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BRIEF REPORT

Folia Microbiol. ** (*), *** *** (**09) http://www.biomed.cas.cz/mbu/folia/

Association of Candidatus Phytoplasma asteris

with Yellowing and Phyllody of Plantago lanceolata

J. FR NOV, M. IMKOV

Department of Plant Virology, Institute of Plant Molecular Biology,

Biology Centre of Academy of Sciences of the Czech Republic, v.v.i., 370 05 esk Bud jovice, Czech Republic

fax +420-***-***-***

e-mail abpnvh@r.postjobfree.com

Received 20 March 2009

Revised version 6 August 2009

ABSTRACT. Long plantains (Plantago lanceolata L.) with symptoms resembling those associated with

phytoplasma infection were observed repeatedly during the period 2000 2008 in southern Bohemia (Czech

Republic). The symptoms of the plants were leaf yellowing, stunted growth, flower phyllody and lack of seed

production. Transmission electron microscopy showed phytoplasmas in the sieve cells of affected plants but

not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain react-

ion using phytoplasma-specific universal ribosomal primers R16F2n/R16R2. An amplification product of the

expected size (1.2 kb) was observed in all samples of the symptomatic long plantains. The restriction pro-

files obtained from digestion of the PCR products with three endonucleases (AluI, HhaI, MseI) showed that

the phytoplasmas infecting long plantains in the Czech Republic were indistinguishable from those belong-

ing to the aster yellows group (subgroup 16SrI-B). Sequence analysis of 1748 bp of the ribosomal operon in-

dicated that the closest related phytoplasma was that associated with Rehmannia glutinosa var. purpurea,

originating also in Bohemia. This is the first report of the natural occurrence of Candidatus Phytoplasma

asteris in plants of P. lanceolata.

Abbreviations

PCR polymerase chain reaction TEM transmission electron microscopy

RFLP restriction fragment length polymorphisms

The long plantain (Plantago lanceolata), a member of the Plantaginaceae family, is an important

medicinal plant used to treat cough and inflammation. There are only a limited number of reports about phy-

toplasma diseases in the family Plantaginaceae, and these are based primarily on the description of symptoms

and evidence from electron microscopy (see review in McCoy et al. 1989; Seem ller et al. 1998; Staniulis

and Genyte 1976). The only report about the molecular identification of a phytoplasma disease in Plantago

coronopus and P. major is from Germany. In that case, phytoplasmas associated with plantain virescence

were classified on the basis of restriction analysis of 16S rDNA digested with AluI and RsaI. The phyto-

plasmas were assigned to group I, which also includes the American aster yellows phytoplasma (Schneider

et al. 1993).

In this paper we report the molecular identification of a phytoplasma in naturally infected long plan-

tain plants showing stunting, yellowing and phyllody. The phytoplasma was identified as a member of the

aster yellows phytoplasma group (16SrI group) (Lee et al. 1998) also known as Candidatus Phytoplasma

asteris (Lee et al. 2004).

Long plantains with flower abnormalities, yellowing, necrosis and stunted growth reminiscent of

phytoplasma infection were repeatedly observed in Jamn (southern Bohemia) during the period 2000 2008.

Early symptoms consisted of leaf vein clearing with a light green mosaic and new leaves that were smaller

and pale green, many leaf-like structures growing from inflorescences and flowers that failed to produce seeds

(Fig. 1). Severely infected long plantains died at the end of autumn or during the winter.

Tissues from footstalks of leaf-like structures from inflorescences and the trunks of nonsymptoma-

tic P. lanceolata plants were processed for TEM analysis. Immediately after collection, samples of 2 mm

size were fixed with 5 % glutaraldehyde in 0.1 mol/L potassium phosphate buffer (pH 7.2) for at least for 2 d

at 4 C and, subsequently, post-fixed in 2 % osmium tetroxide in the same buffer. The specimens were de-

hydrated by an ethanol series in Durcupan resin (Fluka, Switzerland). Ultrathin sections were double-stained

with uranyl acetate in 70 % ethanol and lead citrate, then examined using a JEM 1010 TEM (Jeol 78290,

France). Phytoplasma-like structures were detected in the sieve tube elements of affected plants. They were

observed in mature and immature phloem sieve tubes. The majority of particles were ovoid or spherical, with

470 J. FR NOV and M. IMKOV Vol. 54

sizes that ranged from 50 50 to 460 680 nm in diameter (average, 500 430 nm). Two kinds of par-

ticles were observed in some cells: one with an electron transparent central region and the other with electron-

dense contents (Fig. 2). The prokaryotes were found only in symptomatic plants; no other microorganisms,

bacteria or viruses were observed.

DNA was extracted from flowers and leaf

midrib tissue from three healthy plants, seven di-

seased long plantains, and reference phytoplasma

strains [aster yellows (subgroup 16SrI-B, host: Heli-

chrysum bracteatum), clover phyllody (subgroup

16SrI-C, host: Trifolium repens), apple prolifera-

tion (subgroup 16SrX-A, host: Malus domestica

cv. Mat ino), and stolbur (subgroup 16SrXII-A,

host: Trifolium pratense)] following the procedure

of Lee et al. (1991). The nucleic acid pellet was re-

suspended in 50 L of TE buffer (10 mmol/L Tris-

HCl, 1 mmol/L EDTA, pH 8.0) to a final concen-

tration of 20 ng/ L. DNA extracts were used as

template in PCR reactions to amplify part of the

16S rRNA gene using the phytoplasma universal

primers R16F2n and R16R2 (Schaff et al. 1992).

Reactions lacking DNA template or with DNA

template from symptomless samples were included

Fig. 1. Diseased long plantain showing leaf vein clearing, in each experiment as negative controls. An aliquot

trunk necrosis and flower phyllody.

(6 L) of the PCR products was analysed by

electrophoresis on a 1 % agarose gel, followed by

SYBR Green I staining and visualisation of DNA bands with a UV transilluminator. Using the primer pair

R16F2n/R16R2, DNA fragments of approximately 1.2 kb

from the 16S rRNA gene were detected in all seven samples

of symptomatic long plantains. No PCR product was obtained

from asymptomatic plants or from the control reaction con-

taining water instead of DNA template. Amplification of the

reference phytoplasma strains resulted in a product of the

same size as those in the diseased plantains.

For restriction analysis, 3 L of PCR product (200 ng)

were digested with 3 U each of the restriction endonucleases

AluI, HhaI, and MseI (New England Biolabs, USA) in 20 L

at 37 C overnight. The digest was resolved on a 10 % acryl-

amide gel, stained with ethidium bromide and visualised by

UV transillumination. The gel was photographed using a Kodak

digital camera with a red filter. All phytoplasma-positive

samples from plantains showed the same restriction profiles

with each employed enzyme. These profiles (Fig. 3) were

identical to those of aster yellows phytoplasma (subgroup

16SrI-B) (Lee et al. 1998) also known as Candidatus Phyto-

plasma asteris (Lee et al. 2004).

DNA isolated from one long plantain plant (Fig. 1)

was sequenced. A set of overlapping PCR products from an

infected plant was generated by amplification with the primer

pairs P1/U3 (position 6 1230) (Deng and Hiruki 1991; Lo-

Fig. 2. Ultrathin section of a sieve tube cell

renz et al. 1995), R16F2n/R16R2 (position 152 397) (Gun- showing a large number of phytoplasma bodies.

dersen and Lee 1996; Lee et al. 1993), and 16R758/P7 (posi- Net-like structure of DNA fibrils (NA) and riboso-

tion 758 1818) (Gibb et al. 1995; Schneider et al. 1995). PCR mes (R) are visible inside the phytoplasma partic-

les; thin membrane (M) surrounds the organisms.

products were sequenced using the BIG DYE sequencing

Smaller bodies (SB) with electron-dense contents

terminator kit (PE Biosystems, UK), from both directions.

are also visible. CW cell wall; bar = 500 nm.

Sequencing was performed using an ABI PRISM 310 se-

quencer (PE Applied Biosystems, USA). These reactions pro-

vided the partial sequence of the 16S ribosomal RNA gene, the spacer region between the 16S and 23S

rRNA genes, and the start of the 23S rRNA gene. The final nucleotide sequence data were assembled by

P. lanceolata: A NEW HOST FOR Candidatus PHYTOPLASMA ASTERIS 471

2009

employing the Contig Express (a component of Vector NTI Suite 8.0 software) and deposited in the GenBank

under accession number AY549311 (1748 bp). Comparison with sequences available in GenBank using the

BLASTN 2.2.16 server indicated that the closest related phytoplasma is the Rehmannia glutinosa var.

purpurea phytoplasma (acc. no. AF335107; P ibylov et al. 2001), originating also from Bohemia. A single

base-pair difference was found out of the 1744 bp compared; at position 1639 there is an A in the sequence

from the phytoplasma infecting the long plantain, while there is a nucleotide deletion in the sequence of the

phytoplasma infecting the R. glutinosa var. purpurea plant (nucleotide positions according to the AY549311

sequence).

Fig. 3. Results of RFLP analysis of phytoplasma 16S rDNA sequences amplified using primers R16F2n/R2 from long plantain plants

(1 7) infected with aster yellows (16Sr I-B) and control phytoplasma strains; ST stolbur (16SrXII-A), AP apple proliferation

(16SrX-A), CP clover phyllody (16SrI-C), AY aster yellows (16SrI-B); M 100-bp DNA fragment size in base pairs from top to

bottom: 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 bp; restriction enzymes used are indicated at the top of the figure.

The symptoms observed in long plantains during the present survey are similar to those described

for phytoplasma infection in various host species (Bertaccini 2007; Hogenhout et al. 2008; Mc Coy et al.

1989). Transmission electron microscopy confirmed the presence of phytoplasmas in diseased plants in the

absence of other disease agents and was not found in healthy controls. RFLP analysis of the amplified DNA

fragments with AluI, HhaI and MseI restriction endonucleases indicates that all of the phytoplasmas exami-

ned can be considered members of the aster yellows group, ribosomal subgroup 16SrI-B (Lee et al. 1998).

With more than 100 isolates, the aster yellows phytoplasma group is among the groups containing the large

number of phytoplasma diseases worldwide (Marcone et al. 2000; Seem ller et al. 1998). The discovery of

Candidatus Phytoplasma asteris in P. lanceolata plants showing yellowing and phyllody represents the

first detection of phytoplasma in this host species.

This work was funded as part of a grant of the Grant Agency of the Academy of Sciences of the Czech Republic (1QS500510558,

AV0Z50510513). The authors are deeply indebted to Mrs J. Rakousk for technical assistance.

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