Phytopathogenic Mollicutes doi: 10.5958/2249-4677.2020.00004.

3
Vol. 12 (2), December 2022, 83-87

Research Article

Lantana camara showing little leaf symptoms: a new weed host of

‘Candidatus Phytoplasma trifolii’

Smriti Mall1, Apoorva Srivastava1 and Rajarshi Kumar Gaur2

1
Molecular Plant Pathology Laboratory, Department of Botany, DDU Gorakhpur University Gorakhpur-273009, Uttar
Pradesh, India
2
Department of Biotechnology, DDU Gorakhpur University Gorakhpur-273009, Uttar Pradesh, India

Received: September 09, 2022; Accepted: December 28, 2022

Abstract

Lantana camara is an invasive weed, alien for India, with active phytochemical constituents making it less prone
to diseases. Recently Lantana plants showing little leaf, shortening of internodes and stunted growth symptoms
were detected in Rajahi village near Gorakhpur, Uttar Pradesh, India. These symptoms are indicative of
phytoplasma presence. With the use of PCR and nested PCR assay with primer pairs P1/P7 and R16F2n/R2, the
presence of phytoplasmas in samples from three symptomatic plants was confirmed. Sequence analysis revealed
that the obtained 1.2 kb amplicon sequence shares more than 99% identity with sequences of ‘ Candidatus
Phytoplasma trifolii’ and in the phylogenetic analysis, the sequence clustered with other ‘Ca. P. trifolii’ strains. In
silico RFLP analysis of the 16Sr RNA sequence indicates its inclusion in 16SrVI-D subgroup that is therefore
reported here for the first time in the world in little leaf diseased Lantana plants.

Keywords: phytoplasma disease, 16S rRNA, sequencing, weed, India

Introduction 2019). Garibaldi et al. (2003) reported web blight on

Lantana camara L., commonly known as wild sage or
red sage is a persistent and invasive weed found among
60 countries worldwide with more than 650 varieties.
It belongs to the family Verbenaceae and is native to
the American tropics. The presence of therapeutically
rich phytochemicals makes Lantana resistant to most
microbial infections. Bioactive compounds like
alkaloids, flavonoids, saponins, tannins, germacrene A,
B, D and triterpenes like lantadenes A, B, C, and D are
present in it (Ved et al., 2018). L. camara leaf extracts
exhibited anti-proliferative, antimicrobial, fungicidal,
insecticidal and nematocidal activities (Bashir et al.,
Lantana caused by Rhizoctonia solani in Europe. Weed
diseases associated with phytoplasmas have been
reported predominantly in south-eastern Asian
countries and Europe (Marcone et al., 2016). So far
eighty weed species were reported as phytoplasma
hosts and infected with ‘ Candidatus Phytoplasma
asteris’, ‘Ca. P. aurantifolia’, ‘Ca. P. australasia’, ‘Ca. P.
ziziphi’, ‘Ca. P. trifolii’ and ‘Ca. P. cynodontis’. Most of
them are reported only from India (Rao, 2021). In this
study, phytoplasma presence has been detected in L.
camara plants growing near brinjal fields at Rajahi
village near Gorakhpur, Uttar Pradesh, India based on

Corresponding author e-mail: Smriti Mall (smriti.mall@rediffmail.com)

Smriti Mall et al.
PCR amplification, sequence comparison and in silico
RFLP analysis.
Materials and Methods
Sample collection
L. camara plants exhibiting little leaf, shortening of
internodes and stunted growth symptoms indicating
a possible phytoplasma infection were sampled from
Rajahi village fields near Gorakhpur, Uttar Pradesh,
India in 2021. Samples were collected from three
symptomatic and two asymptomatic plants.
DNA extraction and PCR assays
Leaves (100 mg) of both symptomatic and
asymptomatic plants were used to extract DNA
utilizing the DNeasy Plant Mini Kit (Qiagen, Germany)
as per the instructions provided by the manufacturer.
DNA of a 16SrVI phytoplasma infected brinjal plant
was used as a positive control (Mall et al ., 2021).
Amplification of phytoplasma DNA was carried out
using P1/P7 universal primers (Deng and Hiruki, 1991;
Schneider et al ., 1995) followed by primers 3F/3R
(Manimekalai et al ., 2010). An aliquot of the PCR
product (0.3 µl) was used for nested PCR assay. Gel
electrophoresis of the nested PCR products was
performed in a 1% (w/v) agarose gel, stained with
ethidium bromide and observed under UV
transilluminator (REMI, India).
Sequencing and phylogenetic analysis
Three nested PCR amplicons were purified (Wizard SV
gel kit, Promega) and sequenced (Eurofins, India). The
Figure 1. Healthy lantana
plant on the left and plant
showing little leaf symptoms
on the right.
84
sequences were assembled using BioEdit software (Hall,
1999) and aligned using CLUSTAL W software with
closely related sequences available in NCBI database.
The edited sequences were deposited in NCBI GenBank
and utilised for phylogenetic tree construction via
Neighbour-Joining method with 1,000 replicates for
each bootstrap using MEGAXI software (Tamura et al.,
2021). Acholeplasma laidlawii (GenBank accession
number AM073014) served as the out-group for
rooting the tree. The 16Sr subgroup identification was
done using the iPhyClassifier online tool (Zhao et al.,
2009).
Results and Discussion
The L. camara plants exhibited little leaf, stunted
growth and shortening of internodes (Figure 1). The
diseased plants were easily identifiable from a distance
owing to their distinct appearance. After DNA
extraction, PCR reaction using P1/P7 followed by 3F/
3R primers yielded amplicons of 1.3 kb from all the
symptomatic samples and from the positive control
but not from asymptomatic samples. The 16S rRNA
gene sequences were identical and one of the sequences
was therefore named Lantana little leaf (LLL) and
submitted to GenBank with Accession Number
OL944007. BLASTn analysis for 16S rRNA sequence
comparison showed that LLL sequence shared 99%
sequence identity with the 16S rRNA gene sequence of
‘Candidatus Phytoplasma trifolii’ strains from India
detected in brinjal showing little leaf disease (GenBank
accession numbers KX284700 and MW273758),
Allamanda little leaf (GenBank accession number
Phytopathogenic Mollicutes, Vol. 12 (2), December 2022
Lantana camara showing little leaf symptoms: a new weed host of ‘Candidatus Phytoplasma trifolii’

KX641019), periwinkle little leaf (GenBank accession
number MW648596), brinjal little leaf (GenBank
accession number KX421260), lentils witches’ broom
(GenBank accession number KY439869) and Datura
witches’ broom (GenBank accession number
KY078230) (Table 1). The phylogenetic analysis showed
the closest relationships of LLL phytoplasma with
strains of ‘Ca. P. trifolii’ (Figure 2). LLL strain was found
99.47% (1 SNP and 6 GAP) identical to the strain from
India in Lens culinaris (GenBank accession number
KY439869) enclosed in subgroup 16SrVI-D and showed
in silico RFLP profile identical to the reference pattern
of 16Sr group VI, subgroup D (Figure 3).
On the basis of molecular characterization,
sequencing of partial 16S rRNA gene and virtual RFLP
profiling of LLL strain in the present study it is
confirmed the presence of ‘Ca. P. trifolii’ in L. camara
plants showing little leaf symptoms. This is the first
report of phytoplasma in this plant species in the
world. L. camara is an invasive weed with very powerful

Table 1. Bioinformatic analysis of phytoplasma sequences used in phylogenetic tree and their percentage identity with Lantana little leaf phytoplasma from Gorakhpur
(Uttar Pradesh), India.
Phytoplasma associated disease Host species Country % identity Ribosomal GenBank
(‘Candidatus Phytoplasma’ species) sub-group accession number
Sesame phyllody (‘Ca. P. asteris’) Sesamum indicum Paraguay 90.43 16SrI-B KY933669
Papaya phyllody Carica papaya Pune, India 89.83 16SrII-C MW404675
Melia azedarach decline Melia azedarach Brazil 93.81 16SrIII-B FJ404775
Coconut lethal yellowing (‘Ca. P. palmae’) Cocos nucifera Jamaica 94.34 16SrIV-A AF498307
Alder yellows Corchorus olitorius India 96.77 16SrV-C KF501045
Clover proliferation (‘Ca. P. trifolii’) Solanum tuberosum Turkey 98.71 16SrVI-A MH683601
Potato purple top Solanum tuberosum USA 99.02 16SrVI-A AY496005
Cucumber phyllody Cucumis sativus Iran 99.10 16SrVI-A JF508517
Potato purple top Solanum tuberosum Columbia, USA 99.10 16SrVI-A KR072666
Allamanda little leaf Allamanda cathartica India 99.47 16SrVI-D KX641019
Chrysanthemum stunt Chrysanthemum coronarium India 99.47 16SrVI-D MW648593
Periwinkle little leaf Catharanthus roseus Lucknow, India 99.47 16SrVI-D MW648596
Roselle little leaf Hibiscus sabdariffa India 99.47 16SrVI-D MZ773477
Saponaria stunting Saponaria sp. Pune, India 99.47 16SrVI-D KX641021
Datura witches’ broom Datura stramonium Kushinagar, India 99.47 16SrVI-D KY078230
Lens witches’ broom Lens culinaris India 99.47 16SrVI-D KY439869
Brinjal little leaf Solanum melongena Telangana, India 99.47 16SrVI-D MZ425930
Brinjal little leaf Solanum melongena India 99.47 16SrVI-D KX284698
Brinjal little leaf Solanum melongena Varanasi, India 99.47 16SrVI-D KX421260
Brinjal little leaf Solanum melongena Pune, India 99.47 16SrVI-D KX421253
Brinjal little leaf Solanum melongena India 99.40 16SrVI-D KX284703
Ash yellows (‘Ca. P. fraxini’) Fraxinus sp. USA 96.99 16SrVII-A AF092209
Luffa witches’ broom (‘Ca. P. luffae’) Luffa aegyptica Taiwan 95.48 16SrVIII-A AF248956
Chicory bushy stunt Cichorium intybus Saudi Arabia 93.40 16SrIX-J KY986922
Apple proliferation (‘Ca. P. mali’) Malus domestica Italy 90.57 16SrX-A AJ542541
Triticum yellow stunting (‘Ca. P. sacchari’) Triticum turgidum India 94.34 16SrXI-B LT594113
Strawberry yellows (‘Ca. P. fragariae’) Fragaria x ananassa Lithuania 89.87 16SrXII-E DQ086423
Papaya apical curl necrosis Carica papaya Brazil 90.20 16SrXIII-E JQ792171
Hibiscus witches’ broom (‘Ca. P. brasiliense’) Hibiscus rosa sinensis Brazil 89.65 16SrXV-A AF147708
Papaya bunchy top (‘Ca. P. caricae’) Carica papaya Cuba 86.66 16SrXVII-A AY725234

Phytopathogenic Mollicutes, Vol. 12 (2), December 2022 85

Smriti Mall et al.

Figure 2. Phylogenetic tree

showing the phylogenetic
relationship of 16Sr RNA gene
of Lantana phytoplasma
(GenBank accession number
OL944007) with sequences of
other phytoplasmas. The tree
was constructed using
neighbour joining method and
bootstrap values obtained for
1,000 replicates. Diamond
marks the sequence obtained in
the present study.

Figure 3. Virtual RFLP

pattern derived from the
query 16S rDNA R16F2n/
R2 fragment of the strain
studied (GenBank
accession number
OL944007) that result
identical to the pattern of
16Sr group VI, subgroup D
(GenBank accession
number KY439869).

86 Phytopathogenic Mollicutes, Vol. 12 (2), December 2022

Lantana camara showing little leaf symptoms: a new weed host of ‘Candidatus Phytoplasma trifolii’
bioactive compounds. Due to the presence of active
phytochemicals the species has a strong defence
mechanism and is not very prone to pathogen
infections. Yet the confirmation of phytoplasma
presence suggest that these molecules are not able to
reduce the phytoplasma pathogenic strength.
Lantana little leaf phytoplasma was identified as a
member of 16SrVI-D subgroup the same that is mainly
associated with brinjal little leaf (BLL) disease in India
(Maheshwari et al., 2017). So there lies a possibility that
Lantana, being a noxious weed which grows near the
agricultural fields, may act as the natural reservoir of
BLL phytoplasma during the off-season of brinjal crop.
The phytoplasma may get transmitted to commercial
crops like brinjal during the growing season thereby
facilitating their spread through efficient leafhopper
vectors like Hishimonus phycitis, Exitianus indicus and
Empoasca prima (Gopala et al., 2018). As there is still
a lack proper management measures to tackle
phytoplasma diseases, detection of phytoplasma in
new host plant species which can act as reservoir for
phytoplasma makes the path more challenging.
Acknowledgements
Authors are thankful to UGC CSIR fellowship for
financial support.
References
Bashir S, Jabeen K, Iqbal S, Javed S and Naeem A 2019. Lantana
camara: phytochemical analysis and antifungal prospective.
Planta Daninha, 37: e019193526.
Deng S and Hiruki C 1991. Amplification of 16S rRNA genes from
culturable and non culturable Mollicutes. Journal of
Microbiological Methods, 14: 53–61.
Garibaldi A, Minuto A, Bertetti D, Nicoletti R and Gullino ML
2003. First report of web blight on yellow sage (Lantana
Phytopathogenic Mollicutes, Vol. 12 (2), December 2022
camara) caused by Rhizoctonia solani in Europe. Plant
Disease, 87(7): 875.
Gopala, Khasa E, Rao A, Madhupriya and Rao GP 2018. Molecular
characterization of clover proliferation phytoplasma
subgroup-D (16SrVI-D) associated with vegetables crops in
India. Physiological Molecular Biology of Plants, 24(2): 203-
210.
Hall TA 1999. BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows 95/98/
NT. Nucleic Acids Symposium Series, 41: 95-98.
Maheshwari M, Kumar M, Madhu P and Rao GP 2017.
Identification and characterization of phytoplasma
associated with brinjal little leaf, phyllody and witches’
broom disease in India. Indian Phytopathology, 70: 258-261.
Mall SM, Kumar SH, Jadon VJ and Rao GP 2015. Identification of
phytoplasmas associated with weed species in India. Indian
Phytopathology, 68(4): 449-453.
Marcone C, Bellardi MG and Bertaccini A 2016. Phytoplasma
diseases of medicinal and aromatic plants. Journal of Plant
Pathology, 98: 379-404.
Rao GP 2021. Our understanding about phytoplasma research
scenario in India. Indian Phytopathology, 74: 371–401.
Schneider B, Seemüller E, Smart CD and Kirkpatrick BC 1995.
Phylogenetic classification of plant pathogenic mycoplasma-
like organisms or phytoplasmas. In: Razin S, Tully JG eds.
Molecular and Diagnostic Procedures in Mycoplasmology
(New York, USA: Academic Press), 369-380.
Tamura K, Stecher G and Kumar S 2021. MEGA11: molecular
evolutionary genetics analysis version 11. Molecular Biology
and Evolution, 38(7): 3022–3027.
Ved A, Arsi T, Prakash O and Gupta A 2018. A review on
phytochemistry and pharmacological activity of Lantana
camara Linn. International Journal of Pharmaceutical
Science & Research, 9(1): 37-43.
Zhao Y, Wei W, Lee I-M, Shao J, Suo X and Davis RE 2009.
Construction of an interactive online phytoplasma
classification tool, i PhyClassifier, and its application in
analysis of the peach X-disease phytoplasma group (16SrIII).
International Journal of Systematic and Evolutionary
Microbiology, 59: 2582–2593.
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