Genetic variation in Candidatus Phytoplasma australiense

Plant Pathology (2005) 54, 8–14

Doi: 10.1111/j.1365-3059.2004.01113.x

Genetic variation in Candidatus Phytoplasma australiense Blackwell Publishing, Ltd.

C. Streten*† and K. S. Gibb School of Science, Charles Darwin University, Darwin 0909, NT, Australia

This study examined whether genes that are less conserved than the 16S rRNA gene can distinguish Candidatus Phytoplasma australiense strains that are identical based on their 16S rRNA genes, with a view to providing insight into their origins and distribution, and any patterns of association with particular plant hosts. Sequence analysis of the tuf gene and rp operon showed that Ca. P. australiense strains could be differentiated into four subgroups, named 16SrXII-B (tuf-Australia I; rp-A), 16SrXII-B (tuf-New Zealand I; rp-B), 16SrXII-B (tuf-New Zealand II) and 16SrXII-B (rp-C). Strawberry lethal yellows 1, strawberry green petal, Australian grapevine yellows, pumpkin yellow leaf curl and cottonbush witches’ broom phytoplasmas were designated members of the 16SrXII-B (tuf-Australia I; rp-A) subgroup. The strawberry lethal yellows 2 and cottonbush reduced yellow leaves phytoplasmas were assigned to the 16SrXII (tuf-New Zealand II; rp-B) subgroup. No relationship was observed between these phytoplasma subgroups and collection date, location or host plant. However, the study revealed evolutionary divergence in the 16SrXII group. Keywords: evolutionary diversity, 16S rRNA gene, phytoplasmas, rp operon, tuf gene

Introduction Phytoplasmas affect approximately 1000 plant species worldwide (Seemüller et al., 1998). Phylogeny derived from their 16S rRNA genes shows that phytoplasmas represent a distinct clade within the class Mollicutes (Gundersen et al., 1994). Members of the phytoplasma clade can be differentiated into at least 15 phytoplasma 16Sr DNA groups (16Sr) based on RFLP analysis of the 16S rRNA gene (Lee et al., 2000). These 16Sr groups are considered to represent different species within the phytoplasma clade (Gundersen et al., 1994). A representative strain of each 16Sr DNA group is currently being assigned a Candidatus Phytoplasma species name according to minimal taxonomic standards for uncultivated bacteria (Murray & Schleifer, 1994; IRCPM Phytoplasma /Spiroplasma Working Team, 2004). Differentiation studies of phytoplasmas assigned to the same 16Sr group have shown that a clear relationship between phytoplasma, host plant and symptoms cannot be elucidated using the 16S rRNA gene (Seemüller et al., 1998). The highly conserved nature of the 16S rRNA gene means that this gene may not be useful in identifying and defining subgroups within the phytoplasma 16Sr groups (Gundersen et al., 1996; Marcone et al., 2000). Phyto-

*To whom correspondence should be addressed. †E-mail: [email protected] Accepted 29 August 2004

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plasmas assigned to the same 16Sr group have been differentiated further into subgroups through analysis of more variable genes or regions, such as the ribosomal protein-encoding operon (rps19-rpl22-rps3), the tuf gene and the 16S/23S rRNA intergenic spacer region (Lee et al., 2000). The phytoplasma associated with Australian grapevine yellows (16SrXII-B) was designated Candidatus Phytoplasma australiense (hereafter abbreviated as Ca. P. australiense) by Davis et al. (1997a), based on 16S rDNA sequence. Within Australia, Ca. P. australiense is also implicated in strawberry green petal (SGP; Padovan et al., 2000); papaya dieback (PDB; Gibb et al., 1996; Liu et al., 1996); Australian grapevine yellows (AGY; Padovan et al., 1996); mung bean witches’ broom (MBWB; Davis et al., 1997b); and periwinkle phyllody (Davis et al., 2003) diseases. More recently, Ca. P. australiense has been implicated as a causal agent of pumpkin yellow leaf curl (PYLC), Gomphocarpus physocarpus witches’ broom (CBWB) and yellowing of G. physocarpus (CBRYL). Candidatus P. australiense is also associated with several plant diseases in New Zealand, including strawberry lethal yellows (SLY; Andersen et al., 1998); phormium yellow leaf (PYL; Liefting et al., 1998); Cordyline australis (cabbage tree) sudden decline (CSD; Andersen et al., 2001); and coprosma lethal decline (CLD; Andersen et al., 2001). Although Ca. P. australiense is associated with a diverse range of plant species, in which it causes a range of symptoms, and is geographically widespread throughout Australia and New Zealand, the strains from different hosts are indistinguishable based on restriction fragment © 2005 BSPP

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Table 1 Source, host, symptoms and strain designation for the field samples examined

Host

Disease description

Longitude/ latitude

Location

Date

Number of samples

Strain

Fragaria x ananassa Fragaria x ananassa Fragaria x ananassa Carica papaya Vitis vinifera Catharanthus roseus Phaseolus aureus Cucurbita sp. Gomphocarpus physocarpus Gomphocarpus physocarpus

Strawberry lethal yellows Strawberry lethal yellows Strawberry green petal Papaya dieback Australian grapevine yellows Periwinkle phyllody Mung bean witches’ broom Pumpkin yellow leaf curl Cottonbush witches’ broom Cottonbush reduced yellow leaves

28S, 151E 27S, 152E 27S, 153E 27S, 153E 34S, 140E 17S, 141E 15S, 128E 27S, 152E 28S, 151E 27S, 151E

South Queensland South Queensland South Queensland South Queensland South Australia North Queensland Western Australia South Queensland South Queensland South Queensland

2000 2001 1999 1999 1999 2000 1996 2001 2002 2002

2 1 1 5 5 1 1 1 1 1

SLY 1 SLY 2 SGP PDB AGY PPA MBWB PYLC CBWB CBRYL

length polymorphic (RFLP) analysis of their 16S rRNA genes (Liefting et al., 1998; Padovan et al., 2000). A comparative analysis of 16S rRNA genes of the phytoplasmas associated with SGP, SLY, PDB, AGY and PYL diseases indicated that these sequences share 99·6 –99·8% sequence homology (Padovan et al., 2000). A similar analysis of the 16S−23S spacer region of the Ca. P. australiense strains AGY, PDB, PYL, SLY and SGP showed that the PYL and AGY phytoplasmas can be distinguished from the SLY, PDB and SGP phytoplasmas (Padovan et al., 2000). Schneider et al. (1997) examined genetic variability among members of the aster yellows group using the tuf gene, and included Ca. P. australiense in the study. RFLP analysis of the PYL phytoplasma tuf gene differed from the patterns attributed to the AGY, SLY, SGP and PDB phytoplasmas in a subsequent study (Padovan et al., 2000). It seems plausible that collective data derived from analysis of evolutionarily more variable genes may enable further differentiation of Ca. P. australiense and provide insights into the evolutionary divergence of strains comprising the 16SrXII-B group, host range and phytoplasma dissemination (Padovan et al., 2000). Sequence tags that differentiate closely related phytoplasmas would assist in identification of specific strains and precise determination of the relationships between a phytoplasma associated with a specific crop disease and others associated with host plant species growing nearby. Furthermore, the ability to identify phytoplasma strains accurately should facilitate searches for candidate vector species. Phytoplasmas assigned to group 16SrI (aster yellows) can be further differentiated into nine subgroups based on analysis of their ribosomal protein (rp)-encoding genes (rps19-rpl22-rps3), which indicates that these genes allow finer differentiation than 16S rRNA gene sequences alone (Gundersen et al., 1994). Similarly, members of the 16SrX (apple proliferation) group can be further delineated through analysis of their nitroreductase gene ( Jarausch et al., 1994, 2000). The objectives of this study were to determine if phytoplasma genes that are less conserved than the 16S rRNA gene would enable a more detailed differentiation of Ca. P. australiense strains. © 2005 BSPP Plant Pathology (2005) 54, 8–14

Materials and methods Candidatus Phytoplasma australiense samples and DNA extraction Diseased plant samples that were previously identified as being infected with Ca. P. australiense by 16S rRNA and tuf gene RFLP analysis were used for this study (Table 1). DNA extraction from diseased or healthy plant material was as described by Doyle & Doyle (1990).

PCR primers and amplification conditions Candidatus P. australiense strains were amplified by PCR using phytoplasma-specific tuf gene primer pair ftufAY (5 ′ -GCTAAAAGTAGAGCTTATGA-3 ′ )/rtufAY (5 ′ CGTTGTCACCTGGCATTACC-3′) (Schneider et al., 1997) and ribosomal protein gene primer pair rpF1 (5′GGACATAAGTTAGGTGAATTT-3′)/rpR1 (5′-ACGATATTTAGTTCTTTTTGG-3′) using previously reported conditions (Lim & Sears, 1991). PCR reactions were prepared according to Schneider et al. (1999). A DNA sample of each healthy plant species was included in all amplifications.

RFLP and sequence analysis PCR products amplified using primers rpF1/rpR1 were digested with MseI, AluI or DraI (Gundersen et al., 1996). Digestions were performed according to the manufacturer’s specifications and DNA fragments were separated on a nondenaturing 12% polyacrylamide gel. For sequencing, PCR products were purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturer’s protocol. DNA quantity was determined by comparing purified DNA against a low molecular weight DNA mass ladder (Invitrogen). Samples were sequenced using the Big Dye terminator sequencing kit (Applied Biosystems). Sequencing reactions were separated using automated equipment at the Australian Genome Research Facility. PCR products amplified by tuf gene primers were sequenced using the primers fTufAY, rTufAY, fTufi1 (5′CAAGTTGGTGTTCCAAA-3′) and rTufi1 (5′-GTTGT-

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CACCTGCTTGAGC-3′) (Schneider et al., 1997). The rp operon PCR products were initially sequenced using the rpF1 and rpR1 primers (Lim & Sears, 1991). The internal primers rpF2 (5′-GCAAAGATGGTGAAACTCG-3′) and rpR2 (5′-GAGTTTCACCATCTTTGCC-3′) were designed using the primer3 program (Rozen & Skaletsky, 1998) to complete the rp operon sequence. Candidatus P. australiense nucleotide sequences were characterized using DNA analysis programs accessed through the BioManager website (Entigen Corporation, www.entigen.com). The fasta search engine (Pearson & Lipman, 1988) was used to identify homologous sequences in the GenBank main and SwissPro + TrEMBL databases (Entigen). Similarity between the nucleotide sequences of the rp operon or tuf genes of Ca. P. australiense strains was calculated using the gap program (GCG). Phylogenetic trees were constructed using the sequence analysis programs clustalw (accurate) (Thompson et al., 1994), seqboot, DNApars and consense (Felsenstein, 1989).

Figure 1 Phylogenetic relationship of the tuf gene nucleotide sequences of selected Candidatus Phytoplasma australiense strains, showing boot strapped values. Accession numbers are shown in parentheses. See Tables 1 and 2 for details of strains. (Correspondence for Mark Andersen, [email protected]).

Results Amplification and sequence analysis of the tuf gene PCR primers specific for the tuf gene amplified a product (0·8 kb) from DNA of all Ca. P. australiense strains, except the MBWB phytoplasma, using the published PCR parameters. Phylogenetic analysis of tuf gene sequences indicated that Ca. P. australiense strains could be classified into at least three 16SrXII-B subgroups (Fig. 1; Table 3). The tuf genes of the PYLC, PDB, AGY, CBWB and SLY1 phytoplasmas grouped on branches that arose from a common lineage and were assigned to the 16SrXII-B (tuf-Australia I) subgroup (Fig. 1). The SLY2 and CBRYL phytoplasmas grouped on branches that arose from the midpoint between the PYL and SLY1 [16SrXII-B (tuf-Australia I) subgroup] phytoplasmas, which suggests that the SLY2 and CBRYL phytoplasmas are both equally related to all

other Ca. P. australiense strains and represent a different subgroup (Fig. 1) (www.treebase.org /treebase, accession number SN2014). To explore further the relationship between strains SLY2, CBRYL and PYL and members of the 16SrXII-B (tuf-Australia I) subgroup, tuf gene sequences of all 16SrXII-B phytoplasmas were compared. The nucleotide sequences of the tuf genes amplified from phytoplasmas SLY1, SLY2, SGP, PDB, AGY, PYLC, CBWB and CBRYL shared 98–100% similarity (Table 2). Nucleotide differences between the tuf genes were distributed throughout the nucleotide sequences (data not shown). The tuf gene nucleotide sequences of the SLY2 and CBRYL phytoplasmas shared 98% homology with both the PYL phytoplasma and members of the 16SrXII-B (tuf-Australia I) subgroup (Table 2). These results, combined with tuf gene

Table 2 Pairwise similarities (%) between the tuf gene or ribosomal protein rp gene nucleotide sequences of Candidatus Phytoplasma australiense strains (tuf/rp operon)

SLY1 SLY 2 SGP PDB AGY PPA MBWB PYLC CBWB CBRYL

PYL

CBRYL

CBWB

PYLC

MBWB

PPA

AGY

PDB

SGP

SLY2

98·3/– 98/– 98·3/ 98/– 98·3/– –/– –/– 98·1/– 98·1/– 98/–

98/99 100/99·4 98/99 97·7/– 98·1/98·9 –/98·8 –/98·8 98/98·9 98/98·8

100/99·5 98/98·6 100/99·5 99·5/– 99·5/99·4 –/99·8 –/99·8 100/99·5

100/99·7 98/98·9 100/99·7 99·5/– 99·6/99·6 –/99·5 –/99·7

–/99·7 –/98·8 –/99·7 –/– –/99·3 –/99·5

–/99·7 –/98·8 –/99·7 –/– –/99·3

99·6/98·7 98·1/99·6 99·6/99·6

99·5/– 97·7/– 99·5/–

100/100 98/98·9

98/98·9

Accession numbers for rp operon sequences: SLY1, AY303561; SLY2, AY303562; SGP, AY303570; AGY, AY376666; PPA, AY303564; MBWB, AY303563; PYLC, AY303560; CBWB, AY303558; ABRYL, AY303559.

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Table 3 Subgroup designations for Candidatus Phytoplasma australiense strains based on rp and tuf gene nucleotide sequences

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Ca. P. australiense strain

tuf gene subgroup

rp gene operon subgroup

tuf and rp subgroup

SLY1 SLY 2 SGP PDB AGY PPA MBWB PYLC CBWB CBRYL PYL

tuf Australia I tuf New Zealand I tuf Australia I tuf Australia I tuf Australia I – – tuf Australia I tuf Australia I tuf New Zealand I tuf New Zealand II

rpA rpB rpA – rpA rpA rpC rpA rpA rpB –

tuf Australia I; rpA tuf New Zealand I; rpB tuf Australia I; rpA – tuf Australia I; rpA – – tuf Australia I; rpA tuf Australia I; rpA tuf New Zealand I; rpB –

Figure 2 RFLP banding patterns of the rp operon amplified from Candidatus Phytoplasma australiense strains. See Tables 1 and 2 for details of strains.

phylogeny, indicated that the SLY2, CBRYL and PYL phytoplasmas should be divided into two Ca. P. australiense subgroups (Table 3).

Amplification, RFLP and sequence analysis of the rp operon The ribosomal protein (rp) gene operon (1·1 kb) was amplified from DNA of all Ca. P. australiense strains, except for samples containing the PDB phytoplasma. The RFLP patterns of the rp gene operon PCR products from the SLY1, SGP, PYLC, CBWB, AGY and PPA phytoplasmas were identical when digested separately with AluI, DraI or MseI endonucleases (Fig. 2), which suggests they are all members of the same subgroup (Table 3). The SLY2 and CBRYL phytoplasmas had the same RFLP pattern as the SLY1, PYLC, CBWB, AGY and PPA phytoplasmas when cut with DraI, but not with MseI or AluI (Fig. 2), indicating that they represent another rp subgroup (Table 3). The RFLP banding pattern of the rp gene operon of the MBWB phytoplasma differed from all other 16SrXII-B group phytoplasmas when examined by digestion with DraI, MseI or AluI (Fig. 2), suggesting that this © 2005 BSPP Plant Pathology (2005) 54, 8–14

phytoplasma should be assigned to a third rp subgroup (Table 3). The complete rp gene operons (1100–1152 bp) of the AGY, SGP, SLY1, SLY2, PYLC, CBWB and CBRYL phytoplasmas were sequenced. A nucleotide sequence of only 420 bp was obtained for both MBWB and PPA phytoplasmas. The partial rp gene operon of the strawberry 2 Florida phytoplasma (PSU96617) (phytoplasma group 16SrI) was identified as most similar to the rp gene operon sequences amplified from the CBWB, CBRYL, AGY, SLY1, SGP, SLY2 and PYLC phytoplasmas. The closest match to the rp gene operon of the PPA and MBWB phytoplasmas was a corresponding region from the periwinkle little leaf phytoplasma (AF453327), a member of phytoplasma group 16SrI. Sequence similarity comparisons for the rp gene operon nucleotide ranged from 99 to 100% (Table 2), which supported the assignment of the SGP, SLY1, PPA, PYLC and CBWB phytoplasmas to the 16SrXII-B (rp-A) subgroup, while indicating that the SLY2 and CBRYL phytoplasmas represented the 16SrXII-B (rp-B) subgroup (Table 3). Although the MBWB phytoplasma was identified as representing a third distinct rp subgroup through RFLP analysis, the rp operon gene sequence comparisons

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showed that this phytoplasma should to be assigned the same subgroup as the SGY, SLY1, PPA, PYLC and CBWB phytoplasmas (Table 3) (www.treebase.org/treebase, accession number SN2014).

Discussion A PCR assay employing tuf gene primer pair ftufAY/rtufAY amplified a product from all phytoplasma DNA tested, except for that of the MBWB and PPA phytoplasmas. The tuf gene may not have been amplified from the MBWB phytoplasma because of DNA degradation, the presence of inhibitors in DNA samples or the low titre of the pathogen in host plant tissues. These factors possibly also affected the amplification of the rp operon gene from the papaya dieback phytoplasma. Marcone et al. (2000) reported that, among members of the aster yellows phytoplasma group, the tuf gene was more conserved than the 16Sr RNA gene. This finding was not observed among the Ca. P. australiense strains examined in this study, which were delineated into three 16SrIIX-B subgroups based on tuf gene comparisons, while 16S rRNA gene analysis showed them to be identical. Strains of Ca. P. australiense assigned to the same 16SrXII-B subgroup, however, exhibited the same similarity between their tuf genes as reported for their 16Sr RNA genes (Padovan et al., 2000). This absence of variation between the tuf genes of members assigned to same subgroup is consistent with genetic studies of the aster yellows phytoplasma subgroups (Marcone et al., 2000). The SLY2, CBRYL and PYL phytoplasmas had an identical tuf gene RFLP banding pattern, but combined phylogenetic analysis and sequence comparison of their tuf genes revealed that these phytoplasmas should be assigned to different 16SrXII-B tuf subgroups (Table 3). This is the first example of a phytoplasma identified as Ca. P. australiense according to 16S rRNA gene RFLP analysis, differing from the PYL, AGY, PDB, SLY and SGP phytoplasmas based on combined tuf gene comparisons and phylogeny. These results demonstrate the importance of comparing several gene sequences of closely related phytoplasmas. As sequencing genes from hundreds of closely related phytoplasmas during epidemiology studies is time-consuming and expensive, identification of variable genes that facilitate the differentiation of closely related phytoplasmas based on RFLP analysis could be useful for more informative epidemiology studies. Delineation of Ca. P. australiense strains into subgroups based on rp gene operon analysis was consistent with groupings based on tuf gene sequences, except for the MBWB, PPA and PYL phytoplasmas (Table 3). The rp gene operon of the MBWB phytoplasma produced banding patterns that were distinct from those of other members of the 16SrXII-B group, which suggested that this phytoplasma represented a third 16SrIIX-B rp subgroup (Table 3). Comparative analysis of the MBWB phytoplasma rp gene operon nucleotide sequence with the corresponding operon of other members assigned to the 16SrIIX-B group did not confirm that this phytoplasma represented a third rp

subgroup. Therefore the subgroup designation for this Ca. P. australiense strain was inconclusive. Analysis of the completed rp gene operon for the MBWB phytoplasma may facilitate the assignment of this phytoplasma to a third 16SrXII-B rp subgroup. The 16SrXII-B subgroup names were modified to reflect both rp gene operon and tuf gene analysis (Table 3). This is consistent with previously published subgroup designations (Gundersen et al., 1994; Lee et al., 2000). The rp gene operon subgroup designations did not indicate strain origin, because the PYL phytoplasma rp gene operon product has not been sequenced. Combined tuf gene and rp gene operon analysis revealed that Ca. P. australiense strains could be differentiated into four subgroups not identified using the 16S rRNA gene. These findings agree with previous reports that the 16S rRNA gene is insufficiently variable to distinguish closely related phytoplasmas, and that the inclusion of more genetically variable regions of the genome facilitates finer differentiation of closely related phytoplasmas ( Jarausch et al., 1994; Gundersen et al., 1996; Schneider et al., 1997; Marcone et al., 2000). It is important to consider this issue when examining phytoplasma–host relationships over a number of localities, because studies based entirely on the 16S rRNA gene do not possess sufficient strain-resolving power. There was no relationship between subgroups of Ca. P. australiense strains and collection date, location or host plant. However, the identification of subgroups did reveal which hosts are possibly affected by Ca. P. australiense strains that may be considered identical. The Ca. P. australiense strains SLY2 and CBWB were assigned to the same 16SrXII-B subgroup, which suggests that the same phytoplasma is associated with these diseases. Therefore G. physocarpus plants exhibiting witches’ broom may be a source of inoculum for SLY disease, while SLY-diseased plants may be the source of phytoplasma for CBWB disease. This concept may also be relevant for the other 16SrXII-B subgroups identified. Furthermore, the study showed that the majority of Ca. P. australiense strains are assigned to the 16SrXII-B (tuf Australia I; rpA) subgroup, members of which are widespread in Australia, while members of the 16SrXII-B (tuf New Zealand I; rpB) subgroup were identified only in southern Queensland, and the only member of the 16SrXII-B (rpC) subgroup was collected in Western Australia. This suggests that members of the 16SrXII-B (tuf New Zealand I; rpB) and 16SrXII-B (rpC) subgroups may represent isolated niches in Australia (Gundersen et al., 1996; Lee et al., 2000). Overall, the combined results from rp gene operon and tuf gene analysis allowed greater discrimination between Ca. P. australiense strains, except for the phytoplasmas associated with strawberry green petal and strawberry lethal yellows, whose tuf genes and rp operon nucleotide sequences were identical. The absence of variation between phytoplasmas associated with SLY and SGP means that, based on current sequence data, the different disease symptoms exhibited by the strawberry plants are associated with the same phytoplasma. The fact that different symptoms are apparently induced on strawberry in response to © 2005 BSPP Plant Pathology (2005) 54, 8–14

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infection by the same phytoplasma may be caused by the presence of another pathogen (mixed infection) that was not detected. An inability to detect mixed infections in diseased plant samples by molecular techniques has been reported previously (Lee et al., 1998). While others were able to identify mixed-infection phytoplasmas by nested PCR (Lee et al., 2000), nested amplifications with phytoplasma-specific PCR primers fP1/rP7 and fU5/m23sr and DNA from SLY- and SGP-diseased samples failed to provide any evidence of a mixed infection (C.S., unpublished data). Loi et al. (1995) showed that mixed infections could be detected through dodder transmission of phytoplasmas from field samples to the experimental host periwinkle. Dodder transmission experiments on SLY- and SGP-diseased plants may provide evidence that another pathogen is associated with these diseases.

Acknowledgements This research was supported by the Cooperative Research Center for Tropical Plant Protection (Brisbane, Australia), the Australian Research Council (Canberra, Australia), and the Better Berries Program (Brisbane, Australia). We thank Don Hutton, Geoff Waite, Denis Persley and Mark Herrington for their assistance.

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