Guilt by Association: DNA Barcoding-Based Identification of Potential Plant Hosts of Phytoplasmas from Their Insect Carriers.

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.

Cas12a-Based Diagnostics for Potato Purple Top Disease Complex Associated with Infection by 'Candidatus Phytoplasma trifolii'-Related Strains.

'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.

The agent associated with blue dwarf disease in wheat represents a new phytoplasma taxon, 'Candidatus Phytoplasma tritici'.

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'.

Differential Symptomology, Susceptibility, and Titer Dynamics Manifested by Phytoplasma-Infected Periwinkle and Tomato Plants.

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.

Identification of Phytoplasmas Representing Multiple New Genetic Lineages from Phloem-Feeding Leafhoppers Highlights the Diversity of Phytoplasmas and Their Potential Vectors.

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.

PCR-Based Sequence Analysis on Multiple Genes Other than 16S rRNA Gene for Differentiation of Phytoplasmas.

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.

Draft genome sequence of the New Jersey aster yellows strain of 'Candidatus Phytoplasma asteris'.

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.