R. Jomantiene

Classification of new phytoplasmas associated with diseases of strawberry in Florida, based on analysis of 16s rRNA and ribosomal protein gene operon sequences

Strawberry plants exhibiting symptoms of stunting and abnormally small leaves were observed in production fields in central Florida, USA. Since the symptoms were suggestive of phytoplasma infection, plants were assayed for presence of phytoplasma by PCR amplification of 16S rDNA and ribosomal protein (rp) gene sequences. Amplification of phytoplasma-specific DNA sequences by PCR indicated infection of the diseased strawberry plants by phytoplasmas. RFLP analyses of amplified 16S rDNA revealed that the plants were infected by two mutually distinct phytoplasmas that differed from strawberry green petal phytoplasma (group 16Srl-C). Both phytoplasmas were members of 16S rRNA gene group I (16Srl). Based on RFLP analysis of amplified 16S rDNA and rp gene sequences, one was classified in group 16Srl subgroup I and new rp subgroup 16Srl-l(rp); its 16S rRNA-rp subgroup was designated 16Srl-K(rr-rp). The second phytoplasma represented a previously undescribed subgroup, designated K, in 16S rRNA group I but belonged to rp subgroup 16Srl-J(rp); this phytoplasmas 16S rRNA-rp subgroup was designated 16Srl-J(rr-rp). Results of RFLP analyses agreed with putative restriction site maps based on nucleotide sequences determined for the amplified 16S rDNAs and rp gene operon DNAs. Further evidence indicated that the 16Srl-K(rr-rp) strawberry phytoplasma, Mexican periwinkle virescence phytoplasma and stolbur phytoplasma shared sequence homologies that enabled amplification of DNA from all three by PCR using primers previously designed as stolbur-specific.

Ancient, recurrent phage attacks and recombination shaped dynamic sequence-variable mosaics at the root of phytoplasma genome evolution

Mobile genetic elements have impacted biological evolution across all studied organisms, but evidence for a role in evolutionary emergence of an entire phylogenetic clade has not been forthcoming. We suggest that mobile element predation played a formative role in emergence of the phytoplasma clade. Phytoplasmas are cell wall-less bacteria that cause numerous diseases in plants. Phylogenetic analyses indicate that these transkingdom parasites descended from Gram-positive walled bacteria, but events giving rise to the first phytoplasma have remained unknown. Previously we discovered a unique feature of phytoplasmal genome architecture, genes clustered in sequence-variable mosaics (SVMs), and suggested that such structures formed through recurrent, targeted attacks by mobile elements. In the present study, we discovered that cryptic prophage remnants, originating from phages in the order Caudovirales, formed SVMs and comprised exceptionally large percentages of the chromosomes of ‘Candidatus Phytoplasma asteris’-related strains OYM and AYWB, occupying nearly all major nonsyntenic sections, and accounting for most of the size difference between the two genomes. The clustered phage remnants formed genomic islands exhibiting distinct DNA physical signatures, such as dinucleotide relative abundance and codon position GC values. Phytoplasma strain-specific genes identified as phage morons were located in hypervariable regions within individual SVMs, indicating that prophage remnants played important roles in generating phytoplasma genetic diversity. Because no SVM-like structures could be identified in genomes of ancestral relatives including Acholeplasma spp., we hypothesize that ancient phage attacks leading to SVM formation occurred after divergence of phytoplasmas from acholeplasmas, triggering evolution of the phytoplasma clade.

New Group 16SrIII Phytoplasma Lineages in Lithuania Exhibit rRNA Interoperon Sequence Heterogeneity

Previously undescribed phytoplasmas were detected in diseased plants of dandelion (Taraxacum officinale) exhibiting virescence of flowers, thistle (Cirsium arvense) exhibiting symptoms of white leaf, and a Gaillardia sp. exhibiting symptoms of stunting and phyllody in Lithuania. On the basis of restriction fragment length polymorphism (RFLP) analysis of 16S rDNA amplified in PCR, the dandelion virescence (DanVir), cirsium whiteleaf (CirWL), and gaillardia phyllody (GaiPh) phytoplasmas were classified in phylogenetic group 16SrIII (X-disease phytoplasma group), new subgroups III-P and III-R and subgroup III-B, respectively. RFLP and nucleotide sequence analyses revealed 16S rRNA interoperon sequence heterogeneity in the two rRNA operons, rrnA and rrnB, of both DanVir and CirWL. Results from phylogenetic analysis based on nucleotide sequences of 16S rDNA were consistent with recognition of the two new subgroups as representatives of distinct new lineages within the group 16SrIII phytoplasma subclade. The branching order of rrnA and rrnB sequences in the phylogenetic tree supported this interpretation and indicated recent common ancestry of the two rRNA operons in each of the phytoplasmas exhibiting interoperon heterogeneity.

Folate Biosynthesis Pseudogenes, ΨfolP and ΨfolK, and an O-Sialoglycoprotein Endopeptidase Gene Homolog in the Phytoplasma Genome

Phytoplasmas are wall-less phytopathogenic prokaryotes of small genome sizes that are obligate parasites of insect vectors and plant hosts. We have cloned a clover phyllody (CPh) phytoplasma DNA locus containing five potential coding sequences. Two were identified as pseudogenes (PsifolP and PsifolK) homologous to folP and folK genes, which encode dihydropteroate synthase (DHPS) and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), respectively, in other bacteria. Evolution of the phytoplasma presumably involved loss of functions through the formation of these and other pseudogenes during adaptation to obligate parasitism. The findings suggest that the phytoplasma lacks capacity for de novo folate biosynthesis and possesses a transport system for absorption of preformed folate from host cells. The PsifolP-PsifolK region was flanked by three open reading frames (ORFs) encoding a DegV family protein, a hypothetical protein with a P60-like lipoprotein domain homologous with the P60-like Mycoplasma hominis protein, and a glycoprotease (Gcp) protein that possibly functions as a host adaptation or virulence factor.

Single and Mixed Phytoplasma Infections in Phyllody- and Dwarf-Diseased Clover Plants in Lithuania

Naturally diseased plants of clover (Trifolium spp.) exhibiting symptoms of clover phyllody (virescence and phyllody of flowers) or of clover dwarf (abnormally small leaves, shortened internodes, proliferation of shoots, and dwarf growth habit) were observed in fields in Lithuania. Phytoplasma group-specific polymerase chain reactions (PCRs) and restriction fragment length polymorphism (RFLP) analysis of 16S rDNA revealed that the plants were infected by two mutually distinct phytoplasmas. Clover phyllody-diseased plants were infected by a subgroup 16SrI-C (subgroup I-C) phytoplasma (CPh-L) related to clover phyllody (CPh-C) phytoplasma in Canada. Clover dwarf-diseased plants were infected by both CPh-L and a phytoplasma (CYE-L) related to clover yellow edge (CYE-C) phytoplasma (subgroup 16SrIII-B = III-B) in Canada. A 1.8-kbp fragment of rRNA operon from CYE-L was amplified, cloned, and sequenced, and putative restriction sites mapped. This sequence shared high similarity (99.7%) with that of CYE-C and exhibited no differences from CYE-C in RFLP patterns of 16S rDNA; therefore, we tentatively classified CYE-L in subgroup 16SrIII-B (type strain, CYE = CYE-C phytoplasma) of the X-disease phytoplasma group. These findings extend the known geographical ranges of subgroup I-C and subgroup III-B taxa to the region of northern Europe including Lithuania and suggest a role of the subgroup III-B phytoplasma in clover dwarf disease.

Phytoplasmas infecting sour cherry and lilac represent two distinct lineages having close evolutionary affinities with clover phyllody phytoplasma

Phytoplasmas infecting sour cherry and lilac in Lithuania were found to represent two lineages related to clover phyllody phytoplasma (CPh), a subgroup 16SrI-(R/S)C (formerly 16SrI-C) strain exhibiting rRNA interoperon sequence heterogeneity. 16S rDNAs amplified from the cherry bunchy leaf (ChBL) and lilac little leaf (LcLL) phytoplasmas were identical or nearly identical to those of operon rrnA and operon rrnB, respectively, of CPh. There was no evidence of 16S rRNA interoperon sequence heterogeneity in either LcLL or ChBL phytoplasma. Based on collective RFLP patterns of 16S rDNA, ChBL was classified in subgroup 16SrI-R, and LcLL was classified in new subgroup 16SrI-S. The ribosomal protein (rp) gene sequences from LcLL phytoplasma were identical to those of CPh, and strain LcLL was classified in rp subgroup rpI-C. By contrast, rp gene sequences from ChBL phytoplasma differed from those of subgroup rpI-C; based on RFLP patterns of rp gene sequences, ChBL was classified in new rp subgroup rpI-O. Single nucleotide polymorphisms (SNPs), designated here by a new SNP convention, marked members of rp subgroup rpI-C, and distinguished LcLL and CPh from ChBL and other non-rpI-C phytoplasmas in group 16SrI. The results raise questions concerning phytoplasma biodiversity assessment based on rRNA genes alone and encourage the supplemental use of a single copy gene in phytoplasma identification and classification, while drawing attention to a possible role of horizontal gene transfer in the evolutionary history of these lineages.

First Report of Aster Yellows-Related Subgroup I-A Phytoplasma Strains in Carrot, Phlox, Sea-Lavender, Aconitum, and Hyacinth in Lithuania

Phytoplasma strains that belong to group 16SrI (aster yellows phytoplasma group), subgroup A (I-A, North American tomato big bud phytoplasma subgroup) were discovered in diverse plant species in Lithuania. Plants in which the strains were found exhibited symptoms characteristic of infections by phytoplasma. Carrot (Daucus sativus) with carrot proliferation disease exhibited symptoms of proliferation of the crown, chlorosis of young leaves, and reddening of mature leaves. Diseased phlox (Phlox paniculata) exhibited symptoms of virescence and leaf chlorosis. Diseased sea-lavender (Limonium sinuatum) exhibited abnormal proliferation of shoots, chlorosis of young leaves, reddening of mature leaves, and degeneration of flowers. Diseased hyacinth (Hyacinthus orientalis) exhibited chlorosis of leaves and undeveloped flowers. Diseased Aconitum sp. exhibited proliferation of shoots. Phytoplasma-characteristic ribosomal (r) DNA was detected in the plants by use of the polymerase chain reaction (PCR). The rDNA was amplified in PCR primed by primer pair P1/P7 and reamplified in nested PCR primed by primer pair R16F2n/R16R2 (F2n/R2), as previously described (1). The phytoplasmas were classified through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, amplified in the nested PCR primed by F2n/R2, using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, and Sau3AI. Collective RFLP patterns indicated that all detected phytoplasma strains were affiliated with subgroup I-A. The 16S rDNA amplified from the phytoplasma (CarrP phytoplasma) in diseased carrot was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank data library (GenBank accession no. AF291682). The 16S rDNAs of CarrP and tomato big bud (GenBank acc. no. AF222064) phytoplasmas shared 99.8% nucleotide sequence similarity. Phytoplasmas belonging to group 16SrIII (3), group 16SrV (D. Valiunas, unpublished data), and subgroup I-C in group 16SrI (2,3) occur in Lithuania. This report records the first finding of a subgroup I-A phytoplasma in the Baltic region and expands the known plant host range of this phytoplasma subgroup. References: (1) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (2) Jomantiene et al. Phytopathology 90:S39, 2000. (3) Staniulis et al. Plant Dis. 84:1061, 2000.

Phytoplasma Genomes: Evolution Through Mutually Complementary Mechanisms, Gene Loss and Horizontal Acquisition

Phytoplasmas are cell wall-less bacteria that inhabit phloem sieve cells of infected plants and are transmitted by phloem-feeding insect vectors. Having descended from a Gram-positive, low G+C walled bacterium and more recently from an Acholeplasma-like ancestor, the phytoplasma clade underwent substantial evolutionary genome shrinkage. Yet, phytoplasma genomes contain numerous repeated sequences that appear in genomic islands. These islands, first termed sequence-variable mosaics (SVMs), were formed by recurrent and targeted attacks by ancient phages. This chapter describes distinctive architecture of phytoplasma genomes, genes unique to phytoplasmas, and genes that underwent lineage-specific acquisition and loss. The chapter also discusses essential genes that are missing in phytoplasmas compared with those present in Mycoplasma genitalium, a model free-living bacterium thought to have the minimum gene complement among known cellular organisms. Available genome data indicate that a common genomic thread unites all phytoplasmas: Through the formation of phage-based genomic islands, SVMs, the phytoplasma progenitor acquired new capabilities. Repeated and targeted chromosomal integration of phage genomes, and further gene acquisition through targeted insertion of mobile gene cassette-like elements, shaped the phytoplasma genome. While the loss of genes encoding diverse metabolic pathways must have led to increased host dependence, new capabilities were acquired that enabled and enhanced phytoplasma–host interactions. Thus, two mutually complementary, genome-sculpting mechanisms acting in concert played key roles in phytoplasma evolution. The rapidly evolving nature and lineage-specific adaptation of vertically inherited as well as horizontally acquired phytoplasmal genes are evident and deserve attention in future studies.

Molecular Identification and Classification of Strawberry Phylloid Fruit Phytoplasma in Group 16SrI, New Subgroup

Plants of commercial strawberry (Fragaria × ananassa Duch., cv. Camarosa) exhibiting extensive fruit phyllody (development of leafy structures from achenes) were observed in a winter greenhouse production facility in West Virginia. In July 2001, 95 dormant, cold-stored plants were purchased from a California strawberry nursery, potted and grown in this West Virginia facility. Five of the plants developed fruits with phylloid growths. These fruits were assessed for phytoplasma infection using nested polymerase chain reactions (PCRs) in which initial ribosomal (r) DNA amplification was primed by phytoplasma-universal primer pair P1/P7 (2), and rDNA reamplification was primed by primer pair R16F2n/R16R2 (1). Amplification of phytoplasma-characteristic 1.2-kbp 16S rDNA in the nested reactions primed by R16F2n/R16R2 confirmed that the symptomatic plants were infected by a phytoplasma, termed strawberry phylloid fruit (StrawbPhF) phytoplasma. No phytoplasma DNAs were amplified from healthy plants. Restriction fragment length polymorphism (RFLP) patterns of 16S rDNA digested with AluI, KpnI, HhaI, HaeIII, HpaII, MseI, RsaI, and Sau3A1 restriction endonucleases indicated that StrawbPhF phytoplasma belonged to group 16SrI (group I, aster yellows phytoplasma group) according to the phytoplasma classification system of Lee et al. (4). However, the collective patterns distinguished StrawbPhF from its closest known relative, clover phyllody (CPh) phytoplasma, and from all other phytoplasmas classified in group 16SrI. On the basis of the RFLP patterns of 16S rDNA, the StrawbPhF was classified in group 16SrI, new subgroup R. The StrawbPhF phytoplasma 1.2-kbp 16S rDNA PCR product was cloned in Escherichia coli using TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA), sequenced, and the sequence deposited in GenBank under Accession No. AY102275. The StrawbPhF 16S rDNA sequence shared 99.9 and 99.8% similarity with the two sequence heterogeneous operons, rrnA and rrnB, respectively, of CPh phytoplasma, and shared 99.9% similarity with 16S rDNA of the unclassified cirsium yellows (CirY) phytoplasma (GenBank Accession No. AF200431) reported in Cirsium arvense L. in Lithuania (3). The restriction sites in 16S rDNA of StrawbPhF were identical to those in 16S rDNA of CPh rrnA and CirY. Three restriction sites (AluI, HaeIII, and MseI) and three base substitutions distinguished StrawbPhF 16S rDNA from rrnB of CPh phytoplasma. No evidence was obtained for the presence of a second (sequence heterogeneous) rRNA operon in StrawbPhF phytoplasma, as reported in CPh phytoplasma (4), which clearly distinguishes this phytoplasma from CPh phytoplasma. Future studies on StrawbPhF phytoplasma may provide important information on the evolution of phytoplasmas. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (3) R. Jomantiene et al. Phytopathology 90:S39, 2000. (4) I.-M. Lee et al. Int J. Syst. Bacteriol. 48:1153, 1998.

Molecular identification and classification of a phytoplasma associated with phyllody of strawberry fruit in Maryland.

Several phytoplasmas have been reported to be associated with phyllody of strawberry fruit, including clover yellow edge, clover proliferation, clover phyllody, eastern and western aster yellows, STRAWB2, strawberry multicipita, and Mexican periwinkle virescence phytoplasmas. Plant symptoms in addition to phyllody may include chlorosis, virescence, stunting, or crown proliferation. In this report we describe a new phytoplasma in association with strawberry leafy fruit (SLF) disease in Maryland. Diseased plants exhibited fruit phyllody, floral virescence, leaf chlorosis, and plant stunting. Phytoplasmal 16S rDNA was amplified from SLF diseased plants by using the polymerase chain reaction (PCR) primed by primer pair P1/P7 and was reamplified in nested PCR primed by primer pair R16F2n/R2 (F2n/R2) as previously described (1). These results indicated the presence of a phytoplasma, designated SLF phytoplasma. Identification of SLF phytoplasma was accomplished by restriction fragment length polymorphism (RFLP) analysis of DNA amplified in PCR primed by F2n/R2, using endonuclease enzyme digestion with AluI, HhaI, KpnI, HaeIII, MseI, HpaII, RsaI, and Sau3AI. Phytoplasma classification was done according to the system of Lee et al. (2). RFLP analyses of rDNA amplified in three separate PCRs gave identical patterns. On the basis of collective RFLP patterns of the amplified 16S rDNA, the SLF phytoplasma was classified as a member of group 16SrIII (group III, X-disease phytoplasma group). The HhaI RFLP pattern of SLF 16S rDNA differed from that of the apparently close relative, clover yellow edge (CYE) phytoplasma, and all other phytoplasmas previously described in group III. Based on these results, SLF phytoplasma was classified in a new subgroup, designated subgroup K (III-K), within group III. The 1.2 kbp DNA product of PCR primed by primer pair F2n/R2 was sequenced, and the sequence deposited in GenBank under Accession No. AF 274876. Results from putative restriction site analysis of the sequence were in agreement with the results from actual enzymatic RFLP analysis of rDNA amplified from phylloid strawberry fruit. Although the sequence similarity between the 1.2-kbp fragment from the 16S rDNA of SLF phytoplasma and that of CYE phytoplasma was 99.9%, the Hha1 RFLP pattern of SLF rDNA supports the conclusion that the SLF phytoplasma may be closely related to, but is distinct from, CYE and other strains that are classified in group III. These findings contribute knowledge about the diversity of phytoplasmas affiliated with group III and the diversity of phytoplasmas associated with diseases in strawberry. References: (1) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.