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.

I. Spore Attachment and Invasion.- 1. Adhesion of Fungi to the Plant Surface: Prerequisite for Pathogenesis.- 2. Signaling for Infection Structure Formation in Fungi.- 3. The Plant Cell Wall as a Barrier to Fungal Invasion.- 4. Rust Basidiospore Germlings and Disease Initiation.- 5. Attachment of Mycopathogens to Cuticle: The Initial Event of Mycoses in Arthropod Hosts.- 6. The Fate of Fungal Spores in the Insect Gut.- 7. Candida Blastospore Adhesion, Association, and Invasion of the Gastrointestinal Tract of Vertebrates.- 8. Infectious Propagules of Dermatophytes.- II. Fungal Spore Products and Pathogenesis.- 9. Melanin Biosynthesis: Prerequisite for Successful Invasion of the Plant Host by Appressoria of Colletotrichum and Pyricularia.- 10. The Plant Cuticle: A Barrier to Be Overcome by Fungal Plant Pathogens.- 11. Appearance of Pathogen-Related Proteins in Plant Hosts: Relationships between Compatible and Incompatible Interactions.- 12. The Role of Cuticle-Degrading Enzymes in Fungal Pathogenesis in Insects.- 13. Potential for Penetration of Passive Barriers to Fungal Invasion in Humans.- 14. Dihydroxynaphthalene (DHN) Melanin and Its Relationship with Virulence in the Early Stages of Phaeohyphomycosis.- III. Host Response to Early Fungal Invasion.- 15. Invasion of Plants by Powdery Mildew Fungi, and Cellular Mechanisms of Resistance.- 16. Induced Systemic Resistance in Plants.- 17. The Plant Membrane and Its Response to Disease.- 18. The Fungal Spore: Reservoir of Allergens.- 19. Conidia of Coccidioides immitis: Their Significance in Disease Initiation.- 20. Cell-Mediated Host Response to Fungal Aggression.- 21. Suppression of Phagocytic Cell Responses by Conidia and Conidial Products of Aspergillus fumigatus.- IV. Molecular Aspects of Disease Initiation.- 22. Molecular Approaches to the Analysis of Pathogenicity Genes from Fungi Causing Plant Disease.- 23. Current Status of the Molecular Basis of Candida Pathogenicity.- Taxonomic Index.

The Fungal spore and disease initiation in plants and animals

Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp, than any other phytoplasmas known Within the AY subclade, six distinct phylogenetic lineages were revealed Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences Ten subgroups were differentiated, based on analysis of rp gene sequences It is proposed that AY group phytoplasmas represent at least one novel taxon Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri) virescence in Michigan, USA, was selected as the reference strain for the novel taxon ‘Candidatus Phytoplasma asteris’ A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented

‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxon associated with aster yellows and related diseases

In vitro and in vivo antifungal activities of the essential oils of various plants against tomato grey mould disease agent Botrytis cinerea.

X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described ‘Candidatus
Phytoplasma
’ species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described ‘Candidatus
Phytoplasma
’ species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1R as representative of a novel taxon, ‘Candidatus
Phytoplasma pruni’. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the ‘Ca. Phytoplasma pruni’ lineage. We propose that the term ‘Ca.
Phytoplasma pruni’ be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and - within the tolerance of a single base difference in one unique sequence - peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of ‘Candidatus
Phytoplasma
’ taxa.

'Candidatus Phytoplasma pruni', a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes.

Botrytis cinerea was isolated from infected plants in six greenhouses in Connecticut. Forty-five isolates were evaluated in vitro to determine fungicide sensitivity to benzimidazole (benomyl and thiophanate-methyl) and dicarboximide fungicides (vinclozolin and iprodione). B. cinerea isolates with fungicide resistance were recovered from each greenhouse sampled. Benzimida-zole resistance was more common than dicarboximide resistance (74 to 76% versus 36 to 43%, respectively). Multiple fungicide resistance was common. Nineteen isolates were resistant to both a benzimidazole and a dicarboximide fungicide. The level (EC50) of resistance to dicer-boximides was low compared with resistance to benzimidazoles. Isolate growth rate was not correlated to fungicide sensitivity or EC50. Fungicide resistance was apparently unrelated to the patterns of fungicide use in greenhouses sampled.

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Sensitivity of Botrytis cinerea from Connecticut Greenhouses to Benzimidazole and Dicarboximide Fungicides

In September and October 2011, a new disease was observed on Buxus spp. in North Carolina and Connecticut, respectively. In North Carolina, over 10,000 containerized Buxus sempervirens (American boxwood) were affected at one location. A few weeks later, the disease was found in Connecticut on entire plantings of B. sempervirens ‘Suffruticosa’ (English boxwood) at two residential properties, and shortly thereafter on over 150,000 plants at two production nurseries. Initial foliar symptoms appeared as light to dark brown spots, often with dark borders. Spots enlarged and coalesced, often with a concentric pattern, and black streaks or cankers developed on stems. Infected leaves became brown or straw colored and dropped quickly after foliar symptoms were visible. Branch dieback and plant death were also observed in Connecticut. Cultures were isolated from symptomatic leaves and stems and identified as Cylindrocladium pseudonaviculatum Crous, Groenewald & Hill 2002 (1) (syn. Cylindrocladium buxicola Henricot ...

First Report of Boxwood Blight Caused by Cylindrocladium pseudonaviculatum in the United States.

Frontiers of Plant Science

(...) Of five probes used in Southern blot analyses, one probe containing extrachromosomal DNA from O-1 MLO hybridized with extrachromosomal but not chromosomal DNA of CY MLO. Three probes containing chromosomal DNA of AY MLO and one containing chromosomal DNA of O-1 MLO hybridized with chromosomal DNA of CY MLO. The nucleotide sequence homologies between the probes and nucleic acid of CY MLO are evidence for genetic relatedness of a European MLO and American MLOs and provide diagnostic tools for CY MLO detection

S. M. Douglas

Papers

'Candidatus Phytoplasma pruni', a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes.

Sensitivity of Botrytis cinerea from Connecticut Greenhouses to Benzimidazole and Dicarboximide Fungicides

First Report of Boxwood Blight Caused by Cylindrocladium pseudonaviculatum in the United States.

Frontiers of Plant Science

Detection of chrysanthemum yellows mycoplasmalike organism by dot hybridization and Southern blot analysis.