar and Alsohim 2016), 16SrI from Italy and Canada
(Marcone et al. 1997; Wang and Hiruki 2001), and 16SrXII-
A from Serbia (Pavlovic et al. 2014). Phytoplasmas belonging
to subgroup 16SrII-E were detected in wild calendula
(Asteraceae) showing malformed flowers in Sardinia, Italy
(Tolu et al. 2006). Phytoplasma-infected C. officinalis was
also reported in India (Khurana et al. 1981; Rani et al.
2014). Although different phytoplasma groups have been re-
ported from pot marigold, no difference in the disease symp-
toms were described confirming that different phytoplasmas
may induce identical symptoms in the same plant species
(Marcone et al. 1996). In Iran, the 16SrVI group
phytoplasmas, in addition to pot marigold, were reported in-
fecting cabbage (Salehi et al. 2007b), safflower (Salehi et al.
2009), tomato (Jamshidi et al. 2014), cucumber (Asghari et al.
2010; Esmailzadeh Hosseini et al. 2015; Gharouni-Kardani
and Jamshidi 2018b), maize, tomato, cucumber (Zibadoost
et al. 2016), and cowpea (Gharouni-Kardani and Jamshidi
2018a). However, investigations are still needed to determine
if these phytoplasmas have a common source and a common
insect vector.
The pigeon pea witches’ broom phytoplasma group (16SrIX)
consists of diverse phytoplasma strains associated with 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 (Lee et al. 2012). Nine
16SrIX subgroups (A–I) have been described to date
(Bertaccini and Lee 2018), and “Ca. P. phoenicium” (16SrIX-
B) is the “Candidatus Phytoplasma” species associated with this
group (Verdin et al. 2003). A further 16SrIX-J subgroup infecting
chicory plants in Saudi Arabia is represented by the strain
ChicBS (Pérez-López et al. 2018). The evidence presented here
confirms that a different subgroup or lineage is present in Iran
that is consistent for the sequences and the phylogeny with the
strain described in Onobrychis viciifolia and in other Iranian
strains tentatively assigned to the subgroup 16SrIX-I
(Esmailzadeh Hosseini et al. 2018). Sesame (Salehi et al.
2005), lettuce, and wild lettuce (Salehi et al. 2007a) are other
herbaceous hosts of 16SrIX phytoplasma group in Iran and are
also tentatively assignable to the same subgroup 16SrIX-I based

on the common presence of the same four SNP in the same
position in the 16S ribosomal gene (Esmailzadeh Hosseini
et al. 2018). The lack of the strain in the iPhyClassifier used for
the virtual identification reflects a limitation of the classification
system in which there is no single location for registering new
groups and subgroups of phytoplasmas (Zhao and Davis 2016).
However from the data presented, it appears clear that the
16SrIX-J subgroup can be maintained, but there is the need to
add the 16SrIX-I strain in the iPhyClassifier. There is also the
need of a general a revision of the whole subgrouping of the
16SrIX group since strain classified in subgroup 16SrIX-C and
from different geographic origin result not clustering together. On
the other hand, since only one clone was examined, it was not
possible to verify whether the strain under study was harboring
two heterogeneous genomic copies of 16S rRNA-encoding
genes as reported in some other phytoplasma ribosomal groups
such as 16SrI, 16SrIII, 16SrVI, and 16SrXII (Liefting et al. 1996;
Davis et al. 2003; Duduk et al. 2009; Town et al. 2018).
The 16SrIX phytoplasmas also have been previously detected
in Iran in witches’ broom affected almonds (Salehi et al. 2006)
and grapevine (Salehi et al. 2016) in the Fars province and in
eggplant (Tohidi et al. 2015) in Fars and Hormozgan provinces.
Moreover, subgroup 16SrIX-B (“Ca. P. phoenicium”) in almond
and GF-677 trees was also reported (Motamedi et al. 2016). A
phytoplasma was detected in diseased golden tickseed
(Coreopsis tinctoria Nutt.) and identified as members of 16SrI-
A group in Canada (Wang and Hiruki 2005). In Iran, a 16SrI-B
group phytoplasma was reported to infect lance coreopsis
(Coreopsis lanceolata L.) (Babaie et al. 2007).
As the clover proliferation (16SrVI) and pigeon pea witches’
broom (16SrIX), phytoplasma groups have diverse plant hosts
and vectors, studying the role of insect vectors in the spread of
these diseases or finding the source of infection in Iran’s regions
is necessary. Identification of the associated agent of ornamental
phyllody disease in Iran should facilitate studies concerned epi-
demiological aspects of these diseases, contributing also to the
knowledge of the genetic diversity of phytoplasmas present in
Iran. To the best of the present study’s knowledge, this is the first
report of the tickseed (C. grandiflora) infection with
phytoplasmas; this is also the first study reporting C. officinalis
as a new host species for 16SrVI group phytoplasmas. The re-
search should be continued in order to identify the insect vectors
of these phytoplasmas in the area surveyed in order to be able to
provide appropriate management strategies to reduce the impact
of these diseases in this and other agronomically relevant crops.
Acknowledgments We thank Dr. Narjes Azizi from Forest and
Rangeland Department, Khorasan Razavi Agricultural and Natural
Resource Research and Education center, AREEO, Mashhad, Iran, for
identification of plant species.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Folia Microbiol
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