Phytoplasma Genome Research Articles

Virulence effectors encoded in the rice yellow dwarf phytoplasma genome participate in pathogenesis.

Bacteria-like phytoplasmas alternate between plant and insect hosts, secreting proteins that disrupt host development. In this study, we sequenced the complete genome of 'Candidatus Phytoplasma oryzae' strain HN2022, associated with rice yellow dwarf (RYD) disease, using PacBio HiFi technology. The strain was classified within the 16Sr XI-B subgroup. Through SignalP v5.0 for prediction and subsequent expression analysis of secreted proteins in Nicotiana benthamiana and rice (Oryza sativa L.), we identified the key virulence effector proteins RY348 and RY378. RY348, a homologue of Secreted Aster Yellows Phytoplasma Effector 54 (SAP54), targets and degrades the MADS-box transcription factors MADS1 and MADS15, causing pollen sterility. Meanwhile, RY378 impacts the strigolactone and auxin signaling pathways, substantially increasing tillering. These findings offer insights into the interactions between plants and phytoplasmas.

'Candidatus Phytoplasma vignae', assigning a species description to a long-known phytoplasma occurring in northern Australia.

Gene- and genome-based approaches were used to determine whether Vigna little leaf (ViLL) phytoplasma, which occurs in northern Australia, is a distinct 'Candidatus Phytoplasma' species. The ViLL 16S rRNA gene sequences exhibited the highest known similarity to species in the 16SrXXIX-A and 16SrIX-D subgroups, namely 'Candidatus Phytoplasma omanense' (98.03-98.10%) and 'Candidatus Phytoplasma phoenicium' (96.87-97.20%), respectively. A total of 48 single-copy orthologue genes were identified to be shared among the two draft ViLL phytoplasma genomes, 30 publicly available phytoplasma genomes, and one Acholeplasma laidlawii genome as the outgroup taxon. Phylogenomic assessments using the 48 shared single-copy orthologue genes supported that ViLL and 'Ca. Phytoplasma phoenicium' were closely related yet distinct species. The 16S rRNA gene sequence analysis and phylogenomic assessment indicate that ViLL phytoplasmas are a distinct taxon. As such, a novel species, 'Candidatus Phytoplasma vignae', is proposed. Strain BAWM-336 (genome accession number JAUZLI000000000) detected in Momordica charantia (bitter melon) serves as the reference strain of this species, with infected plant material deposited in the Victorian Plant Pathology Herbarium (VPRI) as VPRI 44369.

A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group ('Ca. Phytoplasma stylosanthis'; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and 'Ca. Phytoplasma stylosanthis'. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of 'Ca. Phytoplasma australasiaticum' and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

PhyEffector, the First Algorithm That Identifies Classical and Non-Classical Effectors in Phytoplasmas.

Phytoplasmas are the causal agents of more than 100 plant diseases in economically important crops. Eleven genomes have been fully sequenced and have allowed us to gain a better understanding of the biology and evolution of phytoplasmas. Effectors are key players in pathogenicity and virulence, and their identification and description are becoming an essential practice in the description of phytoplasma genomes. This is of particular importance because effectors are possible candidates for the development of new strategies for the control of plant diseases. To date, the prediction of effectors in phytoplasmas has been a great challenge; the reliable comparison of effectoromes has been hindered because research teams have used the combination of different programs in their predictions. This is not trivial since significant differences in the results can arise, depending on the predictive pipeline used. Here, we tested different predictive pipelines to create the PhyEffector algorithm; the average value of the F1 score for PhyEffector was 0.9761 when applied to different databases or genomes, demonstrating its robustness as a predictive tool. PhyEffector can recover both classical and non-classical phytoplasma effectors, making it an invaluable tool to accelerate effectoromics in phytoplasmas.

The observation of taxonomic boundaries for the 16SrII and 16SrXXV phytoplasmas using genome-based delimitation.

Within the 16SrII phytoplasma group, subgroups A-X have been classified based on restriction fragment length polymorphism of their 16S rRNA gene, and two species have been described, namely 'Candidatus Phytoplasma aurantifolia' and 'Ca. Phytoplasma australasia'. Strains of 16SrII phytoplasmas are detected across a broad geographic range within Africa, Asia, Australia, Europe and North and South America. Historically, all members of the 16SrII group share ≥97.5 % nucleotide sequence identity of their 16S rRNA gene. In this study, we used whole genome sequences to identify the species boundaries within the 16SrII group. Whole genome analyses were done using 42 phytoplasma strains classified into seven 16SrII subgroups, five 16SrII taxa without official 16Sr subgroup classifications, and one 16SrXXV-A phytoplasma strain used as an outgroup taxon. Based on phylogenomic analyses as well as whole genome average nucleotide and average amino acid identity (ANI and AAI), eight distinct 16SrII taxa equivalent to species were identified, six of which are novel descriptions. Strains within the same species had ANI and AAI values of >97 %, and shared ≥80 % of their genomic segments based on the ANI analysis. Species also had distinct biological and/or ecological features. A 16SrII subgroup often represented a distinct species, e.g., the 16SrII-B subgroup members. Members classified within the 16SrII-A, 16SrII-D, and 16SrII-V subgroups as well as strains classified as sweet potato little leaf phytoplasmas fulfilled criteria to be included as members of a single species, but with subspecies-level relationships with each other. The 16SrXXV-A taxon was also described as a novel phytoplasma species and, based on criteria used for other bacterial families, provided evidence that it could be classified as a distinct genus from the 16SrII phytoplasmas. As more phytoplasma genome sequences become available, the classification system of these bacteria can be further refined at the genus, species, and subspecies taxonomic ranks.

Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing

Obtaining complete phytoplasma genomes is difficult due to the lack of a culture system for these bacteria. To improve genome assembly, a non-ionic, low- and iso-osmotic iodixanol (Optiprep™) density gradient centrifugation method was developed to enrich for phytoplasma cells and deplete plant host tissues prior to deoxyribonucleic acid (DNA) extraction and high-throughput sequencing (HTS). After density gradient enrichment, potato infected with a ‘Candidatus Phytoplasma australasia’-related strain showed a ∼14-fold increase in phytoplasma HTS reads, with a ∼1.7-fold decrease in host genomic reads compared to the DNA extracted from the same sample without density gradient centrifugation enrichment. Additionally, phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment. The method was repeated on capsicum infected with Sweet Potato Little Leaf phytoplasma (‘Ca. Phytoplasma australasia’-related strain) with a lower phytoplasma titer than the potato. In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained, with the genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment. Phytoplasmas from potato and capsicum were both enriched at a density of 1.049–1.058 g/ml. Finally, we present two highly contiguous ‘Ca. Phytoplasma australasia’ phytoplasma reference genomes sequenced from naturally infected Solanaceae hosts in Australia. Obtaining high-quality phytoplasma genomes from naturally infected hosts will improve insights into phytoplasma taxonomy, which will improve their detection and disease management.

The Complete Genome of the "Flavescence Dorée" Phytoplasma Reveals Characteristics of Low Genome Plasticity.

Simple SummaryPhytoplasmas are non-cultivable organisms for which biological and molecular characterization is a challenge. Despite the development of new sequencing technologies, few high- quality phytoplasma genomes are available. This research describes the genome sequencing of the phytoplasma involved in grapevine Flavescence dorée, a devastating disease in European vineyards. Analysis of the gene content and comparison with other representative phytoplasmas revealed specific characteristics of the “Flavescence dorée” phytoplasma chromosome. Their implications for the development of the disease are presented and discussed.Members of the genus ‘Candidatus Phytoplasma’ are obligate intracellular bacteria restricted to phloem sieve elements and are able to colonize several tissues and the hemolymph in their insect vectors. The current unfeasibility of axenic culture and the low complexity of genomic sequences are obstacles in assembling complete chromosomes. Here, a method combining pathogen DNA enrichment from infected insects and dual deep-sequencing technologies was used to obtain the complete genome of a phytoplasma causing Grapevine Flavescence dorée. The de novo assembly generated a circular chromosome of 654,223 bp containing 506 protein-coding genes. Quality assessment of the draft showed a high degree of completeness. Comparative analysis with other phytoplasmas revealed the absence of potential mobile units and a reduced amount of putative phage-derived segments, suggesting a low genome plasticity. Phylogenetic analyses identified Candidatus Phytoplasma ziziphi as the closest fully sequenced relative. The “Flavescence dorée” phytoplasma strain CH genome also encoded for several putative effector proteins potentially playing a role in pathogen virulence. The availability of this genome provides the basis for the study of the pathogenicity mechanisms and evolution of the Flavescence dorée phytoplasma.

Comparative Genome Analysis of 'Candidatus Phytoplasma luffae' Reveals the Influential Roles of Potential Mobile Units in Phytoplasma Evolution.

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of ‘Candidatus Phytoplasma luffae’ strain NCHU2019 that is associated with witches’ broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has a remarkably repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ∼25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for subfunctionalization or neofunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

The Genome Reduction Excludes the Ribosomal Rescue System in Acholeplasmataceae

The trans-translation process is a ribosomal rescue system for stalled ribosomes processing truncated mRNA. The genes ssrA and smpB fulfil the key functions in most bacteria, but some species have either lost these genes or the function of the ribosomal rescue system is taken over by other genes. To date, the ribosomal rescue system has not been analysed in detail for the Acholeplasmataceae. This family, in the Mollicutes class, comprises the genus Acholeplasma and the provisional taxon “Candidatus Phytoplasma”. Despite their monophyletic origin, the two clades can be separated by traits such as not representing primary pathogens for acholeplasmas versus being phytopathogenic for the majority of phytoplasmas. Both taxa share reduced genomes, but only phytoplasma genomes are characterised by a remarkable level of instability and reduction. Despite the general relevance of the ribosomal rescue system, information is lacking on coding, the genomic context and pseudogenisation of smpB and ssrA and their possible application as a phylogenetic marker. Herein, we provide a comprehensive analysis of the ribosomal rescue system in members of Acholeplasmataceae. The examined Acholeplasmataceae genomes encode a ribosomal rescue system, which depends on tmRNA encoded by ssrA acting in combination with its binding protein SmpB. Conserved gene synteny is evident for smpB, while ssrA shows a less conserved genomic context. Analysis of the tmRNA sequences highlights the variability of proteolysis tag sequences and short conserved sites at the 5′- and 3′-ends. Analyses of smpB provided no hints regarding the coding of pseudogenes, but they did suggest its application as a phylogenetic marker of Acholeplasmataceae – in accordance with 16S rDNA topology. Sequence variability of smpB provides sufficient information for species assignment and phylogenetic analysis.

Accelerating Complete Phytoplasma Genome Assembly by Immunoprecipitation-Based Enrichment and MinION-Based DNA Sequencing for Comparative Analyses.

Phytoplasmas are uncultivated plant-pathogenic bacteria with agricultural importance. Those belonging to the 16SrII group, represented by ‘Candidatus P. aurantifolia’, have a wide range of plant hosts and cause significant yield losses in valuable crops, such as pear, sweet potato, peanut, and soybean. In this study, a method that combines immunoprecipitation-based enrichment and MinION long-read DNA sequencing was developed to solve the challenge of phytoplasma genome studies. This approach produced long reads with high mapping rates and high genomic coverage that can be combined with Illumina reads to produce complete genome assemblies with high accuracy. We applied this method to strain NCHU2014 and determined its complete genome sequence, which consists of one circular chromosome with 635,584 bp and one plasmid with 4,224 bp. Although ‘Ca. P. aurantifolia’ NCHU2014 has a small chromosome with only 471 protein-coding genes, it contains 33 transporter genes and 27 putative effector genes, which may contribute to obtaining nutrients from hosts and manipulating host developments for their survival and multiplication. Two effectors, the homologs of SAP11 and SAP54/PHYL1 identified in ‘Ca. P. aurantifolia’ NCHU2014, have the biochemical activities in destabilizing host transcription factors, which can explain the disease symptoms observed in infected plants. Taken together, this study provides the first complete genome available for the 16SrII phytoplasmas and contributes to the understanding of phytoplasma pathogenicity.

Genome-wide identification of jujube witches’ broom phytoplasma effectors revealed the role of SJP3 in inducing phyllody

Jujube witches’ broom (JWB), associated with ‘Candidatus Phytoplasma ziziphi’, is a devastating disease in Chinese jujube (Ziziphus jujuba Mill.). One of the symptoms in plants infected with phytoplasmas is the change of floral organs into leaf-like structures, known as phyllody. Effector proteins secreted by phytoplasmas are key to inducing this and other witches’ broom symptoms. However, the phytoplasma effector proteins that induce phyllody in jujube remain unknown. We identified 43 candidate secreted JWB phytoplasma proteins (SJPs) in the JWB phytoplasma genome. Eight of the predicted candidate effector genes exhibited high transcript levels in infected flower buds. Among the eight candidate effectors, SJP3 experimentally showed the potential to induce phyllody. SJP3 expression in infected jujube was at a high level in young leaves and flowers. Ectopic expression of SJP3 in Arabidopsis induced leaf-like flowers and extra secondary flowers and significantly changed the transcript levels of PISTILLATA (PI), SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). The expression levels of these MADS-box homologous genes were also changed in infected jujube flower buds and all four whorls of the floral organs. The data showed that the effector SJP3 disrupts expression of several MADS-box genes associated with floral organ identity and flowering time and is likely key to inducing phyllody in phytoplasma-infected jujube. This study provides new insight into understanding the pathogenesis of JWB disease induced by phytoplasma.

Molecular biological study on the survival strategy of phytoplasma

Phytoplasmas (genus ‘Candidatus Phytoplasma’) are plant pathogenic bacteria that reside intracellularly within the plant phloem. They infect hundreds of plant species worldwide and induce a wide range of symptoms, including stunting, yellowing, witches’ broom, phyllody, virescence, proliferation, and purple top. The phytoplasma genome has unique features that overturn commonly known wisdom. For example, ATP synthase, which is thought to be essential for life, is not encoded, which provides opportunities for reconsidering the question what is life? Phytoplasmas cause dramatic changes in plant development and parasitizes not only plants but also vector insects by host switching. Therefore, the molecular mechanisms of how phytoplasmas manipulate host plants and how they infect different hosts are of particular interest. This review summarizes the history of phytoplasma discovery, unique survival strategies revealed by genome sequencing, and the molecular mechanisms by which phytoplasmas manipulate host plants.

Intra and extragenomic variation between 16S rRNA genes found in 16SrI‐B‐related phytopathogenic phytoplasma strains

AbstractPhytoplasmas are bacterial pathogens that pose a threat to cereal and vegetable production worldwide. However, there is limited information regarding the occurrences of phytoplasmas in Central Europe. In this study we examined samples taken from crop and ornamental plants showing symptoms indicative of phytoplasma infection. Phytoplasmas related to 'Candidatus Phytoplasma asteris' were detected in eight plant species (carrot, lupine, pea, cauliflower, mustard, maize, juniper and marigold). Based on the restriction fragment length polymorphism analysis of the 16S rDNA, the phytoplasmas were classified as subgroup 16SrI‐B, 16SrI‐L or a complex strain, which harboured the I‐B and I‐L‐type 16S rRNA genes. Individual 16S rDNA sequences were analysed from those heterogeneous strains and as many as five genetic variants were distinguished. By operon‐specific amplification of the 16S rRNA genes with primers complementary to RP166 phytoplasma genome, we detected 16SrI‐L/B phytoplasma strains in the multivariant samples. The I‐L‐type sequences were amplified from rrnA and the I‐B‐type sequences were amplified from rrnB. Molecular phylogeny based on the 16S rRNA showed that all the detected phytoplasma variants were clustered with a 'Ca. Phytoplasma asteris' subclade and were closely related to the 16SrI‐B subgroup. Despite the variability of 16S rDNA, the strains from all the eight crops shared identical tuf gene sequences. We report, for the first time, 16SrI‐B phytoplasma infecting lupine, field pea, cauliflower and mustard, 16SrI‐L phytoplasma infecting maize and field pea and 16SrI‐L/B phytoplasma infecting carrot, lupine and marigold. We further demonstrate variability in the 16S rRNA genes found in the 16SrI‐B‐related phytoplasma genomes worldwide. For clearer and more unambiguous nomenclature, we propose that in the future, the more intuitive subgroup 16SrI‐L/B be used for heterogenic phytoplasma strains instead of 16SrI‐(B/L)L.

Comparison of Current Methods for Signal Peptide Prediction in Phytoplasmas.

Although phytoplasma studies are still hampered by the lack of axenic cultivation methods, the availability of genome sequences allowed dramatic advances in the characterization of the virulence mechanisms deployed by phytoplasmas, and highlighted the detection of signal peptides as a crucial step to identify effectors secreted by phytoplasmas. However, various signal peptide prediction methods have been used to mine phytoplasma genomes, and no general evaluation of these methods is available so far for phytoplasma sequences. In this work, we compared the prediction performance of SignalP versions 3.0, 4.0, 4.1, 5.0 and Phobius on several sequence datasets originating from all deposited phytoplasma sequences. SignalP 4.1 with specific parameters showed the most exhaustive and consistent prediction ability. However, the configuration of SignalP 4.1 for increased sensitivity induced a much higher rate of false positives on transmembrane domains located at N-terminus. Moreover, sensitive signal peptide predictions could similarly be achieved by the transmembrane domain prediction ability of TMHMM and Phobius, due to the relatedness between signal peptides and transmembrane regions. Beyond the results presented herein, the datasets assembled in this study form a valuable benchmark to compare and evaluate signal peptide predictors in a field where experimental evidence of secretion is scarce. Additionally, this study illustrates the utility of comparative genomics to strengthen confidence in bioinformatic predictions.

Enrichment of phytoplasma genome DNA through a methyl-CpG binding domain-mediated method for efficient genome sequencing

Phytoplasmas are obligate intracellular phytopathogenic bacteria infecting many agriculturally important plants. Although the information on the genome sequences is very useful, it is difficult to sequence phytoplasma genomes because pure phytoplasma DNA is unavailable. Here, we explored whether a methyl-CpG binding domain (MBD)-mediated enrichment method for bacterial DNA could facilitate enrichment of phytoplasma DNA from infected plants for next-generation sequencing of phytoplasma genome. Quantitative PCR revealed that phytoplasma DNA was enriched 10- to 30-fold by MBD-mediated enrichment compared to total DNA from the infected plant. Using the enriched DNA of the ‘Candidatus (Ca.) Phytoplasma asteris’ onion yellows strain (OY-M), most genome regions were sequenced by only one million sequence reads generated by an Illumina MiSeq platform. In addition, we present the first draft genome sequence of the ‘Ca. P. asteris’ hydrangea phyllody strain (HP) by using MBD-mediated enrichment from total DNA of the natural host plant. The draft features 36 contigs of total length 597,762 bp with 521 coding sequences, one rRNA operon, and 30 tRNA genes. One contig resembles plasmids of ‘Ca. P. asteris’-related strains. Pairwise alignment with the OY-M genome indicated that most regions of the HP genome except for repetitive regions had been sequenced. Thus, MBD-mediated enrichment greatly aids genome sequencing of phytoplasmas.

Species Boundaries and Molecular Markers for the Classification of 16SrI Phytoplasmas Inferred by Genome Analysis.

Phytoplasmas are plant-pathogenic bacteria that impact agriculture worldwide. The commonly adopted classification system for phytoplasmas is based on the restriction fragment length polymorphism (RFLP) analysis of their 16S rRNA genes. With the increased availability of phytoplasma genome sequences, the classification system can now be refined. This work examined 11 strains in the 16SrI group within the genus ‘Candidatus Phytoplasma’ and investigated the possible species boundaries. We confirmed that the RFLP classification method is problematic due to intragenomic variation of the 16S rRNA genes and uneven weighing of different nucleotide positions. Importantly, our results based on the molecular phylogeny, differentiations in chromosomal segments and gene content, and divergence in homologous sequences, all supported that these strains may be classified into multiple operational taxonomic units (OTUs) equivalent to species. Strains assigned to the same OTU share >97% genome-wide average nucleotide identity (ANI) and >78% of their protein-coding genes. In comparison, strains assigned to different OTUs share < 94% ANI and < 75% of their genes. Reduction in homologous recombination between OTUs is one possible explanation for the discontinuity in genome similarities, and these findings supported the proposal that 95% ANI could serve as a cutoff for distinguishing species in bacteria. Additionally, critical examination of these results and the raw sequencing reads led to the identification of one genome that was presumably mis-assembled by combining two sequencing libraries built from phytoplasmas belonging to different OTUs. This finding provided a cautionary tale for working on uncultivated bacteria. Based on the new understanding of phytoplasma divergence and the current genome availability, we developed five molecular markers that could be used for multilocus sequence analysis (MLSA). By selecting markers that are short yet highly informative, and are distributed evenly across the chromosome, these markers provided a cost-effective system that is robust against recombination. Finally, examination of the effector gene distribution further confirmed the rapid gains and losses of these genes, as well as the involvement of potential mobile units (PMUs) in their molecular evolution. Future improvements on the taxon sampling of phytoplasma genomes will allow further expansions of similar analysis, and thus contribute to phytoplasma taxonomy and diagnostics.

First Report of Association of a ‘Candidatus Phytoplasma aurantifolia’ 16SrII-D Related Phytoplasma with Geranium (Pelargonium hortorum) Little Leaf Disease in Iran

Geranium, an important ornamental plant belong to the family Geraniaceae, is widely cultivated in urban landscapes and private gardens all over the world. During a survey in spring 2019, a few seed-propagated geranium plants exhibiting typical symptoms induced by phytoplasmas, including pale green and little leaves, phyllody, virescence, and witches’-broom symptoms, were observed in Jiroft, Kerman province of Iran. Total genomic DNA extracted from leaf midribs and petioles of the three symptomatic and two asymptomatic plants was used as a template in a nested polymerase chain reaction (PCR) assay using universal phytoplasma primer pairs P1/P7 followed by R16F2n/ R16R2 (Gundersen and Lee 1996) to amplify a part of the 16S rDNA phytoplasma genome as previously described by Azadvar et al. (2011). The generated 1.8- and 1.25-kb amplicons in the first and second round of the nested PCR assay, respectively, from each of the three symptomatic plant samples revealed phytoplasma infection of the plants. No amplicon was generated in PCR assays by using the DNA templates from symptomless plants. The purified PCR products were directly sequenced (Bioneer, South Korea) in both forward and reverse directions. The consensus sequences were analyzed by the iPhyClassifier tool (Zhao et al. 2009) and pDRAW32 software (AcaClone software, http://www.acaclone.com) for 16Sr group/subgroup phytoplasma identification. The 16S rDNA nucleotide sequences from the three diseased plants were identical (GenBank accession no. MT187639). iPhyClassifier analysis showed that the F2nR2 16S rRNA encoding gene sequence of the phytoplasma associated with geranium little leaf (GrLL) disease in Iran was identical (similarity coefficient 1.00) to those of the reference pattern of 16Sr group II subgroup D (GenBank accession no. Y10097) and had 98.7% similarity with the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession U15442). Virtual restriction fragment length polymorphism (in silico RFLP) analysis of the 16S rDNA F2nR2 sequence with the pDRAW32 program using AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, RsaI, Sau3AI, SspI, and TaqI endonucleases further confirmed that the GrLL phytoplasma belong to subgroup II-D. The 16SrII-D subgroup phytoplasma has been previously reported in association with Calendula officinalis (Esmailzadeh Hosseini et al. 2016), Helianthus annuus (Salehi et al. (2015), and Petunia hybrida (Faghihi et al. 2014) ornamental plants in Iran. We also found many petunia (P. hybrida) plants showing little leaf, yellowing, phyllody, and witches’-broom symptoms next to the GrLL-infected plants, which hosted the same phytoplasma (GenBank accession no. MT187637). A 16SrII group-related phytoplasma has previously been reported associated with Pelargonium capitatum weed plant exhibiting severe stunting and necrosis in Australia (Lee et al. 2010). To our knowledge, this is the first report of a ‘Ca. P. aurantifolia’ 16SrII-D subgroup-related phytoplasma associated with geranium (Pelargonium hortorum) little leaf disease in the world.

Genomic Characterization of the Periwinkle Leaf Yellowing (PLY) Phytoplasmas in Taiwan.

The periwinkle leaf yellowing (PLY) disease was first reported in Taiwan in 2005. This disease was caused by an uncultivated bacterium in the genus “Candidatus phytoplasma.” In subsequent years, this bacterium was linked to other plant diseases and caused losses in agriculture. For genomic investigation of this bacterium and its relatives, we conducted whole genome sequencing of a PLY phytoplasma from an infected periwinkle collected in Taoyuan. The de novo genome assembly produced eight contigs with a total length of 824,596 bp. The annotation contains 775 protein-coding genes, 63 pseudogenes, 32 tRNA genes, and two sets of rRNA operons. To characterize the genomic diversity across populations, a second strain that infects green onions in Yilan was collected for re-sequencing analysis. Comparison between these two strains identified 337 sequence polymorphisms and 10 structural variations. The metabolic pathway analysis indicated that the PLY phytoplasma genome contains two regions with highly conserved gene composition for carbohydrate metabolism. Intriguingly, each region contains several pseudogenes and the remaining functional genes in these two regions complement each other, suggesting a case of duplication followed by differential gene losses. Comparative analysis with other available phytoplasma genomes indicated that this PLY phytoplasma belongs to the 16SrI-B subgroup in the genus, with “Candidatus Phytoplasma asteris” that causes the onion yellowing (OY) disease in Japan as the closest known relative. For characterized effectors that these bacteria use to manipulate their plant hosts, the PLY phytoplasma has homologs for SAP11, SAP54/PHYL1, and TENGU. For genome structure comparison, we found that potential mobile unit (PMU) insertions may be the main factor that drives genome rearrangements in these bacteria. A total of 10 PMU-like regions were found in the PLY phytoplasma genome. Two of these PMUs were found to harbor one SAP11 homolog each, with one more similar to the 16SrI-B type and the other more similar to the 16SrI-A type, suggesting possible horizontal transfer. Taken together, this work provided a first look into population genomics of the PLY phytoplasmas in Taiwan, as well as identified several evolutionary processes that contributed to the genetic diversification of these plant-pathogenic bacteria.

Molecular characterization of effector protein SAP54 in Bellis virescence phytoplasma (16SrIII-J)

Phytoplasmas are wall-less bacteria with a parasitic lifestyle responsible for numerous plant diseases worldwide. Since the introduction of next generation sequencing technologies, genome wide studies of these pathogens are flourishing and a handful of phytoplasma genomes are available in public databases. In South America, phytoplasmas from the 16SrIII group (X-disease) are the most widely distributed, but only one draft genome from a 16SrIII-J phytoplasma-infected periwinkle plant has been generated (phytoplasma Vc33). Here, in grafting experiments, we characterized the phenotypic signatures of an Argentinian daisy-derived isolate of a 16SrIII-J phytoplasma (Bellis virescence phytoplasma) infecting periwinkle. Moreover, we applied a pipeline for genome-wide annotation of the Vc33 genome and identified the effector protein SAP54. We then employed the obtained data to amplify, clone, sequence and characterize a SAP54 orthologue protein from the Bellis virescence phytoplasma. Structural analyses suggest that the identified SAP54 effector is highly conserved. The results gathered here could provide the basis for reverse genetics experiments using 16SrIII-J SAP54 proteins to assess their eventual role in pathogenesis.

Assembly of Phytoplasma Genome Drafts from Illumina Reads Using Phytoassembly.

Genome drafts for the phytoplasmas may be rapidly and efficiently assembled from NGS sequence data alone exploiting the proper bioinformatic tools and starting from properly collected samples. Here, we describe the use of the Phytoassembly pipeline ( https://github.com/cpolano/phytoassembly ), a fully automated tool that accepts as input row Illumina data from two samples (a phytoplasma infected sample and a healthy reference sample) to produce a phytoplasma genome draft, using the healthy plant host genome as a filter and profiting from the difference in reads coverage between the genome of the pathogen and that of the host. For phytoplasma infected samples containing >2% of pathogen DNA and an isogenic healthy reference sequence the resulting assemblies span the almost entire genomes.