001_Firrao_249 - SIPaV

Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263

Edizioni ETS Pisa, 2005

249

INVITED REVIEW SHORT TAXONOMIC GUIDE TO THE GENUS ‘CANDIDATUS PHYTOPLASMA’ G. Firrao1, K. Gibb2 and C. Streten2 1 Dipartimento

di Biologia Applicata alla Difesa delle Piante, Università degli Studi, Via delle Scienze 208, 33100 Udine, Italy 2 Faculty of Education, Health and Science, Charles Darwin University, Darwin 0909, NT, Australia

SUMMARY

The category of Candidatus was introduced to allow unambiguous reference to organisms that could not be cultivated in vitro. In plant pathology, a major impact of this novel taxonomic concept was to enable the classification of the diverse group of organisms, morphologically similar to the mycoplasmas, known by the trivial name of phytoplasmas. These plant pathogens were originally named according to the disease they caused. Later, extensive sequence analysis of the ribosomal RNA genes allowed the development of an evolutionbased classification and the grouping of the phytoplasmas into phylogenetically distinct clades. Unfortunately, the phytopathological and the phylogenetic classification did not match and had striking contradictions. With the adoption of the category Candidatus, the description of the genus ‘Ca. Phytoplasma’ and several ‘Ca. Phytoplasma’ species, the scientific community is now attempting to provide a classification that takes into account both the phylogenetic and the biological/ ecological characteristics of the organisms. Here we provide an outline of the characteristics and composition of the genus ‘Ca. Phytoplasma’. Key words: Phytoplasma, Candidatus, taxonomy, yellows.

THE CATEGORY OF CANDIDATUS IN THE TAXONOMY OF NONCULTIVABLE BACTERIAL PLANT PATHOGENS

According to recent estimates, a large fraction of the existing microorganisms cannot be cultivated in vitro. The features of many bacteria, including economically relevant plant pathogens, cannot be investigated using the methodologies that are adopted conventionally for the characterisation and differentiation of cultivable

Corresponding author: G. Firrao Fax: +39.0432.558501 E-mail: [email protected]

prokaryotes. Nevertheless, molecular techniques are now available for the molecular characterisation of organisms without their isolation in pure culture. Limited but precise information on the taxonomy of several non cultivable bacteria has been obtained in recent years, particularly by the sequence analysis of DNA obtained by PCR (Polymerase Chain Reaction)-based amplification of 16S rRNA (ribosomal RNA) genes. Phylogenetically significant information can be obtained by selective amplification of target DNA molecules in the presence of an excess of extraneous nucleic acids, thus circumventing the need for the in vitro cultivation of the organism being studied. In plant pathology, phytoplasmas, a large group of plant pathogens, which, despite several attempts during the last 30 years, still resist cultivation, have been the target of extensive nucleic acid characterization. However, the information obtained is not sufficient for a formal taxonomic description according to the minimum standards for descriptions of new species of the class Mollicutes (International Committee on Systematic Bacteriology - Subcommittee on the Taxonomy of Mollicutes, 1979). Therefore conventional Latin binomial nomenclature cannot be used for those non-cultivable mollicutes, which are defined solely on the basis of their DNA sequences. The lack of a conventional nomenclature has made difficult and confusing references to non-cultivable organisms defined on a molecular basis. For instance, the practice of naming a phytoplasma on the basis of the associated plant syndrome causes confusion because it has been shown that phylogenetically distinct phytoplasmas can cause similar symptoms on the same host plant species. In a taxonomic note published in 1994 by the International Journal of Systematic Bacteriology, Murray and Schleifer (1994) proposed the category Candidatus

(i) The Latin name Candidatus is not declined according to Latin rules when it is included in the organism epithet (i.e.,‘Candidatus Phytoplasma’ is used instead of ‘Candidatum Phytoplasma’) to prevent indexing problems. (ii) Candidata taxa cannot have a “type” as defined by rule 18a of the International Code of Nomenclature of Bacteria (Lapage et al., 1992). For practical purposes of comparison, it was agreed to define a “reference” strain for ‘Ca. Phytoplasma’ species. Some early description may lack “reference” or define, improperly, a “type” strain.

250

Taxonomy of Candidatus phytoplasma

“to provide a proper record of sequence-based potential new taxa”. The introduction and ready acceptance (Murray and Stackebrandt, 1995) of the concept of Candidatus defined a milestone in the nomenclature of bacteria, as unambiguous reference to organisms was no longer hampered by the technical inability to provide them with a suitable artificial environment for growth. With reference to plant pathology, the introduction of the Candidatus category had a major impact on the nomenclature of the phytoplasmas, although its implementation was not obvious. A Candidata species should be defined on the basis of habitat and nucleic acid sequence data. For the phytoplasmas, obligate pathogens, the habitat translates as the host. Although the insect host would have a greater significance, given that the phytoplasma probably originated there, it is more convenient to give the plant host more emphasis in the description of ‘Ca. Phytoplasma’ species. With regard to the nucleic acid sequences, the obvious reference is the 16S rRNA gene, the most studied among the prokaryotes. According to the original proposal (Murray and Schleifer, 1994), a Candidata species description should refer to a unique sequence deposited in public databases and also list unique and characteristic oligonucleotide sequences. However, this approach did not fit particularly well for the phytoplasmas, whose extraordinary diversity was reflected in a large number of unique 16S rDNA sequences, and therefore an impractical number of species. From the several studies on phytoplasma diversity carried out in the last 15 years, it is apparent that some strains are closely related even though their 16S rRNA sequences are not identical and it would not be useful to assign different names to each variant within a 16S rRNA group. On the other hand, the use of the same name to refer to groups of related but different strains would result in a loss of potentially significant biological information. The problems identified stimulated a large debate among the phytoplasmologists. Finally, the ‘phytoplasmas, spiroplasmas, entomoplasmas and mesoplasmas working team’ of the International Research Project for Comparative Mycoplasmology (IRPCM; Anonymous, 2000), in agreement with the International Committee of Systematic Bacteriology Subcommittee for the Taxonomy of Mollicutes (2001), made some recommendations for the description of new ‘Candidatus Phytoplasma’ species. Accordingly, a strain that shares more than 97.5% of its 16S rRNA gene sequence with an already described species should not be described as a new Candidata species unless it is demonstrated that the organism belongs to an ecologically distinct population. Finally, the IRPCM Phytoplasma/ Spiroplasma Working Team Phytoplasma taxonomy group (2004) coded the rules in a paper that describes the properties and the composition of the genus ‘Candidatus Phytoplasma’.

Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263 THE GENUS ‘CANDIDATUS PHYTOPLASMA’

Non helical wall-less bacteria that colonize the plant phloem were first reported almost 40 years ago when Doi et al. (1967) detected by electron microscopy mycoplasma-like bodies in a mulberry plant showing yellows symptoms. About twenty-five years later, it was proposed to rename these mycoplasma-like organisms because they had been shown by molecular analyses not to be phylogenetically related to the genus Mycoplasma. The trivial name phytoplasmas was proposed by B.B. Sears and B.C. Kirkpatrick to the International Committee on Systematic Bacteriology (ICSB) Subcommittee on the Taxonomy of Mollicutes (1993) and subsequently used (Sears and Kirkpatrick, 1994). Later, the ICSB Subcommittee on the Taxonomy of Mollicutes (1995) agreed that the phytoplasmas could be assigned a provisional taxonomic status of Candidatus, since they could only be defined on the basis of habitat, morphology and certain characteristics of the membrane and nucleic acids and not on the basis of biochemical, physiological and cultural characteristics because they could not be cultivated in vitro. Phytoplasmas lack rigid cell walls, are surrounded by a single unit membrane, and are sensitive to the antibiotic tetracycline (Doi et al., 1967). When observed by transmission electron microscopy, they appear as rounded to filamentous pleiomorphic bodies with an average diameter ranging from 200 to 800 nm (Kirkpatrick, 1991). They inhabit the phloem sieve elements of infected plants and the gut, haemolymph, salivary gland and other organs of sap-sucking insect vectors (Kirkpatrick, 1991). The genome of the phytoplasmas has a size of 530-1,350 kbp (Marcone et al., 1999) and a G+C content of 23-29 mol% (Kollar and Seemüller, 1989). The phytoplasmas have a tRNAILE in the spacer region between the 16S and the 23S rRNA genes (Kuske and Kirkpatrick, 1992; Smart et al., 1996). A major molecular characteristic for their definition and identification is the sequence of their 16S rRNA gene. Phylogenies based on this gene showed that the phytoplasmas constitute a monophyletic group within the Mollicutes (Gundersen et al., 1994; Seemüller et al., 1994), Acholeplasma palmae being their closest cultivable relative. Due to the distinctive sequences in their 16S rRNA gene, several phytoplasmaspecific PCR assays have been developed (Ahrens and Seemüller, 1992; Firrao et al., 1993; Lee et al., 1993; Namba et al., 1993; Gibb et al., 1995; Lorenz et al., 1995; Padovan et al., 1995; Smart et al., 1996). The phytoplasmas are a large and diverse group causing several hundreds of plant diseases. More than 200 different, nearly full length sequences of phytoplasma 16S rRNA genes have been obtained to date (Seemüller et al., 1998b; Lee et al., 2000; IRPCM Phytoplasma/Spiroplasma Working Team - Phytoplasma taxonomy group, 2004). Their analysis has delineated less than 20 clusters


of strains at 97.5% similarity. Selected representatives of each cluster are described as ‘Ca. Phytoplasma’ species. However, some groups defined at 97.5% or higher similarity, include phytoplasmas with different biological, phytopathological, and molecular properties. In some cases, phytoplasmas sharing high 16S rDNA sequence similarity may cause different plant diseases. Hence, in addition to the minimum set of species belonging to ‘Ca. Phytoplasma’ defined at the 97.5% similarity of the 16S rDNA sequence, more Candidatae species have been defined to distinguish between those organisms that proved to be significantly different on the basis of biological and genetic properties. In order to prevent nomenclatural confusion arising from the description of poorly differentiated new taxa, the Phytoplasma/Spiroplasma Working Team of the International Research Project for Comparative Mycoplasmology (Anonymous, 2000; IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma taxonomy group, 2004) suggested rules for the description as new Candidatae species of phytoplasmas that share more than 97.5% of their 16S rRNA gene. For such cases, the description of

Firrao et al.

251

two different species is recommended only when the following three conditions apply: (i) the two phytoplasmas are transmitted by different vectors; (ii) the two phytoplasmas have a different natural plant host (or at least their behaviour is significantly different in the same plant host); (iii) there is evidence of significant molecular diversity from either hybridization tests with cloned DNA probes, or serological reactions or PCRbased assays. At the time of writing, 21 Candidatae species have been validly described and 6 additional species have an informal description (Fig. 1). As a consequence of the taxonomic rules adopted for the classification of the phytoplasma, the plant host and the distinctive oligonucleotide signature given in the species description should be verified, but not the identity of the complete 16S rDNA sequence. Given the wide use of the restriction analysis of the 16S rDNA, a presumptive identification of the isolate is most often done by analyzing the 16S rDNA RFLP pattern. The next section summarizes the details needed for the identification of each species presently recognized within the genus ‘Candidatus Phytoplasma’.

Fig. 1. Phylogenetic tree reporting the Candidatae species of the genus ‘Candidatus Phytoplasma’ and their relationships with the groups identified by Lee et al. (1998, 2000) and Seemüller et al. (1994, 1998b). The asterisk (*) marks names which have been proposed at the X International Congress of the International Organization of Mycoplasmology (1994), but not yet formally described, and are reported here as incidental citations which do not constitute prior citations, according to rule 28b of the bacteriological code (Lapage et al., 1992). The tree was constructed using CLUSTALX (Thompson et al., 1997) from the alignment of the Candidatus species 16S rDNA sequences, publically available at TreeBase (http://www.treebase.org/treebase/console.html) as matrix accession M1788.

252



‘CANDIDATUS PHYTOPLASMA’ SPECIES

reference strain. The description of this taxon is based on the strain OAYR (=MIAY), associated with Oenothera virescence in Michigan, USA. This strain belongs to the Aster Yellows group (Seemüller et al., 1994, 1998b) or 16SrI group of Lee et al. (1998, 2000). Phylogenetic studies based on sequences from different genes have shown congruently that this group represents a distinct clade within the phytoplasmas (Lee et al., 1998). ‘Ca. P. asteris’ is the only species described within the 16SrI group. However, this clade comprises a large number of related phytoplasmas worldwide, representing the most diverse and widespread phytoplasma group studied (Lee et al., 2004a). The strains related to ‘Ca. P. asteris’, despite the relatively high similarity in the 16S rDNA sequence, occupy diverse ecological niches and show substantial genetic variations (Gundersen et al., 1994; Seemüller et al., 1994, 1998b; Vibio et al., 1996; Lee et al., 1998, 2000, 2002; Marcone et al., 2000; Jomantiene et al., 2002). According to the rules for the description of new Candidatae species this widely diverse group may consist of more than one species. A specific primer pair, designed by Lee et al. (1994) for the amplification of a ribosomal DNA fragment from strains belonging to the 16SrI group and named R16(I)F1/R1, is presently widely used for the amplification from strains related to ‘Ca. P. asteris’, although the 16S rDNA of strains belonging to groups 16SrII and 16SrXII may also be a target. The 16S rDNA of ‘Ca. P asteris’ has characteristic KpnI sites that distinguish it from other species. Useful diagnostic enzymes for the assignment of isolates to subgroups within the 16SrI group are MseI, HhaI and HpaII for the 16S rDNA and MseI, Tsp509I and AluI for the ribosomal protein gene operon (Lee et al., 1998, 2004a). Most ‘Ca. P. asteris’ strains infect herbaceous dicotyledonous plants. Nevertheless, some strains may infect monocotyledonous as well as woody plants. The host range of this species is extremely large; a list of recognized host species is given below but it may not be comprehensive: Aconitum napellus, Allium cepa, Ambrosia artemisifolia, Anemone coronaria, Anethum graveolens, Aphanostephus skirrhobasis, Apium graveolens, Avena sativa, Brassica napus, Brassica oleracea capitata, Brassica oleracea italica, Brassica oleracea palmifolia, Brassica rapa rapifera, Calendula officinalis, Callistephus chinensis, Carthamus tinctorius, Catharanthus roseus, Celtis australis, Chrysanthemum coronarium, Chrysanthemum frutescens, Cirsium arvense, Corinadrum sativum, Cornus racemosa, Cosmos bippinatus, Cryptotaenia japonica, Cyclamen persicum, Cynodon dactylon, Daucus carota, Delphinium sp., Diplotaxis eucoides, Echinacea purpurea, Epilobium sp., Erigeron canadensis, Eupatorium capillifolium, Fragaria x ananassa, Gladiolus sp., Glycine max, Hyacinthus orientalis, Hydrangea macrophylla, Ipomoea obscura, Lactuca sativa, Limonium sinuatum, Lycopersicon esculentum, Medicago sativa, Morus bombycis, Nasturtium microphyllum, Oenothera hookeri, Olea europaea, Opuntia sp., Pa-

‘Ca. Phytoplasma allocasuarinae’ Marcone, Gibb, Streten & Schneider (Marcone et al., 2003a). This phytoplasma was detected in naturally diseased Allocasuarina muelleriana (Slaty sheoak) plants growing in Australia and expressing a yellows symptom. It is associated with the 16S rRNA gene sequence accession number AY135523, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-TTTATTCGAGAGGGCG-3’. Sequence analysis of the 16S rRNA gene shows that this phytoplasma is more closely related to members of the apple proliferation (AP) group than to other phytoplasma subclades (Gibb et al., 2003). Nucleotide sequence comparisons between the 16S rRNA genes of ‘Ca. P. allocasuarinae’ and other phytoplasmas assigned to the AP group revealed the buckthorn witches’-broom (BWB) agent is most closely related, sharing 96% similarity. The ‘Ca. P. allocasuarinae’ agent differs from the BWB and spartium witches’-broom (SpaWB) agents in 17 to 18% of the nucleotide positions on the 16S rRNA gene. The ‘Ca. P. allocasuarinae’ agent shared 94% similarity with apple proliferation (AP), pear decline (PD), European stone fruit yellows (ESFY) and peach yellow leaf roll (PYLR) phytoplasmas, which are also members of the AP-fruit tree group. ‘Ca. P. allocasuarinae’, ESFY, PD and PYLR phytoplasma have the same banding pattern when digested with restriction enzyme ScfI and ‘Ca. P. allocasuarinae’ has a restriction site for PvuII at position 518 that is unique to members of the AP-group. The ‘Ca. P. allocasuarinae’ 16S rRNA gene lacks restriction sites for SspI and AluI enzymes, which are present in the SpaWB and BWB agents. ‘Ca. P. allocasuarinae’ has an additional HpaII restriction site that is not present in other AP-fruit tree agents. A specific PCR assay for ‘Ca. P. allocasuarinae’ is possible using the AP group-specific primer fO1 (Lorenz et al., 1995) in combination with the reverse primer rAllo1 (5’GAGTCCCCAACTAAATGAT-3’) (Gibb et al., 2003). ‘Ca. P. asteris’ Lee, Gundersen, Rindal, Davis, Bottner, Marcone & Seemüller (Lee et al., 2004a). This phytoplasma is associated with the 16S rRNA gene sequence accession M30790, and to the accession M74770, corresponding to genes for ribosomal proteins rpl23 gene (3’ end), rpl2, rps19, rpl22, and rps3 genes, and rpl16 (5’ end); oligonucleotide sequences of unique regions of 16S rRNA gene are: 5’-GGGAGGA-3’ (positions 226-232), 5’-CTGACGGTACC-3’ (positions 476-485) and 5’CACAGTGGAGGTTATCAGTTG-3’ (positions 10081028); oligonucleotide sequences of unique regions of ribosomal protein genes are 5’-CCGCGAACAACTT-3’ and 5’-AGTAATAACTTCTAGCACAAACTTGC-3’ (positions 218-230 and 338-363) for rpl22 and 5’-AAAGAA GATTTTTTAATTC-3’ and 5’-CTAGAAAATCGTATG -3’ (positions 114-133 and 396-410) for rps3. OAY is the


paver rhoeas, Parthenium hysterophorus, Paulownia sp., Plantago coronopus, Plantago major, Poa pratensis, Populus nigra, Portulaca oleracea, Primula sp., Prunus aremeniaca, Prunus cerasus, Pyrus communis, Quercus robur, Ranunculus asiaticus, Ranunculus sp., Raphanus raphanistrum, Salix sp., Santalum album, Saponaria officinalis, Schizanthus pinnatus, Sechium edule, Sechium tacaco, Solanum melongena, Solanum tuberosum, Sonchus asper, Symphytun sp., Tagetes erecta, Taraxacum officinale, Trifolium repens, Trifolium sativum, Trifolium sp., Vaccinium sp., Valeriana officinalis, Vitis vinifera, Zea mays (refer to Lee et al., 2004a, for details and references). Due to the large host range, ‘Ca. P. asteris’ strains are associated with several diseases worldwide, generally named on the basis of symptoms and host. Typical symptoms caused by infection of ‘Ca. P. asteris’ include virescence (greening of flower petals) and phyllody (development of floral parts into leaf like structures), flower streaking and malformation, yellowing and upright posture of leaves, elongation and etiolation of internodes, excessive branching of axillary shoots, witches’-broom and stunting. However, some infected plants may exhibit only some of these symptoms. Plants infected with some so-called “mild” strains may show no obvious symptoms. ‘Ca. Phytoplasma aurantifolia’ Zreik, Carle, Bové & Garnier (Zreik et al., 1995). This phytoplasma was detected in Citrus aurantifolia affected by the witches’broom disease of small-fruited acid lime. The disease appeared in Oman in the late 1970s, was later reported in the United Arab Emirates and recently also in Iran (Bové et al., 1998). ‘Ca. P. aurantifolia’ is associated with the 16S rRNA gene sequence accession U15442, with oligonucleotide sequence complementary to a unique region of the 16S rRNA, 5’-GCAAGTGGTGAACCATTTGTTT-3’. The genome size is estimated to be 720 kbp. The phylogenetic analysis of the 16S rRNA gene sequence assigns this phytoplasma to the faba bean phyllody (FBP) group (Schneider et al., 1995; Seemüller et al., 1998b) or to the 16SrIIB (according to Lee et al., 2000). The reader is referred to ‘Ca. P. australasiae’ below for details about the identification and differentiation of strains of the FBP group using 16S rRNA gene analysis. ‘Ca. P. aurantifolia’ could also be distinguished from closely related strains by Southern blot – RFLP analysis using cloned chromosomal probes and by monoclonal antibody reactions (Zreik et al., 1995). ‘Ca. Phytoplasma australasiae’ White, Blackall, Scott, & Walsh (White et al., 1998) (revised nomenclature of ‘Ca. P. australasia’). This phytoplasma is associated with the 16S rRNA gene sequence accession Y10097, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-TAAAAGGCATCTTTTATC-3’ and 5’-CAAGGAAGAAAAGCAAATGGCGAACCATTTGTTT-3’. Sequence analy-

Firrao et al.

253

sis of the 16S rRNA gene indicates that ‘Ca. P. australasiae’ and ‘Ca. P. aurantifolia’ are phylogenetically closely related to each other and to other phytoplasmas, collectively known as the Faba bean phyllody (FBP) group (Schneider et al., 1995). The first reported phytoplasma of this group was implicated in naturally diseased tomato showing symptoms of tomato big bud (TBB) disease (Bowyer et al., 1969). In a more recent survey, phytoplasmas related to the TBB phytoplasma were detected in 73 plant host species from 26 families (Davis et al., 1997b; Schneider et al., 1999). Most of the plant host species are members of the Fabaceae (Schneider et al., 1999). RFLP and sequence analysis of ribosomal genes indicate that a close relationship exists between the phytoplasmas associated with TBB, papaya yellow crinkle (PYC), papaya mosaic (PM) and sweet potato little leaf (SPLL) (Gibb et al., 1995, 1996; White et al., 1998). Using these data the TBB type phytoplasmas from Australia were placed in the faba bean phyllody (FBP) strain cluster (Schneider et al., 1995), which includes the type specimen FBP from Sudan, and the lime witches’-broom phytoplasma WBDL), named ‘Ca. Phytoplasma aurantifolia’ by Zreik et al. (1995). White et al. (1998) proposed that the PYC, PM, and TBB phytoplasmas be assigned to a new species, ‘Ca. Phytoplasma australasia’ (sic), not in agreement with the recommended criteria (IRPCM Phytoplasma/Spiroplasma Working Team - Phytoplasma taxonomy group, 2004). Therefore the use of this species name is discouraged. This phytoplasma was detected in Australia associated with diseases of Carica papaya L. named papaya yellow crinkle and mosaic. Since this Candidatus species name was extended by the authors of the original paper (White et al., 1998) to include other phytoplasmas in addition to that associated with the sequence Y10097, phytoplasmas isolated from other hosts might potentially be identified as ‘Ca. P. australasiae’. This is not recommended. ‘Ca. Phytoplasma australiense’ Davis, Dally, Gundersen, Lee & Habili (Davis et al., 1997a). This phytoplasma is associated with the 16S rRNA gene sequence accession L76865, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’TTTATCTTTAAAAGACCTCGCAAGA-3’. According to their phylogenetic studies, Lee et al. (2000) represented the ‘Ca. P. australiense’ as a member of the stolbur 16SrXII group, subgroup B. Diagnostic enzymes for the identification of this phytoplasma by RFLP analysis of amplified 16S rRNA gene are AluI, MseI and TaqI. This phytoplasma was detected in naturally diseased ‘Chardonnay’ grapevines showing symptoms of Australian grapevine yellows (AGY), and later in other grapevine cultivars such as ‘Riesling’ and less frequently ‘Traminer’, ‘Montils’ and ‘Semillon’ (Padovan et al., 1996). The disease looks very similar to European

254


grapevine yellows diseases caused by phylogenetically different phytoplasmas. The diseases known as papaya dieback (Carica papaya L.), Phormium yellow leaf (Phormium tenax J.R. Forst. & G. Forst., P. cookianum Le Jol.) and strawberry lethal yellows (Fragaria x ananassa) were found to be associated with ‘Ca. P. australiense’ (Andersen et al., 1998; Liefting et al., 1998; Padovan et al., 2000). ‘Ca. P. australiense’ is currently restricted to Australia and New Zealand. ‘Ca. Phytoplasma brasiliense’ Montano, Davis, Dally, Hogenhout, Pimentel & Brioso (Montano et al., 2001). This phytoplasma was found in Hibiscus rosasinensis L. showing symptoms of hibiscus witches’broom in Brazil (Montano et al., 2001). It is associated with the 16S rRNA gene sequence accession AF147708, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-GAAAAAGAAAG-3’, 5’TCTTTCTTT-3’, 5’-CAG-3’, 5’-ACTTTG-3’ and 5’GTCAAAAC-3’. Phylogenetic analysis of the hibiscus witches’-broom phytoplasma 16S rDNA sequence showed that it was distinct from any other phytoplasma and group 16SrXV was designated to accommodate it (Montano et al., 2001). Diagnostic enzymes for the identification of this phytoplasma by RFLP analysis of amplified 16S rRNA gene are MseI and HpaII. ‘Ca. Phytoplasma castaneae’ Jung, Sawayangi T, Kakizawa, Nishigawa, Miyata, Oshima K, Ugaki, Lee, Hibi & Namba (Jung et al., 2002). This phytoplasma was found in Castanea sativa showing symptoms of witches’-broom disease of chestnut in Japan. It is associated with the 16S rRNA gene sequence accession AB054986, with oligonucleotide sequences complementary to unique regions of the 16S rRNA 5’-CTAGTTTAAAAACAATGATA-3’ and 5’-CTCATCTTCCTCCAATTC-3’. Sequence analysis revealed the Chestnut witches’-broom (CnWB) phytoplasma shares from 86.8 to 94.9% similarity with other phytoplasmas. Phylogeny and sequence analysis revealed the CnWB phytoplasma is most closely related to the phytoplasmas that affect coconut and it represents a distinct subgroup within the group. The CnWB phytoplasma can be specifically amplified by using the primer pair CnWBF1/CnWBR1 (Jung et al., 2002). The phytoplasma signature sequence ACTGGA (155-160) in the CnWB phytoplasma has a ‘G’ instead of a ‘A” at position 155 and a ‘T’ instead of a ‘C’ at position 156. In addition, the phytoplasma signature sequences GTGT (276-279) and CCC (487-489) have a ‘T’ at nucleotides 278 and 487 in the 16S rRNA gene of the CnWB phytoplasma (Jung et al., 2002). ‘Ca. Phytoplasma cynodontis’ Marcone, Schneider & Seemüller (Marcone et al., 2003b). This phytoplasma was detected in Cynodon dactylon exhibiting symptoms of chlorosis, proliferation of axillary shoots, bushy


growth habit, small leaves, shortened stolons and rhizomes, and stunting. The disease was named Cynodon white leaf (CWL) or Bermuda grass white leaf (BGWL). Its geographical distribution includes several countries in Asia (Chen et al., 1972; Zahoor et al., 1995; Viswanathan, 1997; Sdoodee et al., 1999), Italy (Marcone et al., 1997a), Sudan (Dafalla and Cousin, 1988), and Australia (Tran-Nguyen et al., 2000; Blanche et al., 2003). ‘Ca. P. cynodontis’ is associated with the 16S rRNA gene sequence accession AJ550984, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-AATTAGAAGGCATCTTTTAAT-3’. Phylogenetic studies assigned the Bermuda grass white leaf (BGWL) phytoplasma to the 16SrXIVA group, which includes blue grass (Poa annua) white leaf and Brachiaria distachya white leaf (BraWL) phytoplasmas (Lee et al., 1998, 2000; Seemüller et al., 1998b; Sdoodee et al., 1999). Although members of the 16SrXIV group share greater than 97.5% 16S rDNA similarity, data were provided showing that the group includes ecologically and genetically distinct populations that should correspond to distinct ‘Candidatus Phytoplasma’ species. Phylogeny studies and sequence analysis revealed that 16S rRNA genes of BGWL isolates from Italy are identical while the Thai BGWL strain differed from the Italian strain in four nucleotide positions. The 16S rRNA gene of the CWL phytoplasma differed from the Thai isolate at one nucleotide and from the Italian strains at three nucleotides. The BraWL agent is closely related to the BGWL phytoplasma (99.4-99.7% similarity between 16S rRNA genes). The 16S rRNA gene of SGS phytoplasma shares 98.3-98.5% similarity with the BGWL strains while the Sugarcane White Leaf (SCWL) phytoplasma shares 98.2-98.4% similarity with the BGWL isolates. Phylogeny derived from 16S-23S spacer region sequences confirmed the results of the 16S rRNA gene analysis. Enzymatic RFLP analysis showed that there is no variation between the phytoplasmas associated with BGWL, white leaf agent from annual blue grass, BraWL, carpet grass white leaf (CGWL) and date palm diseases. However, theoretical RFLP banding patterns showed that BGWL phytoplasma lacks a TaqI site at position 1,424 that is present in the BraWL phytoplasma and that the BGWL phytoplasma also has a MaeIII site not present in the BraWL agent. The BGWL phytoplasma can be distinguished from other members of the SCWL group including SCWL, SCGS, and SGS phytoplasmas based on RFLP banding patterns. The BGWL phytoplasma can be specifically amplified using the fSCWL forward primer in combination with the rCWL reverse primer (TranNguyen et al., 2000). The estimated chromosome of the BGWL phytoplasma is 530 kbp (Marcone et al., 1999). Polyclonal antisera raised to the BGWL and SCWL phytoplasmas showed no cross-reactivity (Sarindu and Clark, 1993; Viswanathan, 1997, 2001). The BGWL antisera did not


react with the BraWL or Dactylocetenium aegyptum white leaf (DacWL). The BGWL phytoplasma has not been identified in other plant species and it is not transmitted by Matsumuratettis hiroglyphicus, which is the natural vector for SCWL phytoplasma. Although sequence analysis indicated that the BraWL, CGWL and agents associated with date palms are identical or nearly identical to the BGWL phytoplasma, it has been suggested that these phytoplasmas should not be designated as the same ‘Candidatus Phytoplasma’ species name as the BGWL phytoplasma because there is currently insufficient evidence to confirm this relationship. ‘Ca. Phytoplasma fraxini’ Griffiths, Wayne, Sinclair, Smart & Davis (Griffiths et al., 1999b). This phytoplasma was detected in Fraxinus americana L., Fraxinus velutina Torr., Fraxinus pennsylvanica Marsh., Fraxinus bongeana A. DC., showing symptoms of ash yellows and in Syringa patula (Palibin) Nakai, Syringa x prestoniae McKelv., Syringa x josiflexa Preston ex Pringle with lilac witches’-broom disease. It is restricted to the Americas (Griffiths et al., 1999b, 2001). ‘Ca. P. fraxini’ is associated with the 16S rRNA gene sequence accession AF092209, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-CGGAAACCCCTCAAAGGTTT-3’ and 5’-AGGAAAGTC-3’. In phylogenetic analyses, strains associated with ash yellows and lilac witches’-broom constituted a clearly defined clade within the phytoplasmas (Griffiths et al., 1999b), corresponding to 16SrVII group in Lee et al. (1998, 2000). These strains were collectively named ‘Ca. P. fraxini’, and strain AshY1 was selected as a type strain (Griffiths et al., 1999b), the other strains differ from the AshY1 type by 1 to 3 nucleotides in their 16S rRNA gene sequence. However, the selection of a type strain is not valid (see note 2) and strain AshY1R should more properly referred as “reference strain”. Some recently detected phytoplasmas were assigned to the 16SrVII group (Meneguzzi et al., 2001; Barros et al., 2002; Bruni et al., 2005), but those strains appear to be distinct from ‘Ca. P. fraxini’. The genome size of strain AshY3 is around 645 kbp. Digestion of the PCR-amplified 16SrRNA gene with AluI is diagnostic for the identification of this strain cluster (Lee et al., 1998; Griffiths et al., 1999b). A specific PCR assay for AshY1 and closely related strains uses primers fB1/rAsYs (Smart et al., 1996). ‘Ca. Phytoplasma japonicum’ Sawayanagi, Horikoshi, Kanehira, Sinohara, Bertaccini, Cousin, Hiruki & Namba (Sawayanagi et al., 1999). This phytoplasma was detected in Hydrangea macrophylla (Thunb. ex J. Murr.) Ser. f. normalis, Hydrangea serrata (Thunb.) showing symptoms of Japanese Hydrangea phyllody, in Japan. ‘Ca. P. japonicum’ is associated with the 16S rRNA gene sequence accession AB010425, with oligonucleotide sequence complementary to unique region of

Firrao et al.

255

the 16S rRNA 5’-GTGTAGCCGGGCTGAGAGGTCA-3’ and 5’-TCCAACTCTAGCTAAACAGTTTCTG3’. In the phylogenetic analysis of the 16S rRNA gene sequence (Sawayanagi et al., 1999), this phytoplasma was related to the strains in group 16SrI group but did not cluster to any other strain and was therefore assigned to a new sub-group. The most closely related phytoplasmas were the stolbur phytoplasma and ‘Ca. P. australiense’ (Sawayanagi et al., 1999). Diagnostic enzymes for the identification of this phytoplasma by RFLP analysis of the amplified 16S rRNA gene are KpnI, EcoRI, HaeIII and Tru9I (MseI). A specific amplification of this phytoplasma is achieved by using a nested PCR approach using the primer pair SN910601/SN910502 followed by JHPF1/JHPR1 (Sawayanagi et al., 1999). ‘Ca. Phytoplasma mali’ Seemüller & Schneider (Seemüller and Schneider, 2004). This phytoplasma is associated with the economically important disease named apple proliferation. It occurs in a wide range of species of the genus Malus (Kartte and Seemüller, 1991) and has been detected occasionally in plants such as Pyrus communis, Pyrus pyrifolia, Prunus salicina, Corylus avellana, Crataegus monogyna, Quercus robur, Quercus rubra, Carpinus betulus, Convolvulus arvensis (Lee et al., 1995; Marcone et al., 1996b; Schneider et al., 1997; Del Serrone et al., 1998; Seemüller, 2002) by serological and DNA based techniques. ‘Ca. P. mali’ is restricted to Europe. It is associated with the 16S rRNA gene sequence accession AJ542541, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’AATACTCGAAACCAGTA-3’. The reference strain is isolate AP15, which has a chromosome of 690 kb in length. ‘Ca. P. mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’ are phylogenetically closely related, the interspecific differences in their 16S rDNA sequences range from 1.0 to 1.5%. The group was named ‘Apple proliferation strain cluster’ (Seemüller et al., 1994, 1998b) or group 16SrX (Lee et al., 1998, 2000). The AluI and RsaI restriction patterns of the 16S rDNA are characteristic and diagnostic for this group (Schneider et al., 1993). The ‘Ca. P. mali’ phytoplasma can be distinguished by 16S rDNA digestion with SspI and BsaAI from ‘Ca. P. pyri’, and from ‘Ca. P. prunorum’ with RsaI, BsaAI, SspI and SfcI. Several diagnostic primers based on 16S rDNA sequences for the selective amplification of some or all phytoplasmas of the Apple proliferation group have been developed by Firrao et al. (1994), Jarausch et al. (1994), Lee et al. (1995), Lorenz et al. (1995), Smart et al. (1996) and Baric and Dalla Via (2004). AP-group phytoplasma detection and differentiation can also be carried out using randomly cloned chromosomal fragments, such as fragment IH196 of strain AT (Bonnet et al., 1990). PCR amplification using primers AP9/AP10 followed by digestion with RsaI and HincII allows the differentiation of ‘Ca. P. mali’ from ‘Ca. P. pyri’ and ‘Ca.

256


P. prunorum’ (Jarausch et al., 1994, 2000a, b). ‘Ca. Phytoplasma oryzae’ Jung, Sawayanagi, Wongkaew, Kakizawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Hibi & Namba (Jung et al., 2003b). This phytoplasma was detected in Oryza sativa plants exhibiting symptoms of yellowing, stunted growth and failure to produce rice, a disease named rice yellow dwarf that occurs in Japan and Thailand. ‘Ca. P. oryzae’ is associated with the 16S rRNA gene sequence accession AB052873, with oligonucleotide sequences complementary to unique regions of the 16S rRNA 5’AACTGGATAGGAAATTAAAAGGT-3’ and 5’-ATGAGACTGCCAATA-3’. Phylogenetic analysis showed that the closest relatives to rice yellow dwarf (RYD) agent are the sugarcane white leaf (SCWL), sugarcane grassy shoot (SCGS), annual blue grass white leaf (ABGWL), Bermuda grass white leaf (BGWL) and Brachiana grass white leaf (BraWL) phytoplasmas (Nakashima et al., 1996; Lee et al., 1997; Wongkaew et al., 1997; Tran-Nguyen et al., 2000). Sequence analysis of the RYD phytoplasma 16S rRNA gene indicated the presence of several signature sequences that are unique to members of the RYD group, 5’-AACACTG-3’ and 5’-GCAA-3’, which are not found in phytoplasmas assigned to different groups within the subclade. The sequence 5’-TATCAGACTA-3’ at positions 626-635 is also conserved in the RYD group with the exception of the SCWL and BVK phytoplasmas that have a ‘T’ instead of a ‘C’ at nucleotide 629. An additional sequence, 5’-AAATCTTCGGATTT-3’ (61-75) is also conserved between members of the RYD group, except in RYD isolates which have a ‘T’ instead of ‘C’ at nucleotide 65. The ‘Ca. P. oryzae’ 16S rRNA gene contained additional nucleotide variations at positions 149 (G to A), 181 (C to T), 263 (A/C to T), 1,148, (G to A), 1,409 (A to G) and 1,447 (G to A), which differentiated this isolate from other phytoplasmas including the other members of the RYD group. Sequence analysis of the 16S rRNA genes from ‘Ca. P. oryzae’ and BGWL phytoplasma showed these phytoplasmas share 96.3-97.9% similarity. As also discussed above (see ‘Ca. P. cynodontis’), the data suggested that several ‘Ca. Phytoplasma’ species should be described in this group despite their relatively high 16S rDNA similarity. Southern blot analysis shows chromosomal and extrachromosomal DNA extracted from ‘Ca. P. oryzae’ only hybridises with DNA of RYD isolates and not with that from the closely related SCWL phytoplasma (Nakashima et al., 1993). ‘Ca. P. oryzae’ is transmitted by three species of leafhoppers (Nephotettix spp.) that are found only in Asia (Nakashima et al., 1993). RYD disease has only been detected in Asia and it is thought that rice is the only natural host of this phytoplasma (Nakashima et al., 1993).


‘Ca. Phytoplasma phoenicium’ Verdin, Salar, Danet, Choueiri, Jreijiri, El Zammar, Gélie, Bové, Garnier (Verdin et al., 2003). This phytoplasma was detected in Prunus amygdalus showing symptoms of lethal disease of almond trees in Lebanon and Iran (Abou-Jawdah et al., 2002; Verdin et al., 2003). It is associated with the 16S rRNA gene sequence accession AF515636, with oligonucleotide sequence complementary to the unique region of the 16S rRNA 5’-CCTTTTTCGGAAGGTATG-3’. Phylogenetic analysis showed that this phytoplasma clusters with members of the pigeon pea witches’-broom (PPWB) group (Schneider et al., 1995), known as 16SrIX according to Lee et al. (2000). A primer pair, AlmF1/AlmR1 was defined by Verdin et al. (2003) for the specific amplification of a DNA fragment of about 1.5 kb from the 16S rDNA of ‘Ca. P. phoenicium’. The phytoplasmas associated with Picris echioides yellows (PEY) and Knautia arvensis phyllody (KAP) share 99% identity in their 16S rDNA sequence, and the PPWB phytoplasma shares 98.5% identity. They should therefore be referred to as related to ‘Ca. P. phoenicium’. ‘Ca. Phytoplasma pini’ Schneider, Torres, Martìn, Schröder, Behnke & Seemüller (Schneider et al., 2005). This phytoplasma was detected in Pinus halepensis, Pinus silvestris showing abnormal shoot branching and dwarfed needles in Germany and Spain. ‘Ca. P. pini’ is associated with the 16S rRNA gene sequence accession AJ632155, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’GGAAATCTTTCGGGATTTTAGT-3’ (positions 6788) and 5’-TCTCAGTGCTTAACGCTGTTCT-3’ (positions 603-624). According to the phylogenetic analysis carried out by Schneider et al. (2005), the pine phytoplasma strains form a distinct branch and are only distantly related to other phytoplasmas. The closest relatives, sharing 94.5% or less 16S rRNA gene sequence identity, are the phytoplasmas associated with diseases of rice (‘Ca. P. oryzae’), coconut and chestnut (‘Ca. P. castaneae’), included in the 16SrIV or 16SrXI groups by Lee et al. (1998, 2000). No specific PCR primers have been developed for the selective amplification of ‘Ca. P. pini’, neither has the use of diagnostic restriction enzymes been demonstrated. However, the restriction map of the 16S rDNA gene deduced from the sequence suggests that specific patterns should results from the digestion with the enzymes commonly used for the characterization of phytoplasmas (AluI, RsaI, MseI, HhaI and HinfI) of an amplification product obtained using general phytoplasma-specific primers. The reference strain is Pin127SR from Pinus halepensis. ‘Ca. Phytoplasma prunorum’ Seemüller & Schneider (Seemüller and Schneider, 2004). This phytoplasma is thought to cause all the yellows and decline diseases of


Prunus species in Europe known as apricot chlorotic leaf roll, ‘Molières disease’ of sweet cherry, Japanese plum leptonecrosis, peach yellows (in Europe), peach rosette (in Europe), peach vein clearing, decline of almond and decline of flowering cherry (Lorenz et al., 1994; Jarausch et al., 2000a). Most, if not all Prunus species can support the replication of ‘Ca. P. prunorum’ (Lorenz et al., 1994; Jarausch et al., 2000a), although some species may not show obvious symptoms. In addition, ‘Ca. P. prunorum’ was detected in Fraxinus excelsior, Rosa canina, Celtis australis, Corylus avellana (Marcone et al., 1996b; Jarausch et al., 2001). It is restricted to Europe and Turkey (Jarausch et al., 2000a). ‘Ca. P. prunorum’ is associated with the 16S rRNA gene sequence accession AJ542544, with oligonucleotide sequences complementary to unique region of the 16S rRNA 5’-AATACCCGAAACCAGTA3’ and 5’-TGAAGTTTTGAGGCATCTCGAA-3’. The reference strain is isolate ESFY-G1, which has a chromosome 630 kb in length. ‘Ca. P. prunorum’ is phylogenetically closely related to ‘Ca. P. mali’ and ‘Ca. P. pyri’. The reader is referred to the above description of ‘Ca. P. mali’ for details of the methods used to detect and differentiate this group of organisms. In particular, ‘Ca. P. prunorum’ can be distinguished by 16S rDNA digestion with RsaI, BsaAI, SspI and SfcI from ‘Ca. P. mali’, and from ‘Ca. P. pyri’ by using RsaI and SfcI. ‘Ca. Phytoplasma pyri’ Seemüller & Schneider (Seemüller and Schneider, 2004). This phytoplasma is associated with the pear decline, an economically important disease that affects all Pyrus species (Seemüller et al., 1998a). In addition, ‘Ca. P. pyri’ was also detected in Corylus avellana (Marcone et al., 1996b). ‘Ca. P. pyri’ is spread in Europe, Asia and the Americas. In California, a disease of peach, named Peach Yellow Leaf Roll, may be caused by a phytoplasma closely related to ‘Ca. P. pyri’, but, due to the lack of conclusive data, Seemüller and Schneider (2004) did not include the Peach Yellow Leaf Roll phytoplasma in this species. ‘Ca. P. pyri’ is associated with the 16S rRNA gene sequence accession AJ542543, with oligonucleotide sequences complementary to unique region of the 16S rRNA 5’-AATACTCAAAACCAGTA3’ and 5’-ATACGGCCCAAACTCATACGGA-3’. The reference strain is isolate PD1, which has a chromosome 660 kb in length. ‘Ca. P. pyri’ is phylogenetically closely related to ‘Ca. P. mali’ and ‘Ca. P. prunorum’. The reader is referred to the above description of ‘Ca. P. mali’ for details about the methods used to detect and differentiate this group of organisms. In particular, the ‘Ca. P. pyri’ phytoplasma can be distinguished by 16S rDNA digestion with SspI and BsaAI from ‘Ca. P. mali’, and from ‘Ca. P. prunorum’ by using RsaI and SfcI. ‘Ca. Phytoplasma rhamni’ Marcone, Gibb, Streten & Schneider (Marcone et al., 2003a). This phytoplasma was detected in Rhamnus catharticus affected by witch-

Firrao et al.

257

es’-broom disease of buckthorn in South-western Germany. It is associated with the 16S rRNA gene sequence accession number X76431 and AJ583009, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-CGAAGTATTTCGATAC-3’. Sequence analysis of the Buckthorn witches’-broom (BWB) agent shows that this phytoplasma is more closely related to members of the AP group than to other phytoplasma subclades. The 16S rRNA genes of ‘Ca. P. rhamni’ shares 96% similarity with the AP group phytoplasmas, AP, ESFY, PD and PYLR and 95% similarity with the Spartium witches’-broom (SpaWB) phytoplasma (‘Ca. P. spartii’), which is also a member of the AP group. The 16S-23S spacer region ‘Ca. P. rhamni’ differs from the corresponding region of ‘Ca. P. spartii’ in 16% of the nucleotide positions and in 14-17% of nucleotide positions of the spacer regions from the AP, PD, ESFY and PYLR phytoplasmas. RFLP analysis of the 16S rRNA gene of the BWB agent shows that this phytoplasma is indistinguishable from other members of the AP group when the gene sequence is digested with AluI and RsaI (Maürer and Seemüller, 1996). However, the BWB phytoplasma gene lacks SspI and SfcI restriction sites that are present in the 16S rRNA genes of other AP-group phytoplasmas. Like ‘Ca. P. prunorum’, ‘Ca. P. rhamni’ DNA has an additional BsaAI site at position 428. ‘Ca. P. rhamni’ has additional PvuII and AluI restriction sites that do not occur in the gene sequences of other AP-fruit tree agents. There are currently no BWB phytoplasma-specific primers. ‘Ca. Phytoplasma spartii’ Marcone, Gibb, Streten & Schneider (Marcone et al., 2003a). This phytoplasma was detected in Spartium junceum and Sarothamnus scoparius showing witches’-broom in Italy and Spain. It is associated with the 16S rRNA gene sequence accession number X92869, with oligonucleotide sequence complementary to unique region of the 16S rRNA 5’-TTATCCGCGTTAC-3’. Sequence analysis of the Spartium witches’-broom (SpaWB) agent shows this phytoplasma is more closely related to members of the AP group than to other phytoplasma subclades. The SpaWB phytoplasma shares 97.1-97.2% similarity with the AP, PD, ESFY and PYLR phytoplasmas, which are members of the AP group (Marcone et al., 1996a; Seemüller et al., 1998b). In the 16S-23S spacer region (approximately 210 bp), the SpaWB agent differs from the AP and PD phytoplasmas in 12% of the nucleotide positions. Whereas, the SpaWB phytoplasma differs from ESFY and PYLR phytoplasmas in 13 and 18% of nucleotide positions of the 16S23S spacer regions, respectively (Marcone et al., 1996a; Seemüller et al., 1998b). The SpaWB phytoplasma 16S rRNA gene contains a PuvII site at position 518 that is unique to members of the AP group. RFLP analysis shows that the SpaWB phytoplasma 16S rRNA gene has the same banding pattern as the AP phytoplasma when digested with RsaI, Sau3AI, HhaI and BsaAI. However,

258


the SpaWB and AP phytoplasmas give different banding patterns when digested with AluI and SspI (Marcone et al., 1996a, 1997b). The SpaWB phytoplasma can be distinguished from other phytoplasmas assigned to the AP group based on differences in 16S rRNA gene SspI and SfcI banding patterns. The 16S rRNA genes of SpaWB, PD, ESFY and PYLR phytoplasmas lack an SspI restriction site at nucleotide 419 that is present in the AP phytoplasma. The PD, ESFY and PYLR phytoplasmas have SfcI sites at positions 630 and 1,000, the SpaWB phytoplasma lacks the restriction site at position 1,000 and the AP phytoplasma lacks the SfcI site at position 630. Specific detection of the SpaWB phytoplasma is possible by using either the fU5 or P1 forward primers paired with the rSP reverse primer (5’-GCTAATTAGAAATATCAACTA-3’) (Marcone et al., 1996a). The phytoplasma associated with sarothamnus witches’-broom (SSWB) has the same banding pattern as the SpaWB phytoplasma when digested with AluI, RsaI, Sau3AI, MseI, TaqI, SspI and BsaI but differs from the SpaWB phytoplasma when digested with HhaI (Marcone et al., 1997b). A product can be amplified from the SSWB phytoplasma by using the SpaWB specific primer combinations (fU5/rSP or P1/rSP) (Marcone et al., 1997b). ‘Ca. Phytoplasma trifolii’ Hiruki & Wang (Hiruki and Wang, 2004). This phytoplasma was detected in alsike clover (Trifolium hybridum) plants exhibiting symptoms of virescence and proliferation of shoots (Hiruki and Chen, 1984). ‘Ca. P. trifolii’ is associated with the 16S rRNA gene sequence accession number AY390261, with oligonucleotide sequences complementary to unique regions of the 16S rRNA 5’-TTCTTACGA-3’ and 5’-TAGAGTAAAAGCC-3’. Phylogenetic analysis shows the reference strain, CPR, and its close relatives Brinjal little leaf (BLL), Fragaria multicipita phytoplasma (FM) and Illinois elm yellows (ILEY) phytoplasmas form a subcluster and were different from all other phytoplasmas, with sequence divergences of ≥2.5%. RFLP analysis of 16S rRNA genes from CPR, the Alfalfa witches’-broom (AWB), Beet leafhopper-transmitted virescence (BLTV), Potato witches’-broom (PWB), Tomato big bud from California (TBBc) and Potato yellows (PY) phytoplasmas showed these phytoplasmas shared very similar or identical banding patterns when digested with EcoRII, HaeIII, HhaI, HinfI, HpaII, KpnI, RsaI, Sau3AI and ThaI (Lee et al., 1993, 1998; Khadhair et al., 1997; Wang et al., 1998; Wang and Hiruki, 2001). The AWB, BLTV, CPR, PWB and TBBc phytoplasmas produced unique RFLP profiles with restriction endonucleases AluI and MseI (Lee et al., 1993, 1998; Khadhair et al., 1997; Wang et al., 1998, Wang and Hiruki, 2001). The banding patterns of the 16S rRNA genes of the Fragaria multicipita phytoplasma (FM) and Illinois elm yellows (ILEY) phytoplasmas were only differentiated from the CPR phytoplasma by a single restriction site (AluI for


FM and HhaI for ILEY) (Jomantiene et al., 1998; Jacobs et al., 2003). Based on RFLP banding profiles the phytoplasma strains named CPR, AWB, BLTV, PWB, PY, TBBc, FM, BLL, ILEY, ILEY1, ILEY2 are related to ‘Ca. P. trifolii’. According to Hiruki and Wang (2004), they were designated as different subgroups as follows: the AWB, BLTV, CPR, PWB, PY and TBBc phytoplasmas are assigned to CP subgroup A, the FM phytoplasma is the only member of CP subgroup B, and BLL, ILEY, ILEY1 and ILEY2 phytoplasmas are members of subgroup C. ‘Ca. P. trifolii’ and related strains are reported to occur in Canada, United States of America and India. ‘Ca. Phytoplasma ulmi’ Lee, Martini, Marcone & Zhu (Lee et al., 2004b). This phytoplasma is associated with gene sequence accessions AY197655, AY197675, AY197690, corresponding to 16S rRNA, rpl22-rps3 and secY genes. Oligonucleotide sequences of unique regions are 5’-GGAAA-3’ (positions 827-835) and 5’-CGTTAGTTGCC-3’ positions (1,098-1,108) in the 16S rRNA gene, 5’-TTACGCTTGCC-3’ (positions 284-294), 5’-CATTTAATAAAATTGCTATT-3’ (positions 739758) and 5’-AAATTCTATTTCTATGGGAAT-3’ (positions 910-932) in the rpl22-rps3 gene fragment and 5’TTTGATCCAATGTTAA-3’ (positions 350-365), 5’GTCTTTCGGTCATGGATTGA-3’ (positions 595-614), 5’-ATTTAGTCTAAT-3’ (positions 616-627), 5’-CAAA TAGAACAA-3’ (positions 1,053-1,064) in the secY gene. By phylogenetic analysis of 16S rDNA sequences, a distinct group of related phytoplasmas associated with several destructive diseases of elm, grapevine and several fruit trees was identified and named Elm Yellows strain cluster (Seemüller et al., 1994) or 16SrV (Lee et al., 1998, 2000). The phytoplasmas assigned to 16SrV/ Elm Yellows group include those associated with flavescence dorée (FD) in grapevine (Daire et al., 1993), Rubus stunt (RuS) in blackberries (Mäurer and Seemüller, 1994), cherry lethal yellows (CLY) in cherry (Zhu et al., 1997), jujube witches’-broom (JWB) in Ziziphus spp. (Zhu et al., 1997), alder yellows (ALY) in alder (Mäurer et al., 1993), spartium witches’-broom in Spartium sp. in Italy (SpaWB-EY, Marcone et al., 1996a), eucalyptus little leaf phytoplasma in Eucalyptus sp. in Italy (Marcone et al., 1996c), and the phytoplasmas detected in Apocynum cannabinum (Griffiths et al., 1999a), in Virginia creeper (Parthenocissus quinquefolia) (Harrison et al., 2001) and in tomato (Del Serrone et al., 2001). Although the members of the 16SrV/Elm Yellows group share relatively high similarities in their 16S rDNA sequence (98.6 to 99.9%), they constitute several strain clusters with different ecological niches and geographical distribution. Several ‘Ca. Phytoplasma’ species should therefore be described within the group, and the task has only partially been accomplished at the time of writing. The phytoplasma associated with the elm yellows disease was


Firrao et al.

259

named ‘Ca. P. ulmi’. The other strains belonging to 16SrV could be differentiated from ‘Ca. P. ulmi’ by restriction analysis of 16S rDNA (diagnostic enzymes: RsaI and BfaI), or the rpl22-rps3 ribosomal protein gene fragment (diagnostic enzymes: Tsp509I or MseI). Elm Yellows/16SrV group-specific primer pair were designed for both the 16S rRNA and the ribosomal protein gene fragment. Primers R16(V)R1/F1 (Lee et al., 1994) were used to amplify an 1,100 bp fragment of the 16S rDNA gene. For the ribosomal protein gene operon, Lee et al. (2004b) suggested a nested PCR using the ribosomal protein primer pair rp(V) F1/rpR1 specific to the EY phytoplasma group followed by another EY group-specific primer pair, rp(V)F1A/rp(V)R1A yielding a DNA fragment of about 1.2 kb. ‘Ca. P. ulmi’ is widespread in Europe, Asia and North America.

Ahrens U., Seemüller E., 1992. Detection of DNA of plant pathogenic mycoplasma-like organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82: 828-832.

‘Ca. Phytoplasma ziziphi’ Jung, Sawayanagi, Kakizawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Hibi & Namba (Jung et al., 2003a). This phytoplasma was detected in Zizyphus jujuba affected by Jujube witches’broom (JWB) disease in China, Japan and Korea. ‘Ca. P. ziziphi’ is associated with the 16S rRNA gene sequence accession AB052879, with oligonucleotide sequence complementary to unique regions of the 16S rRNA 5’TAAAAAGGCATCTTTTTGTT-3’ and 5’-AATCCGGACTAAGACTGT-3’. Sequence comparisons showed that the ‘Ca. P. ziziphi’ phytoplasma 16SrDNA sequences were most similar to those of the strains belonging to the Elm Yellows strain cluster or 16SrV group (see above ‘Ca. P. ulmi’). Digestion with either HpaII or RsaI distinguished ‘Ca. P. ziziphi’ phytoplasmas from all other members of the Elm Yellows group. A primer set (JWBF1: 5’TAAAAAGGCATCTTTTTGTT-3’, JWBR1: 5’-AATCCGGACTAAGACTGT-3’) was designed by Jung et al. (2003a) from the JWB phytoplasma 16S rDNA sequence to detect ‘Ca. P. ziziphi’ specifically. PCR using primers JWBF1 and JWBR1 amplified an 1,119 bp DNA fragment.

Barros T.S.L., Davis R.E., Resende R.O., Dally E.L., 2002. Erigeron witches’-broom phytoplasma in Brazil represents new subgroup VII-B in 16S rRNA gene group VII, the ash yellows phytoplasma group. Plant Disease 86: 1142-1148.

ACKNOWLEDGEMENTS

Work on phytoplasmas in the Firrao lab is supported by the “Ministero delle Politiche Agrarie e Forstali”, Italy. Gibb and Streten thank the Cooperative Research Centre Tropical Plant Protection and the Australian Research Council for financial support. The advice on restriction endonucleases by Dr. A. Bertaccini is gratefully acknowledged. REFERENCES Abou-Jawdah Y., Karakashian A., Sobh H., Martini M., Lee I.M., 2002. An epidemic of almond witches’-broom in Lebanon: classification and phylogenetic relationships of the associated phytoplasma. Plant Disease 86: 477-484.

Andersen M.T., Longmore J.L., Liefting L.W., Wood G.A., Sutherland P.W., Beck D.L., Forster R.L.S., 1998. Phormium yellow leaf phytoplasma is associated with strawberry lethal yellows disease in New Zealand. Plant Disease 82: 606-609. Anonymous, 2000. Phytoplasmas, spiroplasmas, entomoplasmas and mesoplasmas working team of the International Research Project for Comparative Mycoplasmology. Report of activity 1998-2000. http://mycoplasmas.vm.iastate.edu/ IOM/irpcm.html. Baric S., Dalla-Via J., 2004. A new approach to apple proliferation detection: a highly sensitive real-time PCR assay. Journal of Microbiological Methods 57: 135-145.

Blanche K.R., Tran-Nguyen L.T.T., Gibb K.S., 2003. Detection, identification and significance of phytoplasmas in grasses in northern Australia. Plant Pathology 52: 505-512. Bonnet F., Saillard C., Kollar A., Seemüller E., Bové J.M., 1990. Detection and differentiation of the mycoplasma-like organism associated with apple proliferation disease using cloned DNA probes. Molecular Plant Microbe Interactions 3: 438-443. Bové J.M., Danet J.L., Bananej K., Hassanzadeh N., Taghizadeh M., Salehi M., Garnier M., 1999. Witches’ broom disease of lime (WBDL) in Iran. In: J.V. da Gracia, R.F. Lee and R.K. Yokomi (eds). Proceedings of the 14th Conference of International Organization of Citrus Virologists (IOCV), Campinas 1998, 207-212. Bowyer J.W., Atherton J.G., Teakle D.S., Ahern G.A., 1969. Mycoplasma-like bodies in plants affected by legume little leaf, tomato big bud, and lucerne witches’ broom disease. Australian Journal of Biological Science 22: 271-274. Bruni R., Pellati F., Bellardi M.G., Benvenuti S., Paltrinieri S., Bertaccini A., Bianchi A., 2005. Herbal drug quality and phytochemical composition of Hypericum perforatum L. affected by ash yellows phytoplasma infection. Journal of Agricultural and Food Chemistry 53: 964-968. Chen T.C., Lee C.S., Chen M.J., 1972. Mycoplasma-like organisms in Cynodon dactylon and Brachiaria distachya affected by white leaf disease. Report of Taiwan Sugar Experiment Station 56: 49-55. Dafalla G.A., Cousin M.T., 1988. Fluorescence and electron microscopy of Cynodon dactylon affected with a white leaf disease in the Sudan. Journal of Phytopathology 122: 25-34. Daire X., Clair D., Larrue J., Boudon-Padieu E., Caudwell A., 1993. Diversity among mycoplasma-like organisms inducing grapevine yellows in France. Vitis 32: 159-163. Davis R.E., Dally E.L., Gundersen D.E., Lee I.-M., Habili N., 1997a. ‘Candidatus Phytoplasma australiense’, a new phytoplasma taxon associated with Australian grapevine yel-

260


lows. International Journal of Systematic Bacteriology 47: 262-269. Davis R.I., Schneider B., Gibb K.S., 1997b. Detection and differentiation of phytoplasmas in Australia. Australian Journal of Agricultural Research 48: 535-544. Del Serrone P., La Starza S., Krystai L., Kolber M., Barba M., 1998. Occurrence of apple proliferation and pear decline phytoplasmas in diseased pear trees in Hungary. Journal of Plant Pathology 80: 53-58. Del Serrone P., Marzachì C., Bragaloni M., Galeffi P., 2001. Phytoplasma infection of tomato in central Italy. Phytopathologia Mediterranea 40: 137-142. Doi Y.M., Teranaka M., Yora K., Asuyama H., 1967. Mycoplasma or PTL-group-like microorganisms found in the phloem elements of plants infected with mulberry dwarf, potato witches’-broom, aster yellows, or paulownia witches’-broom. Annals of the Phytopathological Society of Japan 33: 259-266. Firrao G., Gobbi E., Locci R., 1993. Use of polymerase chain reaction to produce oligonucleotide probes for mycoplasma-like organisms. Phytopathology 83: 602-607. Firrao G., Gobbi E., Locci R., 1994. Rapid diagnosis of apple proliferation mycoplasma-like organism using a polymerase chain reaction procedure. Plant Pathology 43: 669674. Gibb K.S., Padovan A.C., Mogen B.D., 1995. Studies on sweet potato little-leaf phytoplasma detected in sweet potato and other plant species growing in Northern Australia. Phytopathology 85: 169-174. Gibb K.S., Persley D.M., Schneider B., Thomas J.E., 1996. Phytoplasmas associated with papaya diseases in Australia. Plant Disease 80: 174-178. Gibb K.S., Tran-Nguyen L.T.T., Randles J.W., 2003. A new phytoplasma detected in the South Australian native perennial shrub, Allocasuarina muelleriana. Annals of Applied Biology 142: 357-364. Griffiths H.M., Sinclair W.A., Boudon-Padieu E., Daire X., Lee I.-M., Sfalanga A., Bertaccini A., 1999a. Phytoplasmas associated with elm yellows: molecular variability and differentiation from related organisms. Plant Disease 83: 1101-1104. Griffiths H.M., Sinclair W.A., Smart C.D., Davis R.E., 1999b. The phytoplasma associated with ash yellows and lilac witches’-broom: ‘Candidatus Phytoplasma fraxini’. International Journal of Systematic Bacteriology 49: 1605-1614. Griffiths H.M., Boa E.R., Filgueira J.J., 2001. Ash yellows: a new disease of Fraxinus chinensis in Columbia. Phytopathology 91: S32-33. Gundersen D.E., Lee I.-M., Rehner S.A., Davis R.E., Kingsbury D.T., 1994. Phylogeny of mycoplasma-like organisms (phytoplasmas): a basis for their classification. Journal of Bacteriology 176: 5244-5254. Harrison N.A., Griffiths H.M., Carpio M.L., Richardson P.A., 2001. Detection and characterization of an elm yellows (16SrV) group phytoplasma infecting Virginia creeper plants in southern Florida. Plant Disease 85: 1055-1062.

Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263 Hiruki C., Chen M.H., 1984. Plant mycoplasma diseases occurring in Alberta. In: Proceedings of the 7th IUFRO Mycoplasma Conference, Edmonton, Alberta, 1984, 7. Hiruki C., Wang K.R., 2004. Clover proliferation phytoplasma: ‘Candidatus Phytoplasma trifolii’. International Journal of Systematic and Evolutionary Microbiology 54: 1349-1353. International Committee on Systematic Bacteriology - Subcommittee on the Taxonomy of Mollicutes, 1979. Proposals of minimum standards for descriptions of new species of the class Mollicutes. International Journal of Systematic Bacteriology 29: 172-180. International Committee on Systematic Bacteriology - Subcommittee on the Taxonomy of Mollicutes, 1993. Minutes of the interim meeting, 1-2 August 1992, Ames, Iowa. International Journal of Systematic Bacteriology 43: 394-397. International Committee on Systematic Bacteriology - Subcommittee on the taxonomy of Mollicutes, 1995. Minutes of the interim meeting, 17 and 26 July 1994, Bordeaux, France. International Journal of Systematic Bacteriology 45: 415-417. International Committee on Systematic Bacteriology - Subcommittee on the Taxonomy of Mollicutes, 2001. Minutes of the interim meeting, July 2000, Fukuoka, Japan. International Journal of Systematic and Evolutionary Microbiology 51: 2227-2230. IRPCM Phytoplasma/Spiroplasma Working Team - Phytoplasma taxonomy group, 2004. ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. International Journal of Systematic and Evolutionary Microbiology 54: 1243-1255. Jacobs K.A., Lee I.M., Griffiths H.M., Miller F.D., Bottner K.D., 2003. A new member of the clover proliferation phytoplasma group (16SrVI) associated with elm yellows in Illinois. Plant Disease 87: 241-246. Jarausch W., Saillard C., Dosba F., Bové J.M., 1994. Differentiation of mycoplasma-like organisms (MLOs) in European fruit trees by PCR using specific primers derived from sequence of a chromosomal fragment of the apple proliferation MLO. Appied and Enviromental Microbiology 60: 29162923. Jarausch W., Jarausch-Wehrheim B., Danet J.L., Broquaire J.M., Dosba F., Saillard C., Garnier M., 2001. Detection and identification of European stone fruit yellows and other phytoplasmas in wild plants in the surroundings of apricot chlorotic leaf roll-affected orchards in southern France. European Journal of Plant Pathology 107: 209-217. Jarausch W., Saillard C., Broquaire J., Garnier M., Dosba F., 2000a. PCR-RFLP and sequence analysis of a non-ribosomal fragment for genetic characterization of European stone fruit yellows phytoplasmas infecting various Prunus species. Molecular and Cellular Probes 14: 171-179. Jarausch W., Saillard C., Helliot B., Garnier M., Dosba F., 2000b. Genetic variability of apple proliferation phytoplasmas as determined by PCR-RFLP and sequencing of a nonribosomal fragment. Molecular and Cellular Probes 14: 17-24. Jomantiene R., Davis R.E., Dally E.L., Maas J.L., 1998. The distinctive morphology of ‘Fragaria multicipita’ is due to phytoplasma. Hort Science 33: 1069-1072.

Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263 Jomantiene R.R., Maas J.L., Takeda F., Davis R.E., 2002. Molecular identification and classification of strawberry phylloid fruit phytoplasma in group 16SrI, new subgroup. Plant Disease 86: 920. Jung H.Y., Sawayanagi T., Kakizawa S., Nishigawa H., Miyata S.I., Oshima K., Ugaki M., Lee J.T., Hibi T., Namba S., 2002. ‘Candidatus Phytoplasma castaneae’, a novel phytoplasma taxon associated with chestnut witches’ broom disease. International Journal of Systematic and Evolutionary Microbiology 52: 1543-1549. Jung H.Y., Sawayanagi T., Kakizawa S., Nishigawa H., Wei W., Oshima K., Miyata S., Ugaki M., Hibi T., Namba S., 2003a. ‘Candidatus Phytoplasma ziziphi’, a novel phytoplasma taxon associated with jujube witches’-broom disease. International Journal of Systematic and Evolutionary Microbiology 53: 1037-1041. Jung H.Y., Sawayanagi T., Wongkaew P., Kakizawa S., Nishigawa H., Wei W., Oshima K., Miyata S., Ugaki M., Hibi T., Namba S., 2003b. ‘Candidatus Phytoplasma oryzae’, a novel phytoplasma taxon associated with rice yellow dwarf disease. International Journal of Systematic and Evolutionary Microbiology 53: 1925-1929. Kartte S., Seemüller E., 1991. Susceptibility of grafted Malus taxa and hybrids to apple proliferation disease. Journal of Phytopathology 131: 137-148. Khadhair A.H., Hiruki C., Hwang S.F., 1997. Molecular detection of alfalfa witches’-broom phytoplasma in four leafhopper species associated with infected alfalfa plants. Microbiological Research 152: 269-275. Kirkpatrick B.C., 1991. Mycoplasma-like organisms: plant and invertebrate pathogens In: Balows A, Trüper H.G., Dworkin M., Harder W., Schliefer K.H. (eds.). The Prokaryotes, pp. 4050-4067. Springer-Verlag Press, New York, USA. Kollar A., Seemüller, E., 1989. Base composition of the DNA of mycoplasma-like organisms associated with various plant diseases. Journal of Phytopathology 127: 177-186. Kuske C.R., Kirkpatrick B.C., 1992. Phylogenetic relationships between the western aster yellows mycoplasmalike organisms and other prokaryotes established by 16S rRNA gene sequence. International Journal of Systematic Bacteriology 42: 226-33. Lapage S.P., Sneath P.H.A., Lessel E.F., Skerman V.B.D., Seeliger H.P.R., Clarck W.A., 1992. International code of nomenclature of bacteria: bacteriological code. 1990 Revision. American Society of Microbiology, Washington, D.C., USA. Lee I.M., Hammond R.W., Davis R.E., Gundersen-Rindal D.E., 1993. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasma-like organisms. Phytopathology 83: 834-842. Lee I.M., Gundersen-Rindal D.E., Hammond R.W., Davis R.E., 1994. Use of mycoplasma-like organism (MLO) group-specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant. Phytopathology 84: 559-566. Lee I.M., Bertaccini A., Vibio M., Gundersen-Rindal D.E., 1995. Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy. Phytopathology 85: 728-735.

Firrao et al.

261

Lee I.M., Pastore M., Vibio M., Danielli A., Attathom S., Davis R.E., Bertaccini A., 1997. Detection and characterization of phytoplasma associated with annual blue grass (Poa annua) white leaf in southern Italy. European Journal of Plant Pathology 103: 251-254. Lee I.M., Gundersen-Rindal D.E., Davis R.E., Bartoszyk I.M., 1998. Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic Bacteriology 48: 1153-1169. Lee I.M., Davis R.E., Gundersen-Rindal D.E., 2000. Phytoplasma: Phytopathogenic mollicutes. Annual Review of Microbiology 54: 221-255. Lee M.E., Grau C.R., Lukaesko L.A., Lee I.M., 2002. Identification of aster yellows phytoplasmas in soybean in Wisconsin based on RFLP analysis of PCR-amplified products (16S rDNAs). Canadian Journal of Plant Pathology 24: 125-130. Lee I.M., Gundersen-Rindal D.E., Davis R.E., Bottner K.D., Marcone C., Seemüller E., 2004a. ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxon associated with aster yellows and related diseases. International Journal of Systematic and Evolutionary Microbiology 54: 1037-1048. Lee I.M., Martini M., Marcone C., Zhu S.F., 2004b. Classification of phytoplasma strains in the elm yellows group (16SrV) and proposal of ‘Candidatus Phytoplasma ulmi’ for the phytoplasma associated with elm yellows. International Journal of Systematic and Evolutionary Microbiology 54: 337-347. Liefting L.W., Padovan A.C., Gibb K.S., Beever R.E., Andersen M.T., Newcomb R.D., Beck D.L., Forster R.L.S., 1998. ‘Candidatus Phytoplasma australiense’ is the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases. European Journal of Plant Pathology 104: 619-623. Lorenz K.H., Dosba F., Poggi Pollini C., Llacer G., Seemüller E., 1994. Phytoplasma diseases of Prunus species in Europe are caused by genetically similar organisms. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 101: 567-575. Lorenz K.H., Schneider B., Ahrens U., Seemüller E., 1995. Detection of the apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and nonribosomal DNA. Phytopathology 85: 771-776. Marcone C., Ragozzino A., Schneider B., Lauer U., Smart C.D., Seemüller E., 1996a. Genetic characterization and classification of two phytoplasmas associated with spartium witches’-broom disease. Plant Disease 80: 365-371. Marcone C., Ragozzino A., Seemüller E., 1996b. Association of phytoplasmas with the decline of European hazel in southern Italy. Plant Pathology 45: 857-863. Marcone C., Ragozzino A., Seemüller E., 1996c. Detection of an elm yellows-related phytoplasma in eucalyptus trees affected by little-leaf disease in Italy. Plant Disease 80: 669673. Marcone C., Ragozzino A., Seemüller E., 1997a. Detection of Bermuda grass white leaf disease in Italy and genetic characterization of the associated phytoplasma by RFLP analysis. Plant Disease 81: 862-866.

262


Marcone C., Ragozzino A., Seemüller E., 1997b. Witches’ broom of Sarothamnus scoparius: a new disease associated with a phytoplasma related to the spartium witches’ broom agent. Journal of Phytopathology 145: 159-161. Marcone C., Neimark A., Ragozzino A., Lauer U., Seemüller E., 1999. Chromosome sizes of phytoplasmas composing major phylogenetic groups and subgroups. Phytopathology 89: 805-810. Marcone C., Lee I.M., Davis R.E., Ragozzino A., Seemüller E., 2000. Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences. International Journal of Systematic and Evolutionary Microbiology 50: 1703-1713. Marcone C., Gibb K.S., Streten C., Schneider B., 2003a. ‘Candidatus Phytoplasma spartii’, ‘Candidatus Phytoplasma rhamni’ and ‘Candidatus Phytoplasma allocasuarinae’, respectively associated with spartium witches’-broom, buckthorn witches’-broom and allocasuarina yellows diseases. International Journal of Systematic and Evolutionary Microbiology 54: 1025-1029. Marcone C., Schneider B., Seemüller E., 2003b. ‘Candidatus Phytoplasma cynodontis’, the phytoplasma associated with Bermuda grass white leaf disease. International Journal of Systematic and Evolutionary Microbiology 54: 1077-1082. Mäurer R., Seemüller E., 1994. Nature and genetic relatedness of the mycoplasma-like organism causing rubus stunt in Europe. Plant Pathology 44: 244-249. Mäurer R., Seemüller E., 1996. Witches’ broom of Rhamnus catharticus: a new phytoplasma disease. Journal of Phytopathology 144: 221-223. Mäurer R., Seemüller E., Sinclair W.A., 1993. Genetic relatedness of mycoplasma-like organisms affecting elm, alder, and ash in Europe and North America. Phytopathology 83: 971-976. Meneguzzi N., Torres L., Galdeano E., Nome C., Docampo D., Nome S., Conci L., 2001. Phylogenetic analysis of an ash yellows group phytoplasma detected in alfalfa (Medicago sativa L.), in Argentina. Fitopatologia Brasileira 26: 510. Montano H.G., Davis R.E., Dally E.L., Hogenhout S., Pimentel J.P., Brioso P.S.T., 2001. ‘Candidatus Phytoplasma brasiliense’, a new phytoplasma taxon associated with hibiscus witches’ broom disease. International Journal of Systematic and Evolutionary Microbiology 51: 1109-1118. Murray R.G.E., Schleifer K.H., 1994. Taxonomic notes: a proposal for recording the properties of putative taxa of prokaryotes. International Journal of Systematic Bacteriology 44: 174-176. Murray R.G.E., Stackebrandt E., 1995 Implementation of the provisional status Candidatus for incompletely described prokaryotes. International Journal of Systematic Bacteriology 45: 185-186. Nakashima K., Kato S., Iwanami S., Murata N. 1993. DNA probes reveal relatedness of rice yellowdwarf mycoplasmalike organisms (MLOs) and distinguish them from other MLOs. Applied and Environmental Microbiology 59: 12061212. Nakashima K., Hayashi T., Chaleeprom W., Wongkaew P., Sirithorn P., 1996. Complex phytoplasma flora in northeast

Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263 Thailand as revealed by 16S rDNA analysis. Annals of the Phytopathological Society of Japan 62: 57-60. Namba S., Kato S., Iwanami S., Oyaizu H., Shiozawa H., Tsuchizaki T., 1993. Detection and differentiation of plantpathogenic mycoplasma-like organisms using polymerase chain reaction. Phytopathology 83: 786-791. Padovan A.C., Gibb K.S., Bertaccini A., Bonfiglioli R.E., Magnet P.A., Sears B.B. 1995. Molecular detection of the Australian grapevine yellows phytoplasma and comparison with grapevine yellows phytoplasmas from Italy. Australian Journal of Grape and Wine Research 1: 25-31. Padovan A.C., Gibb K.S., Daire X., Boudon-Padieu E., 1996. A comparison of the phytoplasma associated with Australian grapevine yellows to other phytoplasmas in grapevine. Vitis 35: 189-194. Padovan A., Gibb K.S., Persley D., 2000. Association of ‘Candidatus phytoplasma australiense’ with green petal and lethal yellows diseases in strawberry. Plant Pathology 49: 362-369. Sarindu N., Clark M.F., 1993. Antibody production and identity of MLOs associated with sugar-cane whiteleaf disease and bermuda-grass whiteleaf disease from Thailand. Plant Pathology 42: 396-402. Sawayanagi T., Horikoshi N., Kanehira T., Shinohara M., Bertaccini A., Cousin M.T., Hiruki C., Namba S., 1999. ‘Candidatus Phytoplasma japonicum’, a new phytoplasma taxon associated with Japanese Hydrangea phyllody. International Journal of Systematic Bacteriology 49: 1275-1285. Schneider B., Ahrens U., Kirkpatrick B.C., Seemüller E., 1993. Classification of plant pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-amplified 16S rDNA. Journal of General Microbiology 139: 519-527. Schneider B., Cousin M.T., Klinkong S., Seemüller E., 1995. Taxonomic relatedness and phylogenetic positions of phytoplasmas associated with diseases of faba bean, sunhemp, sesame, soybean, and eggplant. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 102: 225-232. Schneider B., Marcone C., Kampmann M., Ragozzino A., Lederer W., Cousin M.T., Seemüller E., 1997. Characterization and classification of phytoplasmas from wild and cultivated plants by RFLP and sequence analysis of ribosomal DNA. European Journal of Plant Pathology 193: 675-686. Schneider B., Gibb K.S., Padovan A., Davis R.I., de la Rue S., 1999. Comparison and characterization of tomato big budand sweet potato little leaf-group phytoplasmas. Journal of Phytopathology 147: 31-40. Schneider B., Torres E., Martìn M.P., Schroder M., Behnke H.D., Seemüller E., 2005. ‘Candidatus Phytoplasma pini’, a novel taxon from Pinus silvestris and Pinus halepensis. International Journal of Systematic and Evolutionary Microbiology 55: 303-307. Sdoodee R., Schneider B., Padovan A., Gibb, K.S., 1999. Detection and genetic relatedness of phytoplasmas associated with plant diseases in Thailand. Journal of Biochemistry and Molecular Biology and Biophysics 3: 133-140. Sears B.B., Kirkpatrick B.C., 1994. Unveiling the evolutionary relationships of plant pathogenic mycoplasma-like organisms. ASM News 60: 307-312.

Journal of Plant Pathology (2005), 87 (4, Special issue), 249-263 Seemüller E., Schneider B., 2004. Taxonomic description of ‘Candidatus Phytoplasma mali’ sp. nov., ‘Candidatus Phytoplasma pyri’ sp. nov. and ‘Candidatus Phytoplasma prunorum’ sp. nov., the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively. International Journal of Systematic and Evolutionary Microbiology 54: 1217-1226. Seemüller E., Schneider B., Mäurer R., Ahrens U., Daire X., Kison H., Lorenz K.H., Firrao G., Avinent L., Sears B.B., Stackebrandt E., 1994. Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA. International Journal of Systematic Bacteriology 44: 440-446.

Firrao et al.

263

Viswanathan R., 1997. Detection of phytoplasmas associated with grassy shoot disease of sugarcane by ELISA techniques. Zeitschrift für Pflanzekrankheit und Pflanzenschutz 104: 9-16. Viswanathan R., 2001. Serodiagnosis of phytoplasmas causing grassy shoot disease in sugarcane. In: Rao G.P., Ford R.E., Tosi¢ M., Teakle D.S. (eds). Sugarcane pathology. Vol. 2. Virus and phytoplasma diseases, pp. 209-220. Science Publishers, Enfield, USA. Wang K., Hiruki C., 2001. Use of heteroduplex mobility assay for identification and differentiation of phytoplasmas in the aster yellows group and the clover proliferation group. Phytopathology 91: 546-552.

Seemüller E., Lorenz K.H., Lauer U., 1998a. Pear decline resistance in Pyrus communis rootstocks and progenies of wild and ornamental Pyrus taxa. Acta Horticulturae 472: 681-690.

Wang K., Hiruki C., Chen M.H., 1998. Identification of a phytoplasma causing yellows of Monarda. Plant Pathology 47: 103-106.

Seemüller E., Marcone C., Lauer U., Ragozzino A., Göschl M., 1998b. Current status of molecular classification of the Phytoplasmas. Journal of Plant Pathology 80: 3-26.

White D.T., Blackall L.L., Scott P.T., Walsh K.B., 1998. Phylogenetic positions of phytoplasmas associated with dieback, yellow crinkle and mosaic diseases of papaya, and their proposed inclusion in ‘Candidatus Phytoplasma australiense’ and a new taxon, ‘Candidatus Phytoplasma australasia’. International Journal of Systematic Bacteriology 48: 941-951.

Smart C.D., Schneider B., Blomquist C.L., Guerra L.J., Harrison N.A., Ahrens U., Lorenz K.H., Seemüller E., Kirkpatrick B., 1996. Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer region. Applied and Environmental Microbiology 62: 2988-2993. Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Higgins D.G., 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24: 48764882. Tran-Nguyen L., Blanche K.R., Egan B., Gibb K.S., 2000. Diversity of phytoplasmas in northern Australian sugarcane and other grasses. Plant Pathology 49: 666-679. Verdin E., Salar P., Danet J.L., Choueiri E., Jreijiri F., El-Zammar S., Gelie B., Bove J.M., Garnier M., 2003. ‘Candidatus Phytoplasma phoenicium’ sp. nov., a novel phytoplasma associated with an emerging lethal disease of almond trees in Lebanon and Iran. International Journal of Systematic and Evolutionary Microbiology 53: 833-838. Vibio M., Bertaccini A., Lee I.M., Davis R.E., Clark M.F., 1996. Differentiation and classification of aster yellows and related European phytoplasmas. Phytopathologia Mediterranea 35: 33-42.

Received 20 September 2005 Accepted 3 November 2005

Wongkaew P., Hanboonsong Y., Sirithorn P., Choosai C., Boonkrong S., 1997. Differentiation of phytoplasmas associated with sugarcane and gramineous weed white leaf disease and sugarcane grassy shoot disease by RFLP and sequencing. Theoretical and Applied Genetics 95: 660-663. Zahoor A., Bashir M., Nakashima K., Mitsueda T., Murata N., 1995. Bermuda grass white leaf caused by phytoplasmas in Pakistan. Pakistan Journal of Botany 27: 251-252. Zhu S.F., Hadidi A., Gundersen D.E., Lee I.-M., Zhang C.L., 1997. Characterization of the phytoplasmas associated with cherry lethal yellows and jujube witches’-broom diseases in China. Acta Horticulturae 472: 701-714. Zreik L., Carle P., Bové J.M., Garnier M., 1995. Characterization of the mycoplasma-like organism associated with witches’-broom disease of lime and proposition of a Candidatus taxon for the organism, ‘Candidatus Phytoplasma aurantifolia’. International Journal Systematic Bacteriology 45: 449-453.