Genomic Diversity and Differentiation among Phytoplasma Strains in 16S rRNA Groups I (Aster Yellows and Related Phytoplasmas) and III (X-Disease and Related Phytoplasmas)

Abstract

Conserved gene sequences, including 16s rRNA and ribosomal protein gene sequences, were used to evaluate genetic variations in phytoplasma strains belonging to 16s rRNA groups I (aster yellows and related phytoplasmas) and I11 (X-disease and related phytoplasmas). We used PCR to amplifjr the sequences of the 16s ribosomal DNA and a segment of the ribosomal protein gene operon (encoding the 3' region of rpsl9, all of rp122, and rps3) from diverse phytoplasma group I and I11 strains. Additional chromosomal gene sequences of group I strains were also amplified. The PCR products amplified from members of each group of phytoplasmas were compared by performing restriction fragment length polymorphism (RFLP) analyses. On the basis of the RFLP patterns observed and similarity coefficients derived from combined RFLP analyses, the phytoplasma strains belonging to groups I and I11 were placed in distinct 16s rRNA, ribosomal protein, and 16s rRNA-ribosomal protein subgroups. Analyses of two or more conserved gene sequences revealed that members of the two groups were more diverse than previously thought. Subgroup differentiation on the basis of our combined analyses of 16s rRNA and ribosomal protein gene sequences seemed to adequately reflect the levels of chromosomal homology determined by DNA-DNA hybridization assays. On the basis of unique RFLP profiles, we identified new, previously unclassified group I phytoplasma strains, including the organisms that are associated with Ipomoea obscura witches'-broom [subgroup 16SrI-F(rr-rp)], maize bushy stunt [subgroup 16SrI-I(rr-rp)], and Mexican periwinkle virescence [subgroup 16SrI-J(rr-rp)], and new, previously unclassified group I11 phytoplasma strains, including the organism that is associated with pecan bunch [subgroup 16SrIII-H(rr-rp)]. On the basis of the results of our analyses of 16s rRNA and ribosomal protein conserved gene sequences, we recognized 9 group I subgroups and eight group I11 subgroups. We propose that phytoplasma strains belonging to each group I and I11 subgroup should be distinguished taxonomically at a level equivalent to the subspecies level. Phytoplasmas (26), which previously have been called mycoplasmalike organisms, are unculturable prokaryotes that are associated with diseases in a wide variety of economically important crop plants (44). In nature, phytoplasmas are carried by different homopterous insect vectors (both monophagous and oligophagous) and are transmitted to different host plants (7-10, 31, 44). Traditionally, phytoplasma-induced diseases have been named and the identities of the associated phytoplasmas have been determined on the basis of the plant hosts and the symptoms induced in the plant hosts. In general, it has been assumed that each disease is caused by a single distinct phytoplasma. These biological properties and vector specificity have been used by workers in some laboratories to identify major phytoplasma groups (10). This system is complicated and laborious and often has resulted in misleading conclusions because the phytopl...

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'

“…Although phytoplasmas have been detected in affected plant tissues and insects with the use of technologies based on the transmission electron microscope, antibodies and nucleic acids (Lee & Davis, 1992), they are currently unable to be cultured in vitro. The association of phytoplasmas with dieback and mosaic in papaya has been based solely on PCR amplification of the 16s rRNA gene and adjacent regions using phytoplasmaspecific primers (Davis et al, 1996;Gibb et al, 1996;Liu et al, 1996). The phytoplasmal origin of PCR amplimers from dieback-, mosaic-and yellow-crinkleaffected papaya has been confirmed by restriction endonuclease and DNA sequence analyses White et al, 1997).…”

“…Based on results of PCR detection, phytoplasmas have also been implicated as the aetiologic agents of papaya dieback and papaya mosaic (Davis et al, 1996;Liu et al, 1996). Phytoplasmas are cell-wall-less bacteria belonging to the class Mollicutes and are the proposed causative agents of diseases in several hundred plant species (McCoy et al, 1989).…”

“…To date, the most reliable classifications of phytoplasmas have been based on DNA restriction analysis and phylogenetic sequence analysis, usually of the PCR-amplified 16s rRNA gene plus 16s-23s rRNA intergenic spacer region (R. E. ; R. I. Gundersen et al, 1994Gundersen et al, , 1996Lee et al, 1993;Namba et al, 1993;Schneider et al, 1993Schneider et al, , 1995aSeemuller et al, 1994). These analyses have allowed the provisional classification of phytoplasmas from Europe, North America, Asia and Australia.…”

The phytoplasma associated with ash yellows and lilac witches'-broom: ‘Candidatus Phytoplasma fraxini’

“…Some of the variability is associated with differing plant hosts or geographic origins of the strains. For example, phytoplasmas related to those associated with X-disease of Prunus (group 16SrIII of Lee et al, 1998) but differing from the reference strains CX and WX have been detected in various plants and assigned to eight sub-groups that are differentiated by RFLPs in 16s rRNA and ribosomal protein genes (Gundersen et al, 1996;Lee et al, 1998). These assignments imply that minor differences in sequence homology in the highly conserved 16s rRNA genes are associated with biologically important differences in other parts of the genome.…”

Polymerase Chain Reaction for Phytoplasmas Detection

“…The use of nested-PCR has been reported for diagnostic purposes particularly in plants when phytoplasmas occur in low titer in the phloem vessels of their host-plants and their concentration may be subjected to seasonal fluctuation (57,75,100,117).…”