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Keywords:

  • Phytoplasmas;
  • Aster yellows;
  • Clover phyllody;
  • PCR/RFLP;
  • Sequencing;
  • Ribosomal operon

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Material and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999–2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S–23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after Tru I digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Material and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Phytoplasmas, wall-less prokaryotes grouped in the class Mollicutes, induce an array of symptoms that suggest profound disturbances in the normal balance of plant hormones or growth regulators. Usually, plants infected by phytoplasmas exhibit virescence, phyllody, yellows, witches'-broom, leaf roll and generalised decline [1].

The fasciation of stems and flower stalks has been reported in many plants [2,3]; in the case of some dicot species, this phenomenon is thought to be genetically determined [4–7]. However, the origin of fasciation is unknown in many vegetatively propagated plants [8]. Analyses of genetic background and of environmental factors showed that stem fasciation of Lilium henryi was associated with the presence of nematodes – predominantly from the Rotylenchus and Ditylenchus species [9]. Rhodococcus fascians carrying the fas-1 gene, which codes for an isopentyl transferase, the key enzyme in cytokinin biosynthesis, was found to be associated to similar symptoms [10–13]. Furthermore, mass-spectrometrical quantifications of the free and conjugated forms of indole-3-acetic acid and cytokinins and several giberellins on one transgenic line of aspen correlate the induced developmental alterations such as stem fasciation to changes in plant hormone metabolism [14].

Recently, phytoplasmas belonging to the aster yellows group, subgroup 16SrI-C were identified in Lilium martagon with flattened stem in the Czech Republic [15]. Since only two samples originating from one plant with flat stem were examined and transmission electron microscopy did not reveal phytoplasma presence, the objective of this study was to verify on a larger scale the presence and the molecular identity of phytoplasmas associated with stem fasciation of naturally infected lilies from different localities in the Czech Republic.

2Material and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Material and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Samples examined

Twenty two samples from seven plants of Lilium sp. showing severe fasciation of stem were collected at different locations in Southern and central Bohemia (Table 1). As negative controls, 9 asymptomatic plants (not listed in Table 1) collected from Southern Bohemia – 3 from České Budějovice, 2 from Třísov, and 4 plants cv. Sunray growing from bulbs bought in a shop, and originating from an unknown foreign country, were used.

Table 1.  Summary of phytoplasma detection in the 7 lily plants with fasciation
Plant number/year/localitySample codesaPhytoplasma identification (PCR/RFLP)
Primers R16F2/R2Primers R16(I)F1/R1  
  1. N, nonfasciated stem.

  2. aNumber on the left (1 to 7) are referred to plants (same number same plant); A, sample fresh or two months frozen; B, sample 4 years frozen.

  3. bSample showing alternative profile after RFLP with Tru I on R16(I)F1/R1 amplicons.

1/1999/Velehrad1-A116SrI-B16SrI-B
1-B1N16SrI-C16SrI-Cb 
1-B216SrI-C16SrI-C 
1-B316SrI-C16SrI-C 
1-B4N16SrI-C16SrI-C 
1-B516SrI-C16SrI-C 
    
2/2000/Velehrad2-A116SrI-B16SrI-B
2-A216SrI-B+16SrI-C16SrI-B+16SrI-C 
2-A316SrI-B+16SrI-C16SrI-B+16SrI-C 
2-B116SrI-C16SrI-C 
2-B216SrI-C16SrI-C 
2-B316SrI-C16SrI-C 
    
3/2000/Velehrad3-A1NegativeNegative
3-A216SrI-B+16SrI-C16SrI-B+16SrI-C 
3-A316SrI-C16SrI-C 
3-B416SrI-C16SrI-C 
3-B516SrI-C16SrI-C 
3-B616SrI-C16SrI-C 
    
4/2001/Roudnice4-A116SrI-B16SrI-B
5/2001/Třebíč5-A116SrI-B16SrI-B
6/2003/Třísov6-A116SrI-C16SrI-C
7/2003/Třísov7-A116SrI-C16SrI-C

2.2Nucleic acid extraction and PCR/RFLP analyses on 16Sr DNA

Phloem tissue from stems and leaf midribs of the seven fasciated lily plants were subjected to DNA extraction according to the procedure described by Lee et al. [16] immediately after collection (6 samples, with code A in Table 1), or separately frozen (−20°C), and analysed after 2 months (5 samples) or after 4 years (11 samples) (code B in Table 1).

The primer pair P1 [17] and P7 [18] was used to prime the amplification of a 1.8 kb product of 16S ribosomal RNA (rRNA) gene, the spacer region between the 16S and 23S rRNA gene, and the start of the 23S rRNA gene regions of the phytoplasma genome. Twenty ng of each DNA preparation was added to the PCR reaction mix [19] in a final reaction volume of 25 μl. The DNA was amplified by 35 cycles in a Perkin Elmer 480 thermal cycler. These PCR products were diluted with sterile distilled water (1:39) prior to amplification by nested PCR using general and group-specific primer pairs R16F2/R2 and R16(I)F1/R1 [20]. One PCR reaction tube containing no sample DNA was included in each amplification run as negative control.

Three to 10 μl (about 200 ng of DNA) of each positive nested PCR product observed after 1% agarose gel electrophoresis were separately digested from both R16F2/R2 and R16(I)F1/R1 amplicons. Digestion with 2.5 U of Alu I, Hha I, Kpn I, Rsa I, and Tru I were undertaken according to the manufacturer (Fermentas, Vilnius, Lithuania). Digestion reactions were left at the required temperature for at least 16 h. The restriction patterns were compared after electrophoresis on a 5% polyacrylamide gel followed by ethidium bromide staining, and photographed under UV at 312 nm using a transilluminator. The same procedure was performed with reference phytoplasma strains belonging to subgroups 16SrI-B, 16SrI-C, 16SrIII-F, 16SrX-A, 16SrXII-A (see description in figure legends), and tissues of the asymptomatic lilies (9 samples).

2.3Ribosomal gene sequencing

The 16Sr DNA and 16/23S rDNA spacer regions amplified from two lily plants showing, respectively, a RFLP variant profile and severe stem fasciation (samples 1-B1 and 2-A3 in Table 1) were sequenced.

The R16F2/R2 amplicon obtained from lily sample 1-B1 (data not shown) was cleaned using the Concert Rapid PCR Purification System (Gibco, BRL); nucleic acid was then quantified from agarose electrophoresis and cloned using the InsT/Aclone PCR Product Cloning Kit (Fermentas, MBI, Vilnius, Lituania). Selection of recombinant colonies was performed on LB medium containing 50 μg ml−1 ampicillin using the IPTG/X-Gal system. White colonies were suspended in 10 μl sterile distilled water, incubated 5 min at 95°C then employed in PCR reactions using universal primers M13f/r [M13pUC sequencing primer (−20), M13pUC reverse sequencing primer (−26), Fermentas, MBI, Vilnius, Lituania] using the following protocol for 29 cycles: 1 min at 94°C, 30 s at 55°C, 2 min at 72°C with a final cycle having a 10 min extension at 72°C. The colonies showing bands of the expected length were then employed for PCR using phytoplasma group-specific primers R16(I)F1/R1 and subjected to RFLP analyses with Tru I. One of the recombinant colonies showing the variant profile was then sequenced using primers M13f/r as described above.

Sequencing of sample 2-A3 was obtained aligning PCR products generated by amplification with primers P1/U3 (position 6–1230), R16F2/R2 (position 152–1397), 16R758/P7 (position 758–1818) [21–23]. PCR products were sequenced in both directions using the BIG DYE sequencing terminator kit (PE Biosystems, Warrington, UK).

Both sequences were aligned with those of phytoplasmas available in GenBank using the World Wide Web service CLUSTALW (http://www2.ebi.ac.uk/clustalw/) [24] or BLAST (http://www.ncbi.nlm.nih.gov).

2.4RFLP analyses of tuf gene

Lily samples together with several phytoplasma reference strains belonging to ribosomal group 16SrI (Fig. 3) were also screened using Tuf1f/r primers in direct PCR followed by fTufu/rTufu [25] in nested PCR to amplify a portion of the tuf gene coding for the elongation factor Ef-Tu. The PCR conditions for tuf gene amplification were: 35 cycles each of 30 s denaturation step at 95°C, 30 s annealing at 55°C (for primers fTufu/rTufu) or at 45°C (for primers Tuf1f/r) and 1 min (10 min for the last cycle) at 72°C primer extension were performed [25]. RFLP analyses on fTufu/rTufu amplicons were then performed with Tru I and Tsp509 I (Fermentas, Vilnius, Lithuania) restriction enzymes.

image

Figure 3. Polyacrylamide gels showing the RFLP patterns of selected lily samples among those listed in Table 1, and from control strains amplified with primers fTufu/rTufu and digested with the enzymes listed. Phytoplasma reference strains and markers are as in Fig. 2.

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3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Material and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Samples examined

Symptoms of stem fasciation of examined lily plants were very severe (Fig. 1); however, flat stem appeared not every year on the same plants. A single plant (No. 1 in Table 1) from Velehrad had one fasciated and one stem of usual habit growing from a shared split bulb. The plants did not show any other abnormalities.

image

Figure 1. Apical portion of lily showing fasciation symptoms such as broadening of the main stem, multiplicity of flower parts, increased volume and weight.

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3.2PCR/RFLP analyses on 16Sr DNA gene

When direct PCR was performed, only reference strains provided positive results; nested PCR with phytoplasma-specific primers R16F2/R2 and R16(I)F1/R1 gave positive results from all 7 lily plants with stem fasciation, regardless of which stem – flat or asymptomatic - was examined, from reference phytoplasmas, and from 3 out of the 4 asymptomatic plants cv. Sunray (data not shown). No direct or nested PCR products were obtained from the remaining 6 asymptomatic lilies and negative controls (data not shown).

RFLP analyses of PCR amplicons (Fig. 2 and data not shown) allowed us to classify the phytoplasmas as members of aster yellows group, 16SrI-B or 16SrI-C subgroups, respectively, in two lilies with fasciated stems (Plants No. 4 and 5 in Table 1, locality Roudnice and Třebíč) and in two plants from locality Třísov (No. 6 and 7 in Table 1).

image

Figure 2. Polyacrylamide gel showing RFLP patterns of 16SrDNA of selected lily samples among those listed in Table 1, of one of the asymptomatic positive lily cv Sunray (Sunray), and from control strains amplified with phytoplasma-specific primers R16(I)F1/R1 digested with Tru I. Control samples in periwinkle: NJAY, New Jersey aster yellows (16SrI-A); AY, aster yellows (16SrI-B); CPh, clover phyllody (16SrI-C), STOL, stolbur (16SrXII-A). P, ΦX174, marker Hae III digested; fragment sizes in base pairs from top to bottom: 1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118 and 72; S, marker pBR322 Msp I digest, fragment sizes in base pairs from top to bottom: 622, 527, 404, 307, 242, 238, 217, 201, 190, 180, 160, 147, 123, 110, 90, 76, 67.

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From 3 lily plants (No. 1, 2 and 3 in Table 1, locality Velehrad) the PCR analyses were repeated 6 times using fresh, 2 month-old frozen and 4 year-frozen material: positive results were obtained in 17 out of the 18 samples examined. A single sample taken from each of lily 1 (sample 1-A1 in Table 1) and 2 (sample 2-A1 in Table 1) used for DNA isolation immediately after collection or after 2 months of storage in the freezer, revealed the presence of phytoplasmas belonging to aster yellows group, subgroup 16SrI-B. Mixed infection by phytoplasmas 16SrI-B and 16SrI-C was detected in two samples isolated from tissues of lily plant No. 2 (sample 2-A2, 2-A3) and in one sample (3-A3) of lily plant No. 3.

Among the enzymes used in RFLP analyses of R16F2/R2 amplicons, Kpn I and Rsa I showed patterns identical to those of phytoplasmas belonging to aster yellows group (16SrI), while Alu I, Hha I, and Tru I collective patterns allowed us to identify phytoplasmas as members of the clover phyllody subgroup (16SrI-C) in 11 single samples from lily plants No. 1, 2 and 3 after 4 years of storage in the freezer, and also in one sample (lily 3-A3), where DNA was isolated from fresh tissues (data not shown). After Tru I digestion of R16F2/R2 amplicons, it was possible to observe that 16SrI-C phytoplasmas showed a different intensity in some of the bands (data not shown).

RFLP analysis of amplified R16(I)F1/R1 products with the same enzyme did not, in the majority of samples, show differences to the CPh control, but clearly disclosed a different pattern in sample 1-B1 (Fig. 2).

Moreover, phytoplasmas belonging to the aster yellows group 16SrI-B were detected in 3 asymptomatic plants cv. Sunray (Fig. 2).

3.3Ribosomal gene sequencing

Seven recombinant clones gave a band of expected size (1.3 kb) when screened by PCR using the M13f/r primer pair. RFLP analyses of the PCR product amplified from the recombinant clones showed that six DNA inserts had the same banding pattern as the 1-B1 R16(I)F1/R1 PCR product, and the other amplified DNA insert gave a banding pattern identical to clover phyllody control (Fig. 2 and data not shown). A 1.2 kb DNA insert from one of the six recombinant clones which had the same RFLP banding pattern as the PCR product amplified from sample 1-B1 was sequenced. Blast searches showed this nucleotide sequence shared 99.9% similarity with clover phyllody phytoplasma (AF222066).

The sequence obtained from the PCR product of lily sample 2-A3 was deposited in the GenBank database under accession number AY839617. This nucleotide sequence (1460 bp) shared 99.8% similarity with 16Sr DNA from operon A of clover phyllody phytoplasma (AF222065) (aster yellows group, 16SrI-C subgroup).

Virtual digestion with Tru I of R16F2/R2 sequences from lily samples 1-B1 and 2-A3 showed correspondence of RFLP profiles with CPh operon B and CPh operon A, respectively (Table 2(b)). Nucleotide differences from both operon A and operon B were detected at positions 1100 and 1217 in both lily samples sequenced; furthermore, at position 117 lily sample 2-A3 showed a C while operon A has T (Table 2).

Table 2.  Panel A: position of differential nucleotides in R16F2/R2 sequences of lily 16S ribosomal gene of samples 1-B1 and 2-A3 compared with published sequences of operon A and operon B of the clover phyllody (CPh) reference phytoplasma. Panel B: fragment sizes after virtual Tru I restriction of R16F2/R2 sequences
 Nucleotide position in 16S gene sequencea
 11749211001217
Panel A
Lily 1-B1CTTAATA
Lily 2-A3CTTAGCT
CPh operon ATTTAGTT
CPh operon BCTTAATT

3.4RFLP analyses of tuf gene

PCR/RFLP analyses of fTufu/rTufu amplicons performed on selected lily samples infected by 16SrI-C phytoplasmas showed no differences to the clover phyllody phytoplasma reference strain with either enzyme used (Fig. 3).

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Material and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

An association between fasciation and phytoplasmas has been reported for several tree species [26,27]. However, the only report showing the presence of phytoplasmas in herbaceous plants with flat stem was by Poncarová-Voráčková et al. [15]. Authors detected clover phyllody phytoplasma (aster yellows group, 16SrI-C subgroup) in Lilium martagon by nested PCR using primers R16F2/R2 and R16(I)F1/R1 [28,29]; on the contrary, in Australia, sesame plants showing fasciated stems were found negative for phytoplasma presence using direct PCR [30]. The necessity of using the nested PCR technique to achieve detection confirms the unusually low concentration of phytoplasmas in infected lily tissues. Intensive observations of ultrathin sections from asymptomatic and fasciated stems and leaf midribs by transmission electron microscopy (TEM) JEM 1010 failed to reveal any phytoplasma-like bodies, bacteria or viruses (data not shown). Similarly, Kaminska et al. [31] did not find any phytoplasma bodies in symptomatic and PCR-positive lilies experimentally infected with aster yellows phytoplasmas. Poncarová-Voráčková et al. [15] also did not detect any phytoplasma bodies in fasciated lily stem by TEM, although they observed numerous spherical bodies by scanning electron microscopy (SEM); DAPI staining revealed blue-white fluorescent particles in the sieve tube elements of this L. martagon plant. It seems that positive DAPI and also SEM are not direct proof of the presence of phytoplasma-bodies. Examples are carrot plants showing severe proliferation symptoms, which revealed blue-white fluorescent areas in sieve tube cells, while TEM revealed rickettsia-like organisms [32]. A similar example is leek with proliferation and phyllody symptoms: numerous round structures were observed in cross sections of sieve tube elements by SEM, while a very low concentration of phytoplasma-like bodies, but a lot of mitochondria-like structures were observed by TEM in comparable tissues. These structures could represent the majority of the numerous particles resembling phytoplasmas observed by SEM [33]. Another example is asymptomatic daphne, where DAPI staining was positive, while TEM revealed the presence of numerous rhabdovirus particles [34, and J. Fránová, unpublished]. To our knowledge, Bertaccini and Marani [35] first observed the phytoplasma bodies in lily plants with leaf and flower malformation and discoloration by TEM. Mixed phytoplasma infection (group 16SrI-B and 16SrXII-A) was identified by PCR amplification of 16Sr DNA and RFLP in seven lily cultivars with stunting and several types of symptoms [36,37]. Very recently, phytoplasmas (16SrI-B group) have been identified in lilies with symptoms of leaf necrosis and malformation, flower bud abscission and flower virescence, distortion and abortion [38].

The 16SrI-C lily phytoplasma sequence analyses presented indicate that the phytoplasmas naturally infecting lilies show the presence of two different operons as described for clover phyllody phytoplasmas in Canada as well as in Lithuania (AF222065 and AF222066) [28,39]. It is important to underline that in this case in nested PCR product RFLP analyses a Tru I restriction profile was observed that has been previously described only for phytoplasmas infecting Metcalfa pruinosa (Say) Homoptera, Flatidae [40]; this fact suggests preferential amplification of operon A with respect to operon B. Although samples 1-B1 and 1-B3 revealed similar profiles after RFLP of R16F2/R2 amplicons (data not shown), we were able to visualize the alternative RFLP profile after R16(I)F1/R1 amplification only in sample 1-B1 (Table 1).

PCR-amplification of conserved phytoplasma 16SrDNA gene fragment with subsequent RFLP analyses and sequencing revealed the presence of aster yellows phytoplasmas of 16SrI-B and/or 16SrI-C subgroups in all the 7 lily plants showing fasciation. Repeated analyses of DNA isolated from 3 lily plants (No. 1, 2, and 3 in Table 1) gave no identical results. Phytoplasmas belonging to 16SrI-B and 16SrI-C alone or both together were identified, when fresh tissues or material frozen for 2 months was used for DNA isolation. Eleven samples from the same plants analysed after long term cryoconservation revealed the presence of phytoplasmas belonging to the 16SrI-C subgroup alone. The explanation for this result could be that different phytoplasmas were present in the examined lily plants in an uneven distribution.

Moreover, 3 asymptomatic plants cv. Sunray tested as fresh material were found to be infected with phytoplasmas of aster yellows 16SrI-B subgroup. To our knowledge, there is only one report of PCR detection of phytoplasmas in asymptomatic lily plant cv. Casablanca [41].

Although there remains still a number of factors that cannot be neglected such as environmental conditions and genetic background, it seems that 16SrI-C phytoplasmas can play a role in the development of symptoms such fasciation of lily. Experiments of phytoplasma transmission to lily seedlings, which are underway, will probably help to clarify the relationship between symptom occurrence and phytoplasma presence.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Material and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The work is a part of research granted by the Grant Agency of the Academy of Sciences of the Czech Republic No. S5051014 and AV0Z50510513. The authors are deeply indebted to Mrs. J. Rakouská for technical assistance and Mr. B. Mičulka for sample collection.

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  2. Abstract
  3. 1Introduction
  4. 2Material and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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