The recent sequencing of the entire genomes of Mycoplasma genitalium and M. pneumoniae has attracted considerable attention to the molecular biology of mycoplasmas, the smallest self-replicating organisms. It appears that we are now much closer to the goal of defining, in molecular terms, the entire machinery of a self-replicating cell. Comparative genomics based on comparison of the genomic makeup of mycoplasmal genomes with those of other bacteria, has opened new ways of looking at the evolutionary history of the mycoplasmas. There is now solid genetic support for the hypothesis that mycoplasmas have evolved as a branch of gram-positive bacteria by a process of reductive evolution. During this process, the mycoplasmas lost considerable portions of their ancestors’ chromosomes but retained the genes essential for life. Thus, the mycoplasmal genomes carry a high percentage of conserved genes, greatly facilitating gene annotation. The significant genome compaction that occurred in mycoplasmas was made possible by adopting a parasitic mode of life. The supply of nutrients from their hosts apparently enabled mycoplasmas to lose, during evolution, the genes for many assimilative processes. During their evolution and adaptation to a parasitic mode of life, the mycoplasmas have developed various genetic systems providing a highly plastic set of variable surface proteins to evade the host immune system. The uniqueness of the mycoplasmal systems is manifested by the presence of highly mutable modules combined with an ability to expand the antigenic repertoire by generating structural alternatives, all compressed into limited genomic sequences. In the absence of a cell wall and a periplasmic space, the majority of surface variable antigens in mycoplasmas are lipoproteins. Apart from providing specific antimycoplasmal defense, the host immune system is also involved in the development of pathogenic lesions and exacerbation of mycoplasma induced diseases. Mycoplasmas are able to stimulate as well as suppress lymphocytes in a nonspecific, polyclonal manner, both in vitro and in vivo. As well as to affecting various subsets of lymphocytes, mycoplasmas and mycoplasma-derived cell components modulate the activities of monocytes/macrophages and NK cells and trigger the production of a wide variety of up-regulating and down-regulating cytokines and chemokines. Mycoplasma-mediated secretion of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6, by macrophages and of up-regulating cytokines by mitogenically stimulated lymphocytes plays a major role in mycoplasma-induced immune system modulation and inflammatory responses.

Molecular Biology and Pathogenicity of Mycoplasmas

RFLP analyses of 16S rDNA nested PCR products from 34 phytoplasma strains with 17 restriction enzymes delineated distinct pattern types. Based on similarity coefficients derived from RFLP analyses, the 34 representative phytoplasma strains were differentiated into 14 major groups (termed 16Sr groups) and 32 sub-groups. The similarity coefficients of RFLP patterns between distinct groups were 90% or below. By including additional groups and sub-groups from which RFLP analyses were not performed but for which 16S rDNA sequence data were available to predict restriction sites, a total of 14 groups and 41 sub-groups were proposed. By combined RFLP analyses of 16S rRNA and ribosomal protein gene sequences, thus far, a total of 46 subgroups have been recognized. The phytoplasma 16Sr groups were consistent with the phylogenetic groups (subclades) defined by phylogenetic analysis of near-full-length 16S rRNA gene sequences, indicating that the RFLP-based groups are phylogenetically valid. The approach using RFLP analyses of PCR-amplified 16S rDNA (and ribosomal protein gene sequences) provides a simple, reliable and rapid means for differentiation and classification of unknown phytoplasmas.

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Revised Classification Scheme of Phytoplasmas based on RFLP Analyses of 16S rRNA and Ribosomal Protein Gene Sequences

▪ Abstract During the past decade, research has yielded new knowledge about the plant and insect host ranges, geographical distribution, and phylogenetic relationships of phytoplasmas, and a taxonomic system has emerged in which distinct phytoplasmas are named as separate “Candidatus phytoplasma species.” In large part, this progress has resulted from the development and use of molecular methods to detect, identify, and classify phytoplasmas. While these advances continue, research has recently begun on the phytoplasma genome, how phytoplasmas cause disease, the role of mixed phytoplasmal infections in plant diseases, and molecular/genetic phenomena that underlie symptom development in plants. These and other recent advances are laying the foundation for future progress in understanding the mechanisms of phytoplasma pathogenicity, organization of the phytoplasma genome, evolution of new phytoplasma strains and emergence of new diseases, bases of insect transmissibility and specificity of transmission, and...

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Phytoplasma: phytopathogenic mollicutes.

Plant diseases caused by, or associated with, phytoplasmas occur in hundreds of commercial and native plants, causing minor to extensive damage. Insect vectors, primarily leafhoppers, planthoppers, and psyllids, have been identified for relatively few phytoplasma diseases, limiting the capacity of managers to make informed decisions to protect crops and endangered indigenous plants. In the past two decades our knowledge of insect vector-phytoplasma interactions has increased dramatically, allowing researchers to make more accurate predictions about the nature and epidemiology of phytoplasma diseases. These better-characterized systems also may provide clues to the identity of insect vectors of other phytoplasma-associated diseases. We review the literature addressing the ecology of insect vectors, phytoplasma-insect ecological and molecular interactions, vector movement and dispersal, and possible management strategies with an emphasis on research from the past 20 years.

Insect vectors of phytoplasmas

The trivial name ‘phytoplasma’ has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were formerly known as mycoplasma-like organisms. Although phytoplasmas have not yet been cultivated in vitro, phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes. It is proposed here to accommodate phytoplasmas within the novel genus ‘Candidatus (Ca.) Phytoplasma’. Given the diversity within ‘Ca. Phytoplasma’, several subtaxa are needed to accommodate organisms that share <97?5 % similarity among their 16S rRNA gene sequences. This report describes the properties of ‘Ca. Phytoplasma’, a taxon that includes the species ‘Ca. Phytoplasma aurantifolia’ (the prokaryote associated with witches’-broom disease of small-fruited acid lime), ‘Ca. Phytoplasma australiense’ (associated with Australian grapevine yellows), ‘Ca. Phytoplasma fraxini’ (associated with ash yellows), ‘Ca. Phytoplasma japonicum’ (associated with Japanese hydrangea phyllody), ‘Ca. Phytoplasma brasiliense’ (associated with hibiscus witches’-broom in Brazil), ‘Ca. Phytoplasma castaneae’ (associated with chestnut witches’-broom in Korea), ‘Ca. Phytoplasma asteris’ (associated with aster yellows), ‘Ca. Phytoplasma mali’ (associated with apple proliferation), ‘Ca. Phytoplasma phoenicium’ (associated with almond lethal disease), ‘Ca. Phytoplasma trifolii’ (associated with clover proliferation), ‘Ca. Phytoplasma cynodontis’ (associated with Bermuda grass white leaf), ‘Ca. Phytoplasma ziziphi’ (associated with jujube witches’-broom), ‘Ca. Phytoplasma oryzae’ (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of

'Candidatus Phytoplasma', a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects

In order to develop a diagnostic tool to identify phytoplasmas and classify them according to their phylogenetic group, we took advantage of the sequence diversity of the 16S-23S intergenic spacer regions (SRs) of phytoplasmas. Ten PCR primers were developed from the SR sequences and were shown to amplify in a group-specific fashion. For some groups of phytoplasmas, such as elm yellows, ash yellows, and pear decline, the SR primer was paired with a specific primer from within the 16S rRNA gene. Each of these primer pairs was specific for a specific phytoplasma group, and they did not produce PCR products of the correct size from any other phytoplasma group. One primer was designed to anneal within the conserved tRNA(Ile) and, when paired with a universal primer, amplified all phytoplasmas tested. None of the primers produced PCR amplification products of the correct size from healthy plant DNA. These primers can serve as effective tools for identifying particular phytoplasmas in field samples.

Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer region.

Ribosomal and nonribosomal sequences were analyzed to design polymerase chain reaction (PCR) primers for detection and identification of the phytoplasmas that cause apple proliferation (AP) and pear decline (PD). A ribosomal primer pair (fU5/rU3) was developed that initiated amplification of the target DNA from all 42 samples from PD-infected pear trees and 36 samples from AP-infected apple trees. These primers also amplified rDNA in all other taxonomically different phytoplasma strains that were tested. A pair of group-specific primers (fO1/rO1) derived from the 16S rRNA gene was identified for detection of the closely related phytoplasmas associated with AP, PD, and European stone fruit yellows. PCR detection of the PD agent with primer pairs fU5/rU3 and fO1/rO1 was considerably more sensitive than microscopic detection using the 4'-6-diamidino-2-phenylindole fluorescence method. A more specific ribosomal primer pair (fPD/rO1) amplified phytoplasma rDNA in all samples from infected pear trees but showed some cross-amplification of AP rDNA. Restriction enzyme analysis of the PCR products obtained with primer pairs fO1/rO1 and fPD/rO1 distinguished the AP and PD phytoplasmas. One pair of ribosomal (fPD/rPDS) primers specifically amplified DNA of the PD phytoplasma but from only about 80% of the infected pear trees. Three pairs of nonribosomal primers amplified phytoplasmal DNA from AP- or PD-infected trees or from both phytoplasmas but failed to detect all strains of either of the two pathogens. These results show that is not possible to detect all strains of the AP and PD phytoplasmas, respectively, with pathogen-specific primers.

K. H. Lorenz

Papers

Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer region.

Detection of the apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and nonribosomal DNA.

Genetic diversity among mycoplasmalike organisms associated with stone fruit diseases