'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 trans-cleavage 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 (rDNA) intergenic transcribed spacer (ITS) 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 reader or lateral flow assay for potentially high-throughput and/or field-deployable disease diagnostics.
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 '
Phytoplasma asteris' reference strain, the WBD phytoplasma clearly represents an ecologically separated lineage: the WBD phytoplasma not only has its unique transmitting vector (
) 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 '
. P. asteris'. Such molecular characteristics include lineage-specific antigenic membrane proteins and a lower than 95 % genome-wide average nucleotide identity score with '
. P. asteris'. These ecological, molecular and genomic evidences justify the recognition of the WBD phytoplasma as a novel taxon, '
Phytoplasma tritici'.
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.
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 (
), 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
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.
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.
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 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.
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 (
sec
Y). The results illustrated the complexity and diversity of phytoplasmas associated with potatoes in China.
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.
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