Momordica charantia, also known as bitter melon, bitter gourd, and bitter squash, is a member of the Cucurbitaceae family and is widely grown in tropical and subtropical regions for its edible fruit and medicinal properties (Alves et al. 2017). In April 2022, bitter melon plants exhibiting stem fasciation and excessive tendril symptoms were observed in a 50-acre vegetable farm in Yijia Village, Weishan Yizu Huizu Autonomous County, Dali, Yunnan Province, China (Fig. 1). The farm primarily grew tomatoes, but around 400 bitter melon plants were planted in spots where tomatoes failed to establish. One plot had a 40% incidence rate, with four out of ten bitter melon plants showing symptoms. Scattered cases were observed in other plots, leading to an overall disease incidence rate of around 2% for the entire farm. Phytoplasma infection was suspected due to symptomatic plants growing in the same province as previously reported cases of phytoplasma diseases, such as happy tree (Camptotheca accuminata) witches'-broom disease, and the presence of phytoplasma-transmitting leafhoppers (Qiao et al. 2023). DNA was extracted from four symptomatic samples and two healthy controls collected from the abovementioned plot with a 40% disease incidence using Bioteke's Plant Genomic DNA Extraction Kit and then tested for phytoplasma infection. A nested PCR assay was conducted using primer pair P1/16S-SR followed by P1A/16S-SR to amplify the near full-length phytoplasma 16S rDNA (about 1.5kb) as previously described (Lee et al. 2004). None of the healthy controls tested positive for phytoplasma infection, while three out of four symptomatic plants showed positive results. The amplicons from the nested PCR were cloned into the pCRII-TOPO vector as previously described (Lee et al. 2004). The resulting clones were sequenced, and the representative sequence was deposited into GenBank (accession number PP489216). The iPhyClassifier (Zhao et al. 2009) was employed to determine the phytoplasma species and group/subgroup associated with the bitter melon stem fasciation (BMSF) disease. The results indicated that the diseased bitter melon plants were infected with a strain related to 'Candidatus Phytoplasma malaysianum' (EU371934), with a 98.07% sequence identity. The similarity coefficient was 1.00 compared to the reference strain of 16SrXXXII-D (GenBank accession: MW138004). The phytoplasma strain associated with BMSF disease was designated as BMSF1. In addition, the same DNA samples underwent further characterization of the BMSF strains. A nested PCR was conducted using primer pair rpL2F3/rpIR1A, followed by rp(III)-FN/rpIR1A to amplify a phytoplasma-specific rp gene segment (about 1.2 kb) (Martini et al. 2007; Davis et al. 2013). Three out of four samples tested positive, consistent with the 16S rRNA gene amplification results. Similarly, a primer pair L15F1/MapR1 followed by secYF1(III)/secYR1(III) was used to amplify a phytoplasma-specific partial spc operon (about 1.7 kb) that includes the complete secY gene and partial rpl15 and map genes, as previously described (Lee et al. 2010). The obtained rp and partial spc amplicons were cloned and sequenced (GenBank accession numbers PP464295 and PP464296). The rp and secY gene sequences were searched against the non-redundant nucleotide collection in the NCBI database using BLASTN. The top hit for the rp gene was 'Ca. Phytoplasma luffae' (CP054393), with 83.24% identity (1068/1283 base-matching). The top hit for the secY gene was also 'Ca. Phytoplasma luffae' (CP054393), with 72.53% identity (1294/1784 base-matching). The percent identity of the BMSF sequences compared to the top hit is low since no other group 16SrXXXII rp and secY gene sequences are available for comparison. A subgroup 16SrXXXII-D phytoplasma strain has been previously reported associated with Camptotheca acuminata witches'-broom (Qiao et al. 2023) and Trema tomentosa witches'-broom (Yu et al. 2021) in China. To our knowledge, bitter melon represents a new host of 'Candidatus Phytoplasma malaysianum'-related strains, and this is the first report of BMSF disease in China. The findings suggest that 'Candidatus Phytoplasma malaysianum'-related strains infect not only ornamental plants but also crops.

Phytoplasmas are intracellular pathogenic bacteria that infect a wide range of plant species, including agriculturally important crops and ornamental trees. However, our understanding of the relationship between symptom severity, disease progression, and phytoplasma concentration remains limited due to the inability to inoculate phytoplasmas mechanically into new plant hosts. The present study investigated phytoplasma titer dynamics and symptom development in periwinkle and tomato, both infected with the same potato purple top (PPT) phytoplasma strain using a small seedling grafting approach. Virescence, phyllody, and witches’-broom (WB) symptoms sequentially developed in periwinkle, while in tomato plants, big bud (BB, a form of phyllody), cauliflower-like inflorescence (CLI), and WB appeared in order. Results from quantitative polymerase chain reaction (qPCR) targeting the PPT phytoplasma’s 16S rRNA gene revealed that in both host species, phytoplasma titers differed significantly at different infection stages. Notably, the highest phytoplasma concentration in periwinkles was observed in samples displaying phyllody symptoms, whereas in tomatoes, the titer peaked at the BB stage. Western blot analysis, utilizing an antibody specific to PPT phytoplasma, confirmed substantial phytoplasma presence in samples displaying phyllody and BB symptoms, consistent with the qPCR results. These findings challenge the conventional understanding that phytoplasma infection dynamics result in a higher titer at later stages, such as WB (excessive vegetative growth), rather than in the early stage, such as phyllody (abnormal reproductive growth). Furthermore, the PPT phytoplasma titer was markedly higher in periwinkles than in tomato plants, indicating differing susceptibilities between the hosts. This study reveals distinct host responses to PPT phytoplasma infection, providing valuable insights into phytoplasma titer dynamics and symptom development, with implications for the future management of agricultural disease.

Phytoplasmas are small, intracellular bacteria that infect a vast range of plant species, causing significant economic losses and impacting agriculture and farmers’ livelihoods. Early and rapid diagnosis of phytoplasma infections is crucial for preventing the spread of these diseases, particularly through early symptom recognition in the field by farmers and growers. A symptom database for phytoplasma infections can assist in recognizing the symptoms and enhance early detection and management. In this study, nearly 35,000 phytoplasma sequence entries were retrieved from the NCBI nucleotide database using the keyword “phytoplasma” and information on phytoplasma disease-associated plant hosts and symptoms was gathered. A total of 945 plant species were identified to be associated with phytoplasma infections. Subsequently, links to symptomatic images of these known susceptible plant species were manually curated, and the Phytoplasma Disease Symptom Database (iPhyDSDB) was established and implemented on a web-based interface using the MySQL Server and PHP programming language. One of the key features of iPhyDSDB is the curated collection of links to symptomatic images representing various phytoplasma-infected plant species, allowing users to easily access the original source of the collected images and detailed disease information. Furthermore, images and descriptive definitions of typical symptoms induced by phytoplasmas were included in iPhyDSDB. The newly developed database and web interface, equipped with advanced search functionality, will help farmers, growers, researchers, and educators to efficiently query the database based on specific categories such as plant host and symptom type. This resource will aid the users in comparing, identifying, and diagnosing phytoplasma-related diseases, enhancing the understanding and management of these infections.

Flavescence dorée (FD) is the most threatening grapevine yellows (GY) disease in Europe. Despite strict control measures, alarming signs of the spread of the disease in viticultural areas continue to be detected. FD is attributed to infection by phytoplasma strains of an incidentally cited species, ‘Candidatus Phytoplasma vitis’. In 2017, a GY field survey was carried out in traditional viticulture areas of Tuscany, central Italy. FD phytoplasma (FDp) was detected in 85 GY symptomatic vines, accounting for 17% of a total of 500 symptomatic samples screened. The FDp-positive vines were scattered in 50 vineyards across seven Tuscan provinces, indicating the distribution of FDp has further extended to central and southwestern parts of Tuscany including Florence and Livorno. Multilocus sequence typing of 15 representative FDp strains from six affected vineyards revealed that the Tuscan FDp strains constitute a highly homogeneous lineage within the subgroup 16SrV–C (FD-C). Single nucleotide polymorphisms (SNPs) were identified in the 16S rRNA, rp, and secY genes of the Tuscan FDp lineage. Such SNP markers provide clues to understanding the genetic relationships among different FDp lineages present in Europe and are useful for searching potential vectors and reservoirs involved in the spread of the FDp in the Tuscan region.

Phytoplasmas are small phloem-restricted and insect-transmissible bacteria that infect many plant species, including important crops and ornamental plants, causing severe economic losses. Our previous studies screened phytoplasmas in hundreds of leafhoppers collected from natural habitats worldwide and identified multiple genetically different phytoplasmas in seven leafhopper species (potential insect vectors). As an initial step toward determining the impact of these phytoplasmas on the ecosystem, ribulose 1,5-biphosphate carboxylase large subunit (rbcL), a commonly used plant DNA barcoding marker, was employed to identify the plant species that the phytoplasma-harboring leafhoppers feed on. The DNA of 17 individual leafhoppers was PCR amplified using universal rbcL primers. PCR products were cloned, and five clones per amplicon were randomly chosen for Sanger sequencing. Moreover, Illumina high-throughput sequencing on selected PCR products was conducted and confirmed no missing targets in Sanger sequencing. The nucleotide BLAST results revealed 14 plant species, including six well-known plant hosts of phytoplasmas such as tomato, alfalfa, and maize. The remaining species have not been documented as phytoplasma hosts, expanding our knowledge of potential plant hosts. Notably, the DNA of tomato and maize (apparently cultivated in well-managed croplands) was detected in some phytoplasma-harboring leafhopper species sampled in non-crop lands, suggesting the spillover/spillback risk of phytoplasma strains between crop and non-crop areas. Furthermore, our results indicate that barcoding (or metabarcoding) is a valuable tool to study the three-way interactions among phytoplasmas, plant hosts, and vectors. The findings contribute to a better understanding of phytoplasma host range, host shift, and disease epidemiology.

HomePlant DiseaseVol. 107, No. 3Draft Genome Sequence Resource of CBPPT1, a 'Candidatus Phytoplasma trifolii'-Related Strain Associated with Potato Purple Top Disease in the Columbia Basin, U.S.A. PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseDraft Genome Sequence Resource of CBPPT1, a 'Candidatus Phytoplasma trifolii'-Related Strain Associated with Potato Purple Top Disease in the Columbia Basin, U.S.A.Wei Wei, Jonathan Shao, Kristi D. Bottner-Parker, and Yan ZhaoWei Weihttps://orcid.org/0000-0003-3561-913XMolecular Plant Pathology Laboratory, USDA-ARS, Beltsville, MD 20705, Jonathan ShaoStatistics Group, Northeast Area Bioinformatics, USDA-ARS, Beltsville, MD 20705, Kristi D. Bottner-ParkerMolecular Plant Pathology Laboratory, USDA-ARS, Beltsville, MD 20705, and Yan Zhao†Corresponding author: Y. Zhao; E-mail Address: [email protected]https://orcid.org/0000-0002-0032-7535Molecular Plant Pathology Laboratory, USDA-ARS, Beltsville, MD 20705AffiliationsAuthors and Affiliations Wei Wei1 Jonathan Shao2 Kristi D. Bottner-Parker1 Yan Zhao1 † 1Molecular Plant Pathology Laboratory, USDA-ARS, Beltsville, MD 20705 2Statistics Group, Northeast Area Bioinformatics, USDA-ARS, Beltsville, MD 20705 Published Online:31 Dec 2022https://doi.org/10.1094/PDIS-08-22-1788-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Phytoplasmas are a large group of phloem-inhabiting, insect-transmitted plant pathogenic bacteria lacking a cell wall. They are etiological agents of diseases affecting more than a thousand plant species (Gasparich et al. 2020). Having descended from an ancestral gram-positive, low G + C walled bacterium, phytoplasmas underwent rapid evolution through substantial genome size reduction and recurrent horizontal gene acquisition (Wei et al. 2008; Zhao et al. 2014). Due to loss of many essential genes encoding diverse biosynthetic pathways (Kube et al. 2012; Oshima et al. 2004), phytoplasmas possess limited metabolic capacities and have a strong host dependency. Despite numerous attempts, pure phytoplasma culture has yet to be established in cell-free media. Although extant phytoplasmas have the same habitat specificity and life mode (Zhao et al. 2015) and comprise a monophyletic clade in 16S rRNA gene sequence-based phylogeny, the breadth of their genetic diversity is remarkable (Zhao et al. 2010). As unculturable bacteria, phytoplasmas are currently accommodated in a provisional genus 'Candidatus Phytoplasma' (The IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group 2004). To date, 48 'Candidatus Phytoplasma' species have been formally described (Wei and Zhao 2022). Phytoplasmas are also classified into groups and subgroups based on collective actual enzymatic (Lee et al. 1993, 1998) or computer-simulated (Wei et al. 2007) restriction fragment length polymorphism (RFLP) profiles of their 16S rRNA genes. Thirty-seven such groups have been established in the classification scheme (Jones et al. 2021; Wei and Zhao 2022), with each group containing at least one 'Candidatus Phytoplasma' species. Potato purple top (PPT) is a worldwide-occurring potato disease complex attributed to infection by phytoplasmas. Common symptoms of the disease include purple discoloration of terminal shoots and formation of aerial tubers. Affected plants often wilt and die prematurely, causing substantial tuber yield and quality losses (Munyaneza et al. 2005). At least five mutually distinct 'Candidatus Phytoplasma' species, including 'Ca. P. asteris', 'Ca. P. aurantifolia', 'Ca. P. pruni', 'Ca. P. trifolii', and 'Ca. P. americanum', have been linked to PPT disease incidences that have occurred in various geographic areas (Lee et al. 2004, 2006; Santos-Cervantes et al. 2010). The Columbia Basin potato purple top (CBPPT) phytoplasma is the etiological agent responsible for the PPT epidemics that repeatedly occurred in the Pacific Northwest region of the U.S. since 2002 (Munyaneza et al. 2005). The CBPPT phytoplasma is a 'Ca. P. trifolii'-related strain and is classified in the clover proliferation phytoplasma (CP) group, subgroup A (16SrVI-A). The phytoplasma has a broad plant host range and is capable of infecting potato, tomato, broccoli, carrot, radish, and many other vegetable crops (Lee et al. 2004, 2006; Santos-Cervantes et al. 2010). Since the CBPPT phytoplasma induces virescence symptoms in several hosts and is transmitted from plant to plant by beet leafhopper (Circulifer tenellus, Munyaneza et al. 2010), it has a trivial name, beet leafhopper-transmitted virescence agent or BLTVA.The CBPPT phytoplasma and its alternative host, tomato, have been used as a model system to study phytoplasma–host interactions. Studies revealed that (i) a single infection of the CBPPT phytoplasma was able to induce multiple mutually distinct symptoms in tomato sequentially and (ii) the symptom type on a given branch varied depending on the developmental stage of the apex when it became affected by the phytoplasma (Wei et al. 2013, 2019). Such findings led to a hypothesis that phytoplasma infection can derail the genetically preprogrammed fate of a developing meristem, thereby changing the growth pattern and morphology of the host (Wei et al. 2013). Conceivably, phytoplasma pathogenesis is a multifaceted process that involves complex interactions between the pathogen and its plant host. Previous studies in our laboratory suggested that, in CBPPT phytoplasma-infected tomato plants, the homeostasis of auxin, cytokinin, and gibberellin was disrupted, as the biosynthesis, signaling, and distribution of these phytohormones were altered. Studies also revealed that CBPPT phytoplasma infection in tomato significantly changed the expression profiles of numerous genes including defense-related genes, key meristem switching genes, bioactive gibberellin synthesis genes, marker genes involved in premature leaf senescence, and axillary bud release (Ding et al. 2013; Wei et al. 2013; Wei and Zhao 2022). However, it remains unknown what pathogenicity factor(s) the CBPPT phytoplasma possesses and how these factors trigger the above- observed host responses. As the first step toward answering these questions, the genome of a representative strain of the CBPPT phytoplasma, CBPPT1, was sequenced.The phytoplasma strain CBPPT1 was originally identified in diseased potato plants growing in Washington State (Lee et al. 2004) and was transmitted to Madagascar periwinkle (Catharanthus roseus) via dodder (Cuscuta campestris), a parasitic vine. Once established in periwinkle, the strain was maintained in a greenhouse via serial shoot graft transmission. Over the years, the phytoplasma has consistently induced the same set of characteristic symptoms in the experimental host, indicating its pathogenicity remains essentially unchanged. Infected periwinkle shoots exhibiting phyllody symptom were used for genomic DNA extraction using a modified DNA extraction protocol as described by Lee et al. (1991). The DNA was sent to BGI-Americas for library construction and sequencing on the Illumina HiSeq 4000 platform. A total of 468,647,184 clean reads, with an average length of 150 bp (Q20 = 96.19%), were obtained. Genome assembly and analysis were performed at the USDA-ARS' in-house bioinformatics facility. The phytoplasma reads were separated from the plant host reads by mapping the reads to available phytoplasma genomes using the bowtie2 program (v2.4.5, Langmead and Salzberg 2012). All completed and draft phytoplasma genomes in the National Center for Biotechnology Information (NCBI) BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) were used in the mapping. The assembly was performed using the Velvet assembler (v1.2.10, Zerbino and Birney 2008) and the SPAdes assembler (v3.14.0, Prjibelski et al. 2020). The phytoplasma contigs were further verified based on their nucleotide sequence identity scores with previously sequenced phytoplasma genomes using the BLASTN program.The assembled draft genome of CBPPT1 consists of 71 contigs totaling 512,902 bp with an average coverage of 1,434 and an N50 value of 78,175. Approximately 90% of the assembled sequences are in the 10 largest contigs. The G + C content of the CBPPT1 draft genome is 22.62%. One RNA operon and 26 tRNA genes were identified using software Barrnap (v0.8) and tRNAscan (v1.4), respectively (Fichant and Burks 1991; Seemann 2013). The rRNA gene sequence of CBPPT1 shares 99.61% sequence identity with that of CP (AY390261), the reference strain of 'Ca. P. trifolii' and the representative strain of group 16SrVI, confirming CBPPT1 is a 'Ca. P. trifolii'-related strain. The CBPPT1 is the first 'Ca. P. trifolii'-related strain and the first 16SrVI phytoplasma whose genome is sequenced.The completeness of the CBPPT1 draft genome assembly was assessed by examining the presence of benchmarking universal single-copy orthologs using software BUSCO v5 (Manni et al. 2021). The obtained BUSCO score, 62.1%, was then compared with those of complete phytoplasma genomes. Currently, there are 12 complete phytoplasma genomes available for comparative analysis, and the BUSCO scores of these genomes range from 58.9 to 63.9%, with an average of 61.2%. It is reasonable to conclude that the CBPPT1 draft genome is near completion as its BUSCO score is above the average score of the 12 complete phytoplasma genomes.Protein coding genes (CDS) were predicted using Genemark (v3.36) and the gene annotation was performed using the BLASTP program against the nr database at the NCBI and the Blast2GO module of the OmicsBox (v2.0.36, BioBam Bioinformatics 2019). A total of 554 CDS were predicted. Among them, 442 were assigned to at least one gene ontology (GO) term, and 402 were assigned to InterPro functional categories.The genome size of different phytoplasmas varies, ranging from 530 to 1,350 kbp (Marcone et al. 1999). Apparently, the genome size of CBPPT1 is at the low end of the spectrum. Consistent with its small size, it has only one rRNA operon and possesses much less repetitive genes when compared with other phytoplasmas such as 'Ca. P. asteris'-related strain OYM (Oshima et al. 2004) and 'Ca. P. australiense'-related strain SLY (Andersen et al. 2013). Nevertheless, the CBPPT1 genome contains multiple putative pathogenicity genes (Table 1) including those encoding homologs of SAP54 and SAP05, which are known phytoplasma virulence factors (effectors) responsible for phyllody and witches'-broom symptoms, respectively (Huang et al. 2021; MacLean et al. 2014). Among other putative CBPPT1 pathogenicity genes are those encoding more than a dozen secretory proteins, most of which belong to the SVM family (Table 1). In genomes of other phytoplasmas, SVM protein genes often reside in prophage-derived pathogenicity islands (Wei et al. 2008).Table 1. Putative effectors and other secretory proteins encoded by the CBPPT1 genomeLocus tagContig no.Length (aa)AnnotationM8044_0001532152Immunodominant membrane proteinM8044_0003626256Hypothetical proteinM8044_0003876200Hypothetical protein (SAP09-like)M8044_000392690SVM family protein (SAP44-like)M8044_0004661043Hypothetical proteinM8044_00048313125Phytoplasma effector causing phyllody symptoms (SAP54-like)M8044_00048413774ATP-dependent Zn proteaseM8044_0004931568SVM family proteinM8044_00049415111SVM family proteinM8044_00050817125SVM family proteinM8044_00050917259SVM family proteinM8044_00051822158Putative secreted protein (SAP42-like)M8044_00051922188SVM family proteinM8044_00053029188SVM family protein (SAP19-like)M8044_0005353687Hypothetical protein (SAP53-like)M8044_00053639125Hypothetical protein (SAP05-like)Table 1. Putative effectors and other secretory proteins encoded by the CBPPT1 genomeView as image HTML Since CBPPT phytoplasma is capable of inducing multiple mutually distinct and developmental stage-dependent symptoms in the host, the phytoplasma may possess multiple pathogenicity factors and each could interact with host component(s) in a stage-specific manner, leading to distinct symptoms as previously postulated (Wei et al. 2019). We hope the draft CBPPT1 genome sequence can serve as a resource for the search for and eventual identification of such pathogenicity factors.Data AvailabilityThe draft genome sequence data of the 'Candidatus Phytoplasma trifolii'-related strain CBPPT1 have been uploaded to the NCBI's BioProject database under accession number PRJNA839414. The annotated sequence has been deposited in the GenBank under accession number JANHJP000000000.The author(s) declare no conflict of interest.Literature CitedAndersen, M. T., Liefting, L. W., Havukkala, I., and Beever, R. 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The American Phytopathological Society, 2022.FundingU.S. Department of Agriculture, Agricultural Research ServiceKeywordsBLTVAclover proliferation groupgenomephytoplasmapotato purple topThe author(s) declare no conflict of interest.PDF download

'Candidatus Phytoplasma trifolii' is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species such as alfalfa, clover, eggplant, pepper, potato, and tomato. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT) and potato witches' broom (PWB) that occurred along the Pacific Coast of the United States since 2002, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive, and rapid detection of the pathogens. Here we report the development of a DNA endonuclease targeted CRISPR trans reporter (DETECTR) assay that couples isothermal amplification and Cas12a transcleavage of fluorescent oligonucleotide reporter for highly sensitive and specific detection of 'Candidatus Phytoplasma trifolii'-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the 16S-23S ribosomal DNA intergenic transcribed spacer sequences from PPT- and PWB-diseased samples at the attomolar sensitivity level. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate readers or lateral flow assays for potentially high-throughput and/or field-deployable disease diagnostics.

Phytoplasmas are obligate transkingdom bacterial parasites that infect a variety of plant species and replicate in phloem-feeding insects in the order Hemiptera, mainly leafhoppers (Cicadellidae). The insect capacity in acquisition, transmission, survival, and host range directly determines the epidemiology of phytoplasmas. However, due to the difficulty of insect sampling and the lack of follow-up transmission trials, the confirmed phytoplasma insect hosts are still limited compared with the identified plant hosts. Recently, quantitative polymerase chain reaction (qPCR)-based quick screening of 227 leafhoppers collected in natural habitats unveiled the presence of previously unknown phytoplasmas in six samples. In the present study, 76 leafhoppers, including the six prescreened positive samples, were further examined to identify and characterize the phytoplasma strains by semi-nested PCR. A total of ten phytoplasma strains were identified in leafhoppers from four countries including South Africa, Kyrgyzstan, Australia, and China. Based on virtual restriction fragment length polymorphism (RFLP) analysis, these ten phytoplasma strains were classified into four distinct ribosomal (16Sr) groups (16SrI, 16SrIII, 16SrXIV, and 16SrXV), representing five new subgroups (16SrI-AO, 16SrXIV-D, 16SrXIV-E, 16SrXIV-F, and 16SrXV-C). The results strongly suggest that the newly identified phytoplasma strains not only represent new genetic subgroup lineages, but also extend previously undiscovered geographical distributions. In addition, ten phytoplasma-harboring leafhoppers belonged to seven known leafhopper species, none of which were previously reported insect vectors of phytoplasmas. The findings from this study provide fresh insight into genetic diversity, geographical distribution, and insect host range of phytoplasmas. Further transmission trials and screening of new potential host plants and weed reservoirs in areas adjacent to collection sites of phytoplasma harboring leafhoppers will contribute to a better understanding of phytoplasma transmission and epidemiology.

Wheat blue dwarf (WBD) is one of the most economically damaging cereal crop diseases in northwestern PR China. The agent associated with the WBD disease is a phytoplasma affiliated with the aster yellows (AY) group, subgroup C (16SrI- C). Since phytoplasma strains within the AY group are ecologically and genetically diverse, it has been conceived that the AY phytoplasma group may consist of more than one species. This communication presents evidence to demonstrate that, while each of the two 16 rRNA genes of the WBD phytoplasma shares >97.5 % sequence similarity with that of the ?Candidatus Phytoplasma asteris? reference strain, the WBD phytoplasma clearly represents an ecologically separated lineage: the WBD phytoplasma not only has its unique transmitting vector (Psammotettix striatus) but also elicits a distinctive symptom in its predominant plant host (wheat). In addition, the WBD phytoplasma possesses molecular characteristics that further manifest its significant divergence from ?Ca. P. asteris?. Such molecular characteristics include lineage- specific antigenic membrane proteins and a lower than 95 % genome- wide average nucleotide identity score with ?Ca. P. asteris?. These ecological, molecular and genomic evidences justify the recognition of the WBD phytoplasma as a novel taxon, ?Candidatus Phytoplasma tritici'.

The phytopathogen Spiroplasma phoeniceum was isolated from diseased plants of Madagascar periwinkle [Catharanthus roseus (L.) G. Don]. Here, we report the nucleotide sequence of the 1,791,576-bp circular chromosome and three plasmids of strain P40T This information serves as a resource for comparative analyses of spiroplasmal adaptations to diverse ecological niches.

Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16S rRNA gene, for which "universal" primers are available. To date, 36 ribosomal (16Sr) groups and more than 150 subgroups have been delineated by RFLP analysis of 16S rRNA gene sequences. However, in recent years, the use of moderately conserved genes as additional genetic markers has enhanced the resolving power in delineating distinct phytoplasma strains among members of some 16Sr subgroups. This chapter describes the methodology of amplification, differentiation, and classification of phytoplasma based on less-conserved non-ribosomal genes, named rp and secY. Actual and virtual RFLP analyses of amplicons obtained by semi-universal or group-specific rp and secY gene-based primers are used for finer differentiation of phytoplasma strains within a given group. The rp and secY gene-based classification not only readily resolves 16Sr subgroups within a given 16Sr group, but also provides finer differentiation of closely related phytoplasma strains within a given 16Sr subgroup.

Phytoplasmas (class Mollicutes) are causal agents of plant diseases with an economic impact on crops or threatening local biodiversity. A survey was conducted from 2012 to 2016 on infected Catharanthus roseus plants that exhibited symptoms reminiscent of phytoplasma infection throughout Costa Rica. A total of 73 plants were collected exhibiting symptoms such as virescence, phyllody, axillary proliferation, little leaf, leaf malformation, chlorosis, or yellowing. All samples were tested by nested PCR using phytoplasma universal and specific primer pairs. Phytoplasma infection was detected in 52 (71.2 %) of the plants collected. Phytoplasmas of six subgroups belonging to 16Sr groups I, III, IX, XIII and XV were identified based on sequencing and in silico RFLP analyses. ´Candidatus Phytoplasma asteris´ (16SrI) was the predominant group among the positive samples (n = 30) showing variety of symptoms and wide distribution from sea level to ca. 1 400 masl in six of the seven Costa Rican provinces. Group 16SrIII was the second most abundant (14 samples); and the remaining three groups were seldom found in C. roseus (8 samples). Moreover, group 16SrXIII phytoplasma was detected for the first time in the country. To the best of our knowledge, this is the first report of natural infection of C. roseus with phytoplasma subgroups 16SrI-B, 16SrI-P, 16SrIII-F, 16SrIX-F, 16SrXIII-A, and 16SrXV-B in Costa Rica and Central America.

The NJAY (New Jersey aster yellows) strain of ‘Candidatus Phytoplasma asteris’ is a significant plant pathogen responsible for causing severe lettuce yellows in the U.S. state of New Jersey. A draft genome sequence was prepared for this organism. A total of 177,847 reads were assembled into 75 contigs > 518 bp with a total base value of 652,092 and an overall [G+C] content of 27.1%. A total of 733 protein coding genes were identified. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MAPF00000000. This draft genome was used for genome- and gene-based comparative phylogenetic analyses with other phytoplasmas, including the closely related ‘Ca. Phytoplasma asteris’ strain, aster yellows witches’- broom (AY-WB). NJAY and AY-WB exhibit approximately 0.5% dissimilarity at the nucleotide level among their shared genomic segments. Evidence indicated that NJAY harbors four plasmids homologous to those known to encode pathogenicity determinants in AY-WB, as well as a chromosome-encoded mobile unit. Apparent NJAY orthologs to the important AY-WB virulence factors, SAP11 and SAP54, were identified. A number of secreted proteins, both membrane-bound and soluble, were encoded, with many bearing similarity to known AY-WB effector molecules and others representing possible secreted proteins that may be novel to the NJAY lineage.

A large scale survey of diseased legume plants (mainly clover and alfalfa in the Fabaceae family) was conducted from 2009 to 2013 in four Economic Regions of Russia, Northern (Arkhangelsk and Vologda oblast), Central (Moscow oblast), Volga (Samara oblast) and West Siberian (Novosibirsk oblast). The majority of infected clover plants exhibited symptoms typical of clover phyllody (CPh), clover yellow edge (CYE), or clover proliferation (CP), and infected alfalfa plants exhibited symptoms typical of alfalfa witches’-broom (AWB). Of 161 symptomatic plants from 22 different legume species, 103 tested positive for phytoplasma infection. Phytoplasmas belonging to four groups and six subgroups were detected, of which 31.1% were group 16SrI, with the majority belonging to subgroup 16SrI-C- (causal agent of CPh disease), two belonging to 16SrI-B and two group 16SrI phytoplasmas not identified to the subgroup level;47.6% were group 16SrIII, with the majority belonging to subgroup 16SrIII-B or 16SrIII-B variant (causal agent of CYE disease), and one strain belonging to16SrIII-F; 8.7% were subgroup 16SrVI-A (causal agent of CP and AWB diseases); 9.7% were subgroup 16SrXII-A (causal agent of AWB disease); and 2.9% were mixed infected with subgroups 16SrIII-B and 16SrI-C. The predominant phytoplasma species detected varied by region. In the Northern and Central Regions, the majority of the phytoplasmas detected belonged to subgroups 16SrI-C and 16SrIII-B. In the West Siberian and the Volga Regions, the phytoplasmas predominately detected belonged to subgroups 16SrVI-A and 16SrXII-A, respectively. Subgroup 16SrIII-F was detected in a single plant in the West Siberian Region and a mixed infection of 16SrIII-B and 16SrI-C was detected in three plants, one in the Northern Region and two in the Central Region. Eleven species of insects of the order Hemiptera, suborder Auchenorrhyncha, were collected from leguminous plants in the Moscow oblast of the Central Region. Euscelis incisus and Aphrodes bicinctus were the most prevalent species and may be potential phytoplasma vectors in the Central Region.

A large scale survey on diseased potato plants that exhibited symptoms similar to those of stolbur, potato purple top wilt, potato witches’-broom, and potato round-leaf in eight Economical Regions of Russia was conducted from 2006 to 2012. A total of 1228 potatoes were collected and the associated phytoplasmas were identified. Phytoplasmas belonging to five 16S rRNA (16Sr) phytoplasma groups (16SrI, 16SrII, 16SrIII, 16SrVI, and 16SrXII) and at least eight subgroups (16SrI-B, 16SrI-C, 16SrI-P, 16SrII-A, 16SrIII-B, 16SrVI-A, 16SrVI-C, and 16SrXII-A) were identified. To our knowledge, subgroup 16SrIII-B and 16SrVI-C phytoplasmas have not been reported to infect potatoes. Based on the results of the survey, the percentages of infection caused by various phytoplasmas were: 40.2 % (16SrXII), 25.5 % (16SrIII), 20.9 % (16SrI), 12.4 % (16SrVI), and 1.2 % (16SrII). The results also indicated potato diseases exhibiting stolbur or similar symptoms could be caused by stolbur phytoplasma (16SrXII-A) alone or by other phytoplasma groups (e.g., 16SrI and 16SrIII). A survey on potential insect vectors was conducted from 2009 to 2012 in the Moscow oblast of the Central Region. A total of 23 different species of Hemiptera insects were screened for the presence of phytoplasmas. Eight leafhopper and three froghopper species carried phytoplasmas. Euscelis incisus and Macrosteles laevis were the most prevalent. The other species were present in very low populations.

In China, potato is widely cultivated economic crop. Recently, potato diseases with characteristic symptoms of phytoplasma infections were found in potato fields. In 2006 and 2007, samples exhibiting symptoms including rosette and upright growth, upward rolling, yellowing and purpling of leaves, shortened and thickened internodes and formation of aerial tubers were collected from plants in Yunnan and Inner Mongolia and analyzed for the presence of phytoplasmas. DNA was extracted from tissues of 63 symptomatic and 10 asymptomatic plants. Phytoplasma 16S rRNA was amplified by PCR with primer pair P1/P7, followed by nested PCR with P1A/P7A, P1A/16S-SR or R16F2n/R16R2n. Twenty nine symptomatic plants (46 %), but no asymptomatic plants, tested positive for phytoplasmas. Nested PCR products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas from diseased potatoes shared 98.1–99.8 % similarity with ‘Candidatus Phytoplasma fragariae’ (16SrXII-E) and other strains in 16SrXII subgroups. RFLP and phylogenetic analyses also indicated that phytoplasmas of group 16SrXII were associated with phytoplasma infected potatoes in China; these strains are most closely related to subgroup 16SrXII-E. Our results showed that five strains belonged to 16SrXII-E; 11 strains were designated as a new 16SrXII subgroup, 16SrXII-I; and subgroup affiliations of all others were not determined. The genetic diversity of the strains was corroborated by sequence analysis of ribosomal protein genes, the elongation factor Tu gene (tuf) and the pre-protein translocase membrane subunit gene (secY). The results illustrated the complexity and diversity of phytoplasmas associated with potatoes in China.